Patent Publication Number: US-2023139821-A1

Title: Flow cells and methods for making the same

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This application claims the benefit of U.S. Provisional Application Ser. No. 63/272,927, filed Oct. 28, 2021, the contents of which is incorporated by reference herein in its entirety. 
    
    
     REFERENCE TO SEQUENCE LISTING 
     The Sequence Listing submitted herewith via EFS-Web is hereby incorporated by reference in its entirety. The name of the file is ILI219B_IP-2182-US_Sequence_Listing_ST25.txt, the size of the file is 3,380 bytes, and the date of creation of the file is May 2, 2022. 
     BACKGROUND 
     Some available platforms for sequencing nucleic acids utilize a sequencing-by-synthesis approach. With this approach, a nascent strand is synthesized, and the addition of each monomer (e.g., nucleotide) to the growing strand is detected optically and/or electronically. Because a template strand directs synthesis of the nascent strand, one can infer the sequence of the template DNA from the series of nucleotide monomers that were added to the growing strand during the synthesis. In some examples, sequential paired-end sequencing may be used, where forward strands are sequenced and removed, and then reverse strands are constructed and sequenced. In other examples, simultaneous paired-end sequencing may be used, where forward strands and reverse strands are sequenced at the same time. 
     SUMMARY 
     For simultaneous paired-end sequencing, different primer sets are attached to different regions within each depression and/or on each protrusion of a flow cell surface. These primer sets are attached through polymeric hydrogel(s). Several example methods are described herein to place the primers sets in the desired regions such that, during optical imaging, the signals from one region do not deleteriously affect the signals from another region. In particular, the methods reduce or eliminate the occurrence of one region and primer set surrounding another region and primer set in a padlock like conformation or configuration. It has been found that by reducing the padlock like conformation, signal resolution from each of the regions is improved. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Features of examples of the present disclosure will become apparent by reference to the following detailed description and drawings, in which like reference numerals correspond to similar, though perhaps not identical, components. For the sake of brevity, reference numerals or features having a previously described function may or may not be described in connection with other drawings in which they appear. 
         FIG.  1 A  shows a top view of an example depression with a padlock conformation; 
         FIG.  1 B  and  FIG.  1 C  show top views of example depressions without a padlock like conformation; 
         FIG.  2 A  is a top view of an example flow cell; 
         FIG.  2 B  through  FIG.  2 E  are enlarged, and partially cutaway views of different examples of a flow channel of the flow cell; 
         FIG.  3 A  through  FIG.  3 D  are schematic views of different examples of primer sets that are used in some examples of the flow cells disclosed herein; 
         FIG.  4 A  through  FIG.  4 D  are schematic views that together illustrate an example of a method to pattern a functionalized layer in a deep portion of a multi-depth depression; 
         FIG.  5 A  through  FIG.  5 E  are schematic views that together with  FIG.  4 A  through  FIG.  4 D  depict an example of a method to generate a flow cell surface; 
         FIG.  6 A  through  FIG.  6 F  are schematic views that together with  FIG.  4 A  through  FIG.  4 D  depict an example of a method to generate a flow cell surface; 
         FIG.  7 A  through  FIG.  7 F  are schematic views that together with  FIG.  4 A  through  FIG.  4 D  depict an example of a method to generate a flow cell surface; 
         FIG.  7 A  through  FIG.  7 D  and  FIG.  7 G  are schematic views that together with  FIG.  4 A  through  FIG.  4 D  depict an example of a method to generate a flow cell surface; 
         FIG.  7 A  through  FIG.  7 D  and  FIG.  7 H  through  FIG.  7 L  are schematic views that together with  FIG.  4 A  through  FIG.  4 D  depict an example of a method to generate a flow cell surface; 
         FIG.  8 A  through  FIG.  8 G  are schematic views that together with  FIG.  4 A  through  FIG.  4 D  depict an example of a method to generate a flow cell surface; 
         FIG.  9 A  through  FIG.  9 H  are schematic views that depict another example of a method to generate a flow cell surface; 
         FIG.  10 A  through  FIG.  10 D  are schematic views that together illustrate an example of a method to pattern a metal film in a deep portion of a multi-depth depression; 
         FIG.  11 A  through  FIG.  11 E  are schematic views that together with  FIG.  10 A  through  FIG.  10 D  depict an example of a method to generate a flow cell surface; 
         FIG.  12 A  through  FIG.  12 G  are schematic views that together with  FIG.  10 A  through  FIG.  10 D  depict an example of a method to generate a flow cell surface; 
         FIG.  12 A  through  FIG.  12 D  and  FIG.  12 H  through  FIG.  12 J  are schematic views that together with  FIG.  10 A  through  FIG.  10 D  depict an example of a method to generate a flow cell surface; 
         FIG.  13 A  through  FIG.  13 I  are schematic views that together with  FIG.  10 A  through  FIG.  10 D  depict an example of a method to generate a flow cell surface; 
         FIG.  14 A  through  FIG.  14 I  are schematic views that together with  FIG.  10 A  through  FIG.  10 D  depict an example of a method to generate a flow cell surface; 
         FIG.  15 A  through  FIG.  15 F  are schematic views that together with  FIG.  10 A  through  FIG.  10 D  depict an example of a method to generate a flow cell surface; 
         FIG.  16 A  through  FIG.  16 M  are schematic views that together depict an example of a method to generate a flow cell surface; 
         FIG.  17 A  through  FIG.  17 K  are schematic views that together depict an example of a method to generate a flow cell surface; 
         FIG.  18 A  through  FIG.  18 I  are schematic views that together depict an example of a method to generate a flow cell surface; 
         FIG.  19 A  through  FIG.  19 K  are top views that also depict the method shown in  FIG.  18 A  through  FIG.  18 I ; 
         FIG.  20    is a scanning electron micrograph (SEM) image of a cross-section of a multi-depth depression having a photoresist therein; 
         FIG.  21    is a SEM image of the multi-depth depression of  FIG.  20    after the resin is etched around the photoresist; 
         FIG.  22 A  is a SEM image of a top view of multi-depth depressions having a photoresist therein; and 
         FIG.  22 B  is a SEM image of the multi-depth depressions of  FIG.  22 A  after the photoresist is developed and soluble portions are removed. 
     
    
    
     DETAILED DESCRIPTION 
     Examples of the flow cells disclosed herein may be used for sequencing, examples of which include simultaneous paired-end nucleic acid sequencing. 
     For simultaneous paired-end sequencing, different primer sets are attached to different regions within each depression and/or on each protrusion of the flow cell. In these examples, the primer sets may be controlled so that the cleaving (linearization) chemistry is orthogonal in the different regions. In these examples, orthogonal cleaving chemistry may be realized through identical cleavage sites that are attached to different primers in the different sets, or through different cleavage sites that are attached to different primers in the different sets. This enables a cluster of forward strands to be generated in one region and a cluster of reverse strands to be generated in another region. In an example, the regions are directly adjacent to one another. In another example, any space between the regions is small enough that clustering can span the two regions. In any of these examples, the forward and reverse strands are spatially separate, which separates the fluorescence signals from both reads while allowing for simultaneous base calling of each read. 
     It has been found that some methods used to produce the spatially separate regions where the primer sets (and ultimately the forward and reverse strands) are attached generate a padlock like conformation where, from a top view, one region is surrounded by the other region within the depression. An example of this padlock like conformation is shown in  FIG.  1 A , which depicts the top view of one depression  20 ,  20 ′. As shown in  FIG.  1 A , the depression  20 ,  20 ′ of the flow cell includes adjacent functionalized layers  24 ,  26 , which define the regions where the different primer sets (not shown) are respectively attached. In this example, the functionalized layer  26  is formed in part  31 A of the depression  20 ,  20 ′, and it is desirable for the other functionalized layer  24  to be formed in the adjacent part  31 B of the depression  20 ,  20 ′. However, as a result of the method used, the functionalized layer  26  is applied along sidewall(s)  29  of the depression  20 ,  20 ′ in the adjacent part  31 B. In the depression  20  (having a single depth), the sidewall  29  is a perimeter P of the depression  20 . In the multi-depth depression  20 ′, the sidewalls  29  include the perimeter P and an internal wall I that separates the multiple depths (reference numerals  48  and  50 , see, e.g.,  FIG.  2 C  and  FIG.  4 A ). In either depression  20 ,  20 ′, the functionalized layer  26  may align the perimeter  29 , P, and surround the functionalized layer  24 , generating the padlock like conformation  33 . Additionally, in the multi-depth depression  20 ′, the functionalized layer  26  may also align portions of the internal wall  29 , I. Forward or reverse strands will form during amplification on the functionalized layer  26  in the padlock like conformation  33 , and during sequencing, the signals from these strands may contaminate the signals from the strands formed on the functionalized layer  24 . In some of the examples disclosed herein, the methods reduce the padlock like conformation  33  (e.g., as shown in  FIG.  1 B ) because at least a portion of the functionalized layer  26  that is present in the part  31 B of the depression  20 ,  20 ′ is reduced. In other examples disclosed herein, the methods eliminate the padlock like conformation  33  (e.g., as shown in  FIG.  1 C ) because the functionalized layer  26  is no longer present in the part  31 B of the depression  20 ,  20 ′. 
     Definitions 
     It is to be understood that terms used herein will take on their ordinary meaning in the relevant art unless specified otherwise. Several terms used herein and their meanings are set forth below. 
     The singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. 
     The terms comprising, including, containing and various forms of these terms are synonymous with each other and are meant to be equally broad. 
     The terms top, bottom, lower, upper, on, etc. are used herein to describe the flow cell and/or the various components of the flow cell. It is to be understood that these directional terms are not meant to imply a specific orientation, but are used to designate relative orientation between components. The use of directional terms should not be interpreted to limit the examples disclosed herein to any specific orientation(s). 
     The terms first, second, etc. also are not meant to imply a specific orientation or order, but rather are used to distinguish one component from another. 
     It is to be understood that the ranges provided herein include the stated range and any value or sub-range within the stated range, as if such values or sub-ranges were explicitly recited. For example, a range of about 400 nm to about 1 μm (1000 nm), should be interpreted to include not only the explicitly recited limits of about 400 nm to about 1 μm, but also to include individual values, such as about 708 nm, about 945.5 nm, etc., and sub-ranges, such as from about 425 nm to about 825 nm, from about 550 nm to about 940 nm, etc. Furthermore, when “about” and/or “substantially” are/is utilized to describe a value, they are meant to encompass minor variations (up to +/−10%) from the stated value. 
     An “acrylamide monomer” is a monomer with the structure 
     
       
         
         
             
             
         
       
     
     or a monomer including an acrylamide group. Examples of the monomer including an acrylamide group include azido acetamido pentyl acrylamide: 
     
       
         
         
             
             
         
       
     
     and N-isopropylacrylamide: 
     
       
         
         
             
             
         
       
     
     Other acrylamide monomers may be used. 
     The term “activation,” as used herein, refers to a process that generates reactive groups at the surface of a base support or an outermost layer of a multi-layered structure. Activation may be accomplished using silanization or plasma ashing. While the figures do not depict a separate silanized layer or hydroxyl (—OH groups) from plasma ashing, it is to be understood that activation generates a silanized layer or —OH groups at the surface of the activated support or layer to covalently attach the functionalized layers to the underlying support or layer. 
     An aldehyde, as used herein, is an organic compound containing a functional group with the structure —CHO, which includes a carbonyl center (i.e., a carbon double-bonded to oxygen) with the carbon atom also bonded to hydrogen and an R group, such as an alkyl or other side chain. The general structure of an aldehyde is: 
     
       
         
         
             
             
         
       
     
     As used herein, “alkyl” refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds). The alkyl group may have 1 to 20 carbon atoms. Example alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like. As an example, the designation “C1-4 alkyl” indicates that there are one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, isobutyl, sec-butyl, and t-butyl. 
     As used herein, “alkenyl” refers to a straight or branched hydrocarbon chain containing one or more double bonds. The alkenyl group may have 2 to 20 carbon atoms. Example alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and the like. 
     As used herein, “alkyne” or “alkynyl” refers to a straight or branched hydrocarbon chain containing one or more triple bonds. The alkynyl group may have 2 to 20 carbon atoms. 
     As used herein, “aryl” refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent carbon atoms) containing only carbon in the ring backbone. When the aryl is a ring system, every ring in the system is aromatic. The aryl group may have 6 to 18 carbon atoms. Examples of aryl groups include phenyl, naphthyl, azulenyl, and anthracenyl. 
     An “amine” or “amino” functional group refers to an —NR a R b  group, where R a  and R b  are each independently selected from hydrogen 
     
       
         
         
             
             
         
       
     
     C1-6 (or C1-C6) alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocycle, C6-10 aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein. 
     As used herein, the term “attached” refers to the state of two things being joined, fastened, adhered, connected or bound to each other, either directly or indirectly. For example, a nucleic acid can be attached to a functionalized polymer by a covalent or non-covalent bond. A covalent bond is characterized by the sharing of pairs of electrons between atoms. A non-covalent bond is a physical bond that does not involve the sharing of pairs of electrons and can include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions and hydrophobic interactions. 
     An “azide” or “azido” functional group refers to —N 3 . 
     As used herein, a “bonding region” refers to an area of a patterned structure that is to be bonded to another material, which may be, as examples, a spacer layer, a lid, another patterned structure, etc., or combinations thereof (e.g., a spacer layer and a lid, or a spacer layer and another patterned structure). The bond that is formed at the bonding region may be a chemical bond (as described herein), or a mechanical bond (e.g., using a fastener, etc.). 
     As used herein, “carbocycle” means a non-aromatic cyclic ring or ring system containing only carbon atoms in the ring system backbone. When the carbocycle is a ring system, two or more rings may be joined together in a fused, bridged or spiro-connected fashion. Carbocycles may have any degree of saturation, provided that at least one ring in a ring system is not aromatic. Thus, carbocycles include cycloalkyls, cycloalkenyls, and cycloalkynyls. The carbocycle group may have 3 to 20 carbon atoms. Examples of carbocycle rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicyclo[2.2.2]octanyl, adamantyl, and spiro[4.4]nonanyl. 
     As used herein, the term “carboxylic acid” or “carboxyl” as used herein refers to —COON. 
     As used herein, “cycloalkylene” means a fully saturated carbocycle ring or ring system that is attached to the rest of the molecule via two points of attachment. 
     As used herein, “cycloalkenyl” or “cycloalkene” means a carbocycle ring or ring system having at least one double bond, wherein no ring in the ring system is aromatic. Examples include cyclohexenyl or cyclohexene and norbornenyl or norbornene. Also as used herein, “heterocycloalkenyl” or “heterocycloalkene” means a carbocycle ring or ring system with at least one heteroatom in ring backbone, having at least one double bond, wherein no ring in the ring system is aromatic. 
     As used herein, “cycloalkynyl” or “cycloalkyne” means a carbocycle ring or ring system having at least one triple bond, wherein no ring in the ring system is aromatic. An example is cyclooctyne. Another example is bicyclononyne. Also as used herein, “heterocycloalkynyl” or “heterocycloalkyne” means a carbocycle ring or ring system with at least one heteroatom in ring backbone, having at least one triple bond, wherein no ring in the ring system is aromatic. 
     As used herein, the terms “deep portion” and “shallow portion” refer to three-dimensional (3D) spaces within a multi-depth depression or the multi-depth trench. In the multi-depth depression or trench, the deep portion has a greater depth than the shallow portion, as measured, e.g., from an opening of the multi-depth depression or trench. In some examples of the method disclosed herein, the material that defines the multi-depth depression is processed, and the configurations of the deep and/or shallow portions may change as a result of this processing. In these instances, the terms deep portion and shallow portion may be used to orient the areas of the original multi-depth depression that are being processed, but may no longer be the respective three-dimensional (3D) spaces within the multi-depth depression. As one example, a resin layer that defines a multi-depth depression may be etched to create a multi-step protrusion having surfaces at different heights that correspond with the location, respectively, of the original shallow and deep portions. 
     The term “depositing,” as used herein, refers to any suitable application technique, which may be manual or automated, and, in some instances, results in modification of the surface properties. Generally, depositing may be performed using vapor deposition techniques, coating techniques, grafting techniques, or the like. Some specific examples include chemical vapor deposition (CVD), spray coating (e.g., ultrasonic spray coating), spin coating, dunk or dip coating, doctor blade coating, puddle dispensing, flow through coating, aerosol printing, screen printing, microcontact printing, inkjet printing, or the like. 
     As used herein, the term “depression” refers to a discrete concave feature in a base support or a layer of a multi-layer stack having a surface opening that is at least partially surrounded by interstitial region(s) of the base support or a layer of a multi-layer stack. Depressions can have any of a variety of shapes at their opening in a surface including, as examples, round, elliptical, square, polygonal, star shaped (with any number of vertices), etc. The cross-section of a depression taken orthogonally with the surface can be curved, square, polygonal, hyperbolic, conical, angular, etc. As examples, the depression can be a well or two interconnected wells. The depression may also have more complex architectures, such as ridges, step features, etc. An example of a depression having a step feature is referred to herein as a multi-depth depression, where the step feature defines the shallow portion. 
     The term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection, but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise. 
     The term “epoxy” (also referred to as a glycidyl or oxirane group) as used herein refers to 
     
       
         
         
             
             
         
       
     
     As used herein, the term “flow cell” is intended to mean a vessel having a flow channel where a reaction can be carried out, an inlet for delivering reagent(s) to the flow channel, and an outlet for removing reagent(s) from the flow channel. In some examples, the flow cell accommodates the detection of the reaction that occurs in the flow cell. For example, the flow cell can include one or more transparent surfaces allowing for the optical detection of arrays, optically labeled molecules, or the like. 
     As used herein, a “flow channel” or “channel” may be an area defined between two bonded components, which can selectively receive a liquid sample. In some examples, the flow channel may be defined between two patterned structures, and thus may be in fluid communication with surface chemistry of the patterned structures. In other examples, the flow channel may be defined between a patterned structure and a lid, and thus may be in fluid communication with surface chemistry of the patterned structures. 
     As used herein, a “functionalized layer” refers to a gel material that is applied over at least a portion of a flow cell substrate. The gel material includes functional group(s) that can attach to primer(s). The functionalized layer may be positioned within a portion of a depression defined in the substrate. The functionalized layer may alternatively be positioned on a portion of a protrusion defined in the substrate. The term “functionalized layer” also refers to the gel material that is applied over all or a portion of the substrate, and that is exposed to further processing to define the functionalized layer in the portion of the depression, or the functionalized layer protrusion on the substantially flat substrate surface. 
     As used herein, “heteroaryl” refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent atoms) that contain(s) one or more heteroatoms, that is, an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur, in the ring backbone. When the heteroaryl is a ring system, every ring in the system is aromatic. The heteroaryl group may have 5-18 ring members. 
     As used herein, “heterocycle” means a non-aromatic cyclic ring or ring system containing at least one heteroatom in the ring backbone. Heterocycles may be joined together in a fused, bridged or spiro-connected fashion. Heterocycles may have any degree of saturation provided that at least one ring in the ring system is not aromatic. In the ring system, the heteroatom(s) may be present in either a non-aromatic or aromatic ring. The heterocycle group may have 3 to 20 ring members (i.e., the number of atoms making up the ring backbone, including carbon atoms and heteroatoms). In some examples, the heteroatom(s) are O, N, or S. 
     The term “hydrazine” or “hydrazinyl” as used herein refers to a —NHNH 2  group. 
     As used herein, the term “hydrazone” or “hydrazonyl” as used herein refers to a 
     
       
         
         
             
             
         
       
     
     group in which R a  and R b  are each independently selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocycle, C6-10 aryl, 5-10 membered heteroaryl, and 5-10 membered heterocycle, as defined herein. 
     As used herein, “hydroxy” or “hydroxyl” refers to an —OH group. 
     As used herein, the term “interstitial region” refers to an area, e.g., of a base support or a layer of a multi-layer stack that separates depressions (concave regions). For example, an interstitial region can separate one depression of an array from another depression of the array. The two depressions that are separated from each other can be discrete, i.e., lacking physical contact with each other. In many examples, the interstitial region is continuous, whereas the depressions are discrete, for example, as is the case for a plurality of depressions defined in an otherwise continuous surface. In other examples, the interstitial regions and the features are discrete, for example, as is the case for a plurality of depressions in the shape of trenches, which are separated by respective interstitial regions. The separation provided by an interstitial region can be partial or full separation. Interstitial regions may have a surface material that differs from the surface material of the depressions. For example, depressions can have a polymer and primer set(s) therein, and the interstitial regions can be free of polymer and primer set(s). 
     As used herein, a “negative photoresist” refers to a light sensitive material in which a portion that is exposed to light of particular wavelength(s) becomes insoluble to a developer. In these examples, the insoluble negative photoresist has less than 5% solubility in the developer. With the negative photoresist, the light exposure changes the chemical structure so that the exposed portions of the material becomes less soluble (than non-exposed portions) in the developer. While not soluble in the developer, the insoluble negative photoresist may be at least 99% soluble in a remover that is different from the developer. The remover may be a solvent or solvent mixture used, e.g., in a lift-off process. 
     In contrast to the insoluble negative photoresist, any portion of the negative photoresist that is not exposed to light is at least 95% soluble in the developer. This portion may be referred to as a “soluble negative photoresist”. In some examples, the soluble negative photoresist is at least 98%, e.g., 99%, 99.5%, 100%, soluble in the developer. 
     “Nitrile oxide,” as used herein, means a “R a C≡N + O − ” group in which R a  is defined herein. Examples of preparing nitrile oxide include in situ generation from aldoximes by treatment with chloramide-T or through action of base on imidoyl chlorides [RC(Cl)=NOH] or from the reaction between hydroxylamine and an aldehyde. 
     “Nitrone,” as used herein, means a 
     
       
         
         
             
             
         
       
     
     group in which R 1 , R 2 , and R 3  may be any of the R a  and R b  groups defined herein, except that R 3  is not hydrogen (H). 
     As used herein, a “nucleotide” includes a nitrogen containing heterocyclic base, a sugar, and one or more phosphate groups. Nucleotides are monomeric units of a nucleic acid sequence. In RNA, the sugar is a ribose, and in DNA, the sugar is a deoxyribose, i.e. a sugar lacking a hydroxyl group that is present at the 2′ position in ribose. The nitrogen containing heterocyclic base (i.e., nucleobase) can be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G), and modified derivatives or analogs thereof. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and modified derivatives or analogs thereof. The C-1 atom of deoxyribose is bonded to N-1 of a pyrimidine or N-9 of a purine. A nucleic acid analog may have any of the phosphate backbone, the sugar, or the nucleobase altered. Examples of nucleic acid analogs include, for example, universal bases or phosphate-sugar backbone analogs, such as peptide nucleic acid (PNA). 
     In some examples, the term “over” may mean that one component or material is positioned directly on another component or material. When one is directly on another, the two are in contact with each other. In  FIG.  2 C , the resin layer  18 ,  18 ′ may be applied over the base support  17 ,  17 ′ so that it is directly on and in contact with the base support  17 ,  17 ′. 
     In other examples, the term “over” may mean that one component or material is positioned indirectly on another component or material. By indirectly on, it is meant that a gap or an additional component or material may be positioned between the two components or materials. In  FIG.  2 E , the functionalized layers  24 ,  26  are positioned over the base support  17 ,  17 ′ such that the two are in indirect contact. The resin layer  18 ,  18 ′ is positioned therebetween. 
     A “patterned structure” refers to a single layer base support that includes, or a multi-layer stack with a layer that includes surface chemistry in a pattern, e.g., in depressions or otherwise positioned on the support or layer surface. The surface chemistry may include a functionalized layer and primers (e.g., used for library template capture and amplification). In some examples, the single layer base support or the layer of the multi-layer stack has been exposed to patterning techniques (e.g., etching, lithography, etc.) in order to generate the pattern for the surface chemistry. However, the term “patterned structure” is not intended to imply that such patterning techniques have to be used to generate the pattern. The patterned structure may be generated via any of the methods disclosed herein. 
     As used herein, the term “polyhedral oligomeric silsesquioxane” refers to a chemical composition that is a hybrid intermediate (e.g., RSiO 1.5 ) between that of silica (SiO 2 ) and silicone (R 2 SiO). An example of polyhedral oligomeric silsesquioxane may be that described in Kehagias et al., Microelectronic Engineering 86 (2009), pp. 776-778, which is incorporated by reference in its entirety. In an example, the composition is an organosilicon compound with the chemical formula [RSiO 3/2 ] n , where the R groups can be the same or different. Example R groups for polyhedral oligomeric silsesquioxane include epoxy, azide/azido, a thiol, a poly(ethylene glycol), a norbornene, a tetrazine, acrylates, and/or methacrylates, or further, for example, alkyl, aryl, alkoxy, and/or haloalkyl groups. 
     As used herein, a “positive photoresist” refers to a light sensitive material in which a portion that is exposed to light of particular wavelength(s) becomes soluble to a developer. In these examples, any portion of the positive photoresist exposed to light is at least 95% soluble in the developer. This portion may be referred to herein as a “soluble positive photoresist”. In some examples, the portion of the positive photoresist exposed to light (i.e., the soluble photoresist), is at least 98%, e.g., 99%, 99.5%, 100%, soluble in the developer. With the positive photoresist, the light exposure changes the chemical structure so that the exposed portions of the material become more soluble (than non-exposed portions) in the developer. 
     In contrast to the soluble positive photoresist, any portion of the positive photoresist not exposed to light is insoluble (less than 5% soluble) in the developer. This portion may be referred to as an “insoluble positive photoresist”. While not soluble in the developer, the insoluble positive photoresist may be at least 99% soluble in a remover that is different from the developer. In some examples, the insoluble positive photoresist is at least 98%, e.g., 99%, 99.5%, 100%, soluble in the remover. The remover may be a solvent or solvent mixture used in a lift-off process. 
     As used herein, the “primer” is defined as a single stranded nucleic acid sequence (e.g., single strand DNA). Some primers, referred to herein as amplification primers, serve as a starting point for template amplification and cluster generation. Other primers, referred to herein as sequencing primers, serve as a starting point for DNA synthesis. The 5′ terminus of the primer may be modified to allow a coupling reaction with a functional group of a polymer. The primer length can be any number of bases long and can include a variety of non-natural nucleotides. In an example, the sequencing primer is a short strand, ranging from 10 to 60 bases, or from 20 to 40 bases. 
     A “spacer layer,” as used herein refers to a material that bonds two components together. In some examples, the spacer layer can be a radiation absorbing material that aids in bonding, or can be put into contact with a radiation absorbing material that aids in bonding. 
     The term “substrate” refers to the single layer base support or a multi-layer structure upon which surface chemistry is introduced. In the examples of the method that utilize a metal film for patterning, the single layer base support or the layers of the multi-layer structure are capable of transmitting ultraviolet light that is used to pattern a photoresist and that is used in nucleic acid sequencing. In the examples of the method that utilize varying thicknesses of a resin layer for patterning, the resin layer (which may be a single layer base support or one layer of the multi-layer structure) is capable of transmitting ultraviolet light at thinner portions and absorbing ultraviolet light at thicker portions. When the resin layer is used in a multi-layer structure, the other layer(s) of the multi-layer structure are capable of transmitting the ultraviolet light that is used to pattern the photoresist and that is used in nucleic acid sequencing. 
     The term “tantalum pentoxide” refers to the inorganic compound with the formula Ta 2 O 5 . This compound is transparent, having a transmittance ranging from about 0.25 (25%) to 1 (100%), to wavelengths ranging from about 0.35 μm (350 nm) to at least 1.8 μm (1800 nm). A “tantalum pentoxide base support” or “tantalum pentoxide layer” may comprise, consist essentially of, or consist of Ta 2 O 5 . In examples where it is desirable for the tantalum pentoxide base support or the tantalum pentoxide layer to transmit electromagnetic energy having any of these wavelengths, the base support or layer may consist of Ta 2 O 5  or may comprise or consist essentially of Ta 2 O 5  and other components that will not interfere with the desired transmittance of the base support or layer. 
     A “thiol” functional group refers to —SH. 
     As used herein, the terms “tetrazine” and “tetrazinyl” refer to six-membered heteroaryl group comprising four nitrogen atoms. Tetrazine can be optionally substituted. 
     “Tetrazole,” as used herein, refer to five-membered heterocyclic group including four nitrogen atoms. Tetrazole can be optionally substituted. 
     The term “transparent” refers to a material, e.g., in the form of a base support or layer, that is capable of transmitting a particular wavelength or range of wavelengths. For example, the material may be transparent to wavelength(s) that are used to chemically change a positive or negative photoresist. Transparency may be quantified using transmittance, i.e., the ratio of light energy falling on a body to that transmitted through the body. The transmittance of a transparent base support or a transparent layer will depend upon the thickness of the base support or layer, the wavelength of light, and the dosage of the light to which it is exposed. In the examples disclosed herein, the transmittance of the transparent base support or the transparent layer may range from 0.25 (25%) to 1 (100%). The material of the base support or layer may be a pure material, a material with some impurities, or a mixture of materials, as long as the resulting base support or layer is capable of the desired transmittance. Additionally, depending upon the transmittance of the base support or layer, the time for light exposure and/or the output power of the light source may be increased or decreased to deliver a suitable dose of light energy through the transparent base support and/or layer to achieve the desired effect (e.g., generating a soluble or insoluble photoresist). 
     Flow Cells 
     An example of the flow cell for simultaneous paired-end sequencing generally includes a patterned structure, which includes a substrate; two functionalized layers over at least two different portions of the substrate; and different primer sets attached to the two functionalized layers. 
     One example of the flow cell  10  is shown in  FIG.  2 A  from a top view. The flow cell  10  may include two patterned structures bonded together or one patterned structure bonded to a lid. Between the two patterned structures or the one patterned structure and the lid is a flow channel  12 . The example shown in  FIG.  2 A  includes eight flow channels  12 . While eight flow channels  12  are shown, it is to be understood that any number of flow channels  12  may be included in the flow cell  10  (e.g., a single flow channel  12 , four flow channels  12 , etc.). Each flow channel  12  may be isolated from another flow channel  12  so that fluid introduced into a flow channel  12  does not flow into adjacent flow channel(s)  12 . Some examples of the fluids introduced into the flow channel  12  may introduce reaction components (e.g., DNA sample, polymerases, sequencing primers, nucleotides, etc.), washing solutions, deblocking agents, etc. 
     Each flow channel  12  is in fluid communication with an inlet and an outlet (not shown). The inlet and outlet of each flow channel  12  may be positioned at opposed ends of the flow cell  10 . The inlets and outlets of the respective flow channels  12  may alternatively be positioned anywhere along the length and width of the flow channel  12  that enables desirable fluid flow. 
     The inlet allows fluids to be introduced into the flow channel  12 , and the outlet allows fluid to be extracted from the flow channel  12 . Each of the inlets and outlets is fluidly connected to a fluidic control system (including, e.g., reservoirs, pumps, valves, waste containers, and the like) which controls fluid introduction and expulsion. 
     The flow channel  12  is at least partially defined by a patterned structure. The patterned structure may include a substrate, such as a single layer base support  14  or  14 ′ (as shown in  FIG.  2 B  and  FIG.  2 D ), or a multi-layered structure  16 ,  16 ′ (as shown in  FIG.  2 C  and  FIG.  2 E ). 
     In examples of the method that utilize a metal film (see, e.g.,  FIG.  9 A ) for patterning, the single layer base support  14  may be any material that is capable of transmitting the light that is used to pattern a photoresist (e.g., ultraviolet light). In these particular examples, suitable materials include siloxanes, glass, modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polyethylene terephthalate (PET), polycarbonate, cyclic olefin copolymer (COC), some polyamides, silica or silicon oxide (SiO 2 ), fused silica, silica-based materials, silicon nitride (Si 3 N 4 ), inorganic glasses, resins, or the like. Examples of resins that can transmit UV light include inorganic oxides, such as tantalum pentoxide (e.g., Ta 2 O 5 ) or other tantalum oxide(s) (TaO x ), aluminum oxide (e.g., Al 2 O 3 ), silicon oxide (e.g., SiO 2 ), hafnium oxide (e.g., HfO 2 ), indium tin oxide, titanium dioxide, etc., or polymeric resins, such as a polyhedral oligomeric silsesquioxane based resin (e.g., POSS® from Hybrid Plastics), a non-polyhedral oligomeric silsesquioxane epoxy resin, a poly(ethylene glycol) resin, a polyether resin (e.g., ring opened epoxies), an acrylic resin, an acrylate resin, a methacrylate resin, an amorphous fluoropolymer resin (e.g., CYTOP® from Bellex), and combinations thereof. In some examples, the resin used has a UV transmittance (at the predetermined UV dosage being used) that ranges from about 0.5 to about 1, e.g., from about 0.75 to about 1, from about 0.9 to about 0.99. The thickness of the resin that is used in combination with the metal film can be adjusted so that the entire resin exhibits the desired UV transmittance for the UV dosage being used. In some instances, the resin thickness is 150 nm or less. 
     In examples of the method that utilize the metal film for patterning, the multi-layer structure  16  may include a base support  17  and a resin layer  18  on the base support  17 . In this example, any of the materials for the single layer base support  14  may be used as the base support  17 , and any of the resins set forth herein for the single layer base support  14  may be used for the resin layer  18 . 
     In the examples of the method that utilize varying resin layer thickness for patterning, the single layer base support  14 ′ may be any resin material whose UV absorbance, when exposed to a particular UV light dosage, can be altered by adjusting its thickness. Any of the previously listed resins may be used so long as thicker portions absorb the UV light and thinner portions transmit a desirable amount of UV light for patterning when the resin is exposed to a predetermined UV light dosage. In one example, a polyhedral oligomeric silsesquioxane based resin having thicker portions of about 500 nm and thinner portions of about 150 nm will respectively and effectively absorb and transmit UV light when exposed to a dosage ranging from about 30 mJ/cm 2  to about 60 mJ/cm 2 . Other thicknesses may be used, and the UV dosage may be adjusted accordingly to achieve the desired absorption in thicker areas and transmittance in thinner areas. 
     In examples of the method that utilize varying resin layer thickness for patterning, the multi-layer structure  16 ′ may include a base support  17 ′ and a resin layer  18 ′ on the base support  17 ′ ( FIG.  2 C ). In this example, any of the materials set forth herein that are suitable for use as the single layer base support  14  may be used as the base support  17 ′, and any of the resins set forth herein that are suitable for use as the single layer base support  14 ′ may be used for the resin layer  18 ′. In this example, the thick and thin portions of the resin layer  18 ′ are adjusted to achieve the desired absorption and transmittance. 
     The correlation between UV dose, UV absorption constant, and resin layer thickness can be expressed as: 
         D   0   =D ×exp(− kd )
 
     where D 0  is the required UV dose to pattern resin layer, D is the actual UV dose which has to be applied to the resin, k is the absorption constant, and d is the thickness of thinner portion of resin. Thus, the actual UV dose (D) can be expressed as: 
         D=D   0 /exp(− kd )
 
     In one example, the single layer base support  14 ′ or the resin layer  18 ′ is the negative photoresist NR9-1000P (from Futurrex), D 0 =19 mJ/cm 2  at 0.9 μm of thickness, the UV absorption constant (k) of the photoresist is 3×10 4  cm −1 , the thickness of the thinner portion of photoresist is 150 nm, and D is about 30 mJ/cm 2 . 
     In some of the examples set forth herein, the single layer base support  14 ,  14 ′ or the resin layer  18 ,  18 ′ is patterned with depressions  20  (shown in  FIG.  2 B ), or multi-depth depressions  20 ′ (shown in  FIG.  2 C ). 
     Some example materials (e.g., inorganic oxides) can be selectively applied via vapor deposition, aerosol printing, or inkjet printing and the depressions  20  or multi-depth depressions  20 ′ can be formed during this process. Other example materials, e.g., the polymeric resins, may be applied and then patterned to form the depressions  20  or multi-depth depressions  20 ′. For example, the polymeric resins may be deposited using a suitable technique, such as chemical vapor deposition, dip coating, dunk coating, spin coating, spray coating, puddle dispensing, ultrasonic spray coating, doctor blade coating, aerosol printing, screen printing, microcontact printing, etc. Suitable patterning techniques include photolithography, nanoimprint lithography (NIL), stamping techniques, embossing techniques, molding techniques, microetching techniques, etc. 
     The single layer base support  14 ,  14 ′ or the base support  17 ,  17 ′ may be a circular sheet, a panel, a wafer, a die etc. having a diameter ranging from about 2 mm to about 300 mm, e.g., from about 200 mm to about 300 mm, or may be a rectangular sheet, panel, wafer, die etc. having its largest dimension up to about 10 feet (˜3 meters). As one example, a die may have a width ranging from about 0.1 mm to about 10 mm. While example dimensions have been provided, it is to be understood that the single layer base support  14 ,  14 ′ or the base support  17 ,  17 ′ may have any suitable dimensions. 
     In an example, the flow channel  12  has a substantially rectangular configuration (e.g., with slightly bent and curved ends as shown in  FIG.  2 A ). The length and width of the flow channel  12  may be selected so a portion of the single layer base support  14 ,  14 ′ or the resin layer  18 ,  18 ′ of the multi-layered structure  16 ,  16 ′ surrounds the flow channel  12  and is available for attachment to a lid (not shown) or another patterned structure. 
     The depth of the flow channel  12  can be as small as a monolayer thick when microcontact, aerosol, or inkjet printing is used to deposit a separate material that defines the flow channel  12  walls. For other examples, the depth of the flow channel  12  can be about 1 μm, about 10 μm, about 50 μm, about 100 μm, or more. In an example, the depth may range from about 10 μm to about 100 μm. In another example, the depth may range from about 10 μm to about 30 μm. In still another example, the depth is about 5 μm or less. It is to be understood that the depth of the flow channel  12  may be greater than, less than or between the values specified herein. 
       FIG.  2 B ,  FIG.  2 C ,  FIG.  2 D , and  FIG.  2 E  depict examples of the architecture within the flow channel  12 . As shown in  FIG.  2 B , the architecture includes depressions  20  of the same depth separated by interstitial regions  22 . In this example, functionalized layers  24 ,  26  are formed in each depression  20 . As shown in  FIG.  2 C , the architecture includes multi-depth depressions  20 ′ separated by interstitial regions  22 . In this example, functionalized layers  24 ,  26  are formed in different portions of the multi-depth depressions  20 ′. As shown in  FIG.  2 D , the architecture includes multi-depth trenches  21  separated by interstitial regions  22 , and isolated areas of the functionalized layers  24 ,  26  formed on difference surfaces (e.g.,  64 ′,  66 ′) of the multi-depth trenches  21 . As shown in  FIG.  2 E , a multi-step protrusion  28  is formed in the resin layer  18  of the multi-layered structure  16 . As shown in  FIG.  2 E , the architecture includes a plurality of the protrusions  28  across a substantially planar surface of the base support  17 . 
     Many different layouts of the depressions  20 ,  20 ′, and the multi-step protrusion  28  may be envisaged, including regular, repeating, and non-regular patterns. In an example, the depressions  20 ,  20 ′ and/or the multi-step protrusions  28  are disposed in a hexagonal grid for close packing and improved density. Other layouts may include, for example, rectilinear (rectangular) layouts, triangular layouts, and so forth. In some examples, the layout or pattern can be an x-y format in rows and columns. In some other examples, the layout or pattern can be a repeating arrangement of the depressions  20 ,  20 ′ and/or the protrusions  28  and the interstitial regions  22 . In still other examples, the layout or pattern can be a random arrangement of the depressions  20 ,  20 ′ and/or the protrusions  28 , and the interstitial regions  22 . The layout or pattern may be characterized with respect to the density (number) of the depressions  20 ,  20 ′ and/or the protrusions  28  in a defined area. For example, the depressions  20 ,  20 ′ and/or the protrusions  28  may be present at a density of approximately 2 million per mm 2 . The density may be tuned to different densities including, for example, a density of about 100 per mm 2 , about 1,000 per mm 2 , about 0.1 million per mm 2 , about 1 million per mm 2 , about 2 million per mm 2 , about 5 million per mm 2 , about 10 million per mm 2 , about 50 million per mm 2 , or more, or less. It is to be further understood that the density can be between one of the lower values and one of the upper values selected from the ranges above, or that other densities (outside of the given ranges) may be used. As examples, a high density array may be characterized as having the depressions  20 ,  20 ′ and/or the protrusions  28  separated by less than about 100 nm, a medium density array may be characterized as having the depressions  20 ,  20 ′ and/or the protrusions  28  separated by about 400 nm to about 1 μm, and a low density array may be characterized as having the depressions  20 ,  20 ′ and/or the protrusions  28  separated by greater than about 1 μm. 
     The layout or pattern of the depressions  20 ,  20 ′ and/or the protrusions  28  may also or alternatively be characterized in terms of the average pitch, or the spacing from the center of one depression  20 ,  20 ′ and/or protrusion  28  to the center of an adjacent depression  20 ,  20 ′ and/or protrusion  28  (center-to-center spacing) or from the right edge of one depression  20 ,  20 ′ and/or protrusion  28  to the left edge of an adjacent depression  20 ,  20 ′ and/or protrusion  28  (edge-to-edge spacing). The pattern can be regular, such that the coefficient of variation around the average pitch is small, or the pattern can be non-regular in which case the coefficient of variation can be relatively large. In either case, the average pitch can be, for example, about 50 nm, about 0.15 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 100 μm, or more or less. The average pitch for a particular pattern of can be between one of the lower values and one of the upper values selected from the ranges herein. In an example, the depressions  20  have a pitch (center-to-center spacing) of about 1.5 μm. While example average pitch values have been provided, it is to be understood that other average pitch values may be used. 
     The size of each depression  20 ,  20 ′ may be characterized by its volume, opening area, depth, and/or diameter. For example, the volume can range from about 1×10 −3  μm 3  to about 100 μm 3 , e.g., about 1×10 −2  μm 3 , about 0.1 μm 3 , about 1 μm 3 , about 10 μm 3 , or more, or less. For another example, the opening area can range from about 1×10 −3  μm 2  to about 100 μm 2 , e.g., about 1×10 −2  μm 2 , about 0.1 μm 2 , about 1 μm 2 , at least about 10 μm 2 , or more, or less. For still another example, the depth can range from about 0.1 μm to about 100 μm, e.g., about 0.5 μm, about 1 μm, about 10 μm, or more, or less. For another example, the depth can range from about 0.1 μm to about 100 μm, e.g., about 0.5 μm, about 1 μm, about 10 μm, or more, or less. For yet another example, the diameter or length and width can range from about 0.1 μm to about 100 μm, e.g., about 0.5 μm, about 1 μm, about 10 μm, or more, or less. 
     In the multi-depth depression  20 ′, it is to be understood that the depth of the deep portion (reference number  48 , see  FIG.  4 A ) and the depth of the shallow portion (reference number  50 , see  FIG.  4 A ) are each within the ranges provided, with the caveat that the depth of the deep portion  48  is greater than the depth of the shallow portion  50 . It is to be understood that the height of the internal wall  29 , I (see  FIG.  1 A  and  FIG.  4 A ) will vary depending upon the different depths of the deep and shallow portions  48 ,  50 . In some examples, it is desirable that the height of the internal wall  29 , I be substantially equivalent to (e.g., +/−5%) the thickness of the depth of the shallow portion  50 . These dimensions may be desirable, e.g., when the layer in which the multi-depth depression  20 ′ is etched back to form a depression  20  (see, e.g.,  FIG.  10 C  and  FIG.  11 A ) or a protrusion  28  (see, e.g.,  FIGS.  7 C and  7 D ). 
     The size of each protrusion  28  may be characterized by its top surface areas, heights, and/or diameter (if circular in shape) or length and width. The protrusion  28  is a multi-height pad, as shown in  FIG.  1 E , which includes two top surfaces  27 ,  27 ′ ( FIG.  2 E ) at different heights with respect to the surface of the base support  17 . The top surfaces  27 ,  27 ′ are separated by a sidewall  29 ′. In an example, each of the top surfaces  27 ,  27 ′ has a surface area ranging from about 1×10 −3  μm 2  to about 100 μm 2 , e.g., about 1×10 −2  μm 2 , about 0.1 μm 2 , about 1 μm 2 , at least about 10 μm 2 , or more, or less. For still another example, each of the heights can range from about 0.1 μm to about 100 μm, e.g., about 0.5 μm, about 1 μm, about 10 μm, or more, or less, as long as the two heights are different. For yet another example, the diameter or length and width of protrusion  28  can range from about 0.1 μm to about 100 μm, e.g., about 0.5 μm, about 1 μm, about 10 μm, or more, or less. 
     In one example, the layout of the multi-depth trenches  21  is such that the length of each trench  21  is parallel to the length of the flow channel  12  in which the trench  21  is formed. Each flow channel  12  includes two or more trenches  21 , and with this layout, each multi-depth trench  21  extends the length of the flow channel  12 , as represented in  FIG.  2 D , and thus are parallel to each other. In another example, the layout of the multi-depth trenches  21  is such that the length of each trench  21  is perpendicular to the length of the flow channel  12  in which the trench  21  is formed. In this other example, the two or more trenches  21  would be parallel to one another, but would extend the width of the flow channel  12  (as opposed to the length of the flow channel  12 ). 
     Each trench  21  has opposed sidewalls  29 , E 1  and  29 , E 2  that define the edges of the trench  21 , and each trench  21  is separated from an adjacent trench  21  by an interstitial region  22 . The interstitial regions  22  between adjacent trenches  21  may have a width of 150 nm or more. In an example, the width between adjacent trenches  21  may each range from about 150 nm to about 100 μm, e.g., about 0.5 μm, about 2 μm, about 10 μm, or more. In an example, the width of the interstitial regions  22  between adjacent trenches  21  may be about 0.3 μm. 
     The width of each section of the multi-depth trench  21 , e.g., from sidewall  29 , E 1  to internal wall  29 , I and from internal wall  29 , I to sidewall  29 , E 2 , may range from about 300 nm to about 500 nm, and thus the total width of the multi-depth trench  21  may range from about 600 nm to about 1000 nm. 
     The size of each multi-depth trench  21  may be characterized by its volume, opening area, and/or depths. For example, the volume can range from about 0.1 μm 3  to about 0.4 μm 3  per unit area of 1 μm 2 . For another example, the opening area for all of the trenches  21  can range from about 40% to about 80% of the total area of the substrate in which the trenches  21  are formed. 
     Each trench  21  includes a deep portion  48 ′ and a shallow portion  50 ′, and the depth of the trench  21  varies at these portions  48 ′,  50 ′. The depth at the respective portions  48 ′,  50 ′ can range from about 0.1 μm to about 100 μm, e.g., about 0.5 μm, about 1 μm, about 10 μm, or more, or less, with the caveat that the depth of the deep portion  48 ′ is greater than the depth of the shallow portion  50 ′. It is to be understood that the height of the sidewall  29 , E 1  adjacent to the deep portion  48 ′ may be equivalent to the depth of the deep portion  48 ′, and the height of the sidewall  29 , E 2  adjacent to the shallow portion  50 ′ may be equivalent to the depth from the interstitial region  22  to a surface  66 ′ that defines the bottom of the shallow portion  50 ′. The height of the internal wall  29 , I (see  FIG.  2 D  and  FIG.  18 A ) will vary depending upon the different depths of the deep and shallow portions  48 ′,  50 ′. 
     Each of the architectures also includes the functionalized layers  24 ,  26 . In each example, the functionalized layers  24 ,  26  represent areas that have a primer set attached thereto. Some examples of the primer set  30  ( FIGS.  2 B,  2 C,  2 D, and  2 E ) include two different primers  34 ,  36 . Some examples of the primer set  32  ( FIGS.  2 B,  2 C,  2 D, and  2 E ) include two different primer sets  38 ,  40 . The primer sets  30 ,  32  are used in simultaneous paired-end sequencing. It is to be understood that primer set  30  may be attached to functionalized layer  24  or functionalized layer  26 , so long as the primer set  32  is attached to the other of the functionalized layers  26 ,  24 . 
     In some of the examples disclosed herein, the functionalized layers  24 ,  26  are chemically the same, and any of the techniques disclosed herein may be used to immobilize the primer sets  30 ,  32  to the desired layer  24 ,  26 . In other examples disclosed herein, the functionalized layers  24 ,  26  are chemically different (e.g., include different functional groups for respective primer set  30 ,  32  attachment), and any of the techniques disclosed herein may be used to immobilize the primer sets  30 ,  32  to the respective layers  24 ,  26 . In other examples disclosed herein, the materials applied to form the functionalized layers  24 ,  26  may have the respective primer sets  30 ,  32  pre-grafted thereto, and thus the immobilization chemistries of the functionalized layers  24 ,  26  may be the same or different. 
     In some examples, the functionalized layers  24 ,  26  may be any gel material that can swell when liquid is taken up and can contract when liquid is removed, e.g., by drying. In an example, the gel material is a polymeric hydrogel. In an example, the polymeric hydrogel includes an acrylamide copolymer. Some examples of the acrylamide copolymer are represented by the following structure (I): 
     
       
         
         
             
             
         
       
     
     wherein: 
     R A  is selected from the group consisting of azido, optionally substituted amino, optionally substituted alkenyl, optionally substituted alkyne, halogen, optionally substituted hydrazone, optionally substituted hydrazine, carboxyl, hydroxy, optionally substituted tetrazole, optionally substituted tetrazine, nitrile oxide, nitrone, sulfate, and thiol; 
     R B  is H or optionally substituted alkyl; 
     R C , R D , and R E  are each independently selected from the group consisting of H and optionally substituted alkyl; 
     each of the —(CH 2 ) p — can be optionally substituted; 
     p is an integer in the range of 1 to 50; 
     n is an integer in the range of 1 to 50,000; and 
     m is an integer in the range of 1 to 1010,000. 
     One specific example of the acrylamide copolymer represented by structure (I) is poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide, PAZAM. 
     One of ordinary skill in the art will recognize that the arrangement of the recurring “n” and “m” features in structure (I) are representative, and the monomeric subunits may be present in any order in the polymer structure (e.g., random, block, patterned, or a combination thereof). 
     The molecular weight of the acrylamide copolymer may range from about 5 kDa to about 1500 kDa or from about 10 kDa to about 1000 kDa, or may be, in a specific example, about 312 kDa. 
     In some examples, the acrylamide copolymer is a linear polymer. In some other examples, the acrylamide copolymer is a lightly cross-linked polymer. 
     In other examples, the gel material may be a variation of structure (I). In one example, the acrylamide unit may be replaced with N,N-dimethylacrylamide 
     
       
         
         
             
             
         
       
     
     In this example, the acrylamide unit in structure (I) may be replaced with, 
     
       
         
         
             
             
         
       
     
     where R D , R E , and R F  are each H or a C1-C6 alkyl, and R G  and R H  are each a C1-C6 alkyl (instead of H as is the case with the acrylamide). In this example, q may be an integer in the range of 1 to 100,000. In another example, the N,N-dimethylacrylamide may be used in addition to the acrylamide unit. In this example, structure (I) may include 
     
       
         
         
             
             
         
       
     
     in addition to the recurring “n” and “m” features, where R D , R E , and R F  are each H or a C1-C6 alkyl, and R G  and R H  are each a C1-C6 alkyl. In this example, q may be an integer in the range of 1 to 100,000. 
     As another example of the polymeric hydrogel, the recurring “n” feature in structure (I) may be replaced with a monomer including a heterocyclic azido group having structure (II): 
     
       
         
         
             
             
         
       
     
     wherein R 1  is H or a C1-C6 alkyl; R 2  is H or a C1-C6 alkyl; L is a linker including a linear chain with 2 to 20 atoms selected from the group consisting of carbon, oxygen, and nitrogen and 10 optional substituents on the carbon and any nitrogen atoms in the chain; E is a linear chain including 1 to 4 atoms selected from the group consisting of carbon, oxygen and nitrogen, and optional substituents on the carbon and any nitrogen atoms in the chain; A is an N substituted amide with an H or a C1-C4 alkyl attached to the N; and Z is a nitrogen containing heterocycle. Examples of Z include 5 to 10 carbon-containing ring members present as a single cyclic structure or a fused structure. Some specific examples of Z include pyrrolidinyl, pyridinyl, or pyrimidinyl. As still another example, the gel material may include a recurring unit of each of structure (III) and (IV): 
     
       
         
         
             
             
         
       
     
     wherein each of R 1a , R 2a , R 1b  and R 2b  is independently selected from hydrogen, an optionally substituted alkyl or optionally substituted phenyl; each of R 3a  and R 3b  is independently selected from hydrogen, an optionally substituted alkyl, an optionally substituted phenyl, or an optionally substituted C7-C14 aralkyl; and each L 1  and L 2  is independently selected from an optionally substituted alkylene linker or an optionally substituted heteroalkylene linker. 
     In still another example, the acrylamide copolymer is formed using nitroxide mediated polymerization, and thus at least some of the copolymer chains have an alkoxyamine end group. In the copolymer chain, the term “alkoxyamine end group” refers to the dormant species —ONR 1 R 2 , where each of R 1  and R 2  may be the same or different, and may independently be a linear or branched alkyl, or a ring structure, and where the oxygen atom is attached to the rest of the copolymer chain. In some examples, the alkoxyamine may also be introduced into some of the recurring acrylamide monomers, e.g., at position R A  in structure (I). As such, in one example, structure (I) includes an alkoxyamine end group; and in another example, structure (I) includes an alkoxyamine end group and alkoxyamine groups in at least some of the side chains. 
     It is to be understood that other molecules may be used to form the functionalized layer  24 ,  26 , as long as they are capable of being functionalized with the desired chemistry, e.g., primer set(s)  30 ,  32 . Some examples of suitable materials for the functionalized layer  24 ,  26  include functionalized silanes, such as norbornene silane, azido silane, alkyne functionalized silane, amine functionalized silane, maleimide silane, or any other silane having functional groups that can respectively attach the desired chemistry. Still other examples of suitable materials for the functionalized layer  24 ,  26  include those having a colloidal structure, such as agarose; or a polymer mesh structure, such as gelatin; or a cross-linked polymer structure, such as polyacrylamide polymers and copolymers, silane free acrylamide (SFA), or an azidolyzed version of SFA. Examples of suitable polyacrylamide polymers may be synthesized from acrylamide and an acrylic acid or an acrylic acid containing a vinyl group, or from monomers that form [2+2] photo-cycloaddition reactions. Still other examples of suitable materials for the functionalized layer  24 ,  26  include mixed copolymers of acrylam ides and acrylates. A variety of polymer architectures containing acrylic monomers (e.g., acrylamides, acrylates etc.) may be utilized in the examples disclosed herein, such as branched polymers, including dendrimers (e.g., multi-arm or star polymers), star-shaped or star-block polymers, and the like. For example, the monomers (e.g., acrylamide, acrylamide containing the catalyst, etc.) may be incorporated, either randomly or in block, into the branches (arms) of a dendrimer. 
     The gel material for the functionalized layer  24 ,  26  may be formed using any suitable copolymerization process, such as nitroxide mediated polymerization (NMP), reversible addition-fragmentation chain-transfer (RAFT) polymerization, etc. 
     It is to be understood that in any of the examples shown in  FIG.  2 B  through  FIG.  2 E , the positioning of the functionalized layer  24  and the functionalized layer  26  may be reversed. In an example, in  FIG.  2 B , the functionalized layers  24 ,  26  may be in either position within the depression  20 , as long as the functionalized layers  24 ,  26  are adjacent to one another. 
     The attachment of the functionalized layers  24 ,  26  to the underlying base support  14 ,  14 ′ or resin layer  18 ,  18 ′ may be through covalent bonding. In some instances, the underlying base support  14 ,  14 ′ or resin layer  18 ,  18 ′ may first be activated, e.g., through silanization or plasma ashing. Covalent linking is helpful for maintaining the primer set(s)  30 ,  32  in the desired regions throughout the lifetime of the flow cell  10  during a variety of uses. 
     In the examples set forth herein, the flow cell  10  includes one primer set  30 ,  32  attached to one of the functionalized layers  24 ,  26  and a different primer set  30 ,  32  attached to another of the functionalized layers  24 ,  26 . The different primers sets  30 ,  32  are related in that one set includes an un-cleavable first primer and a cleavable second primer, and the other set includes a cleavable first primer and an un-cleavable second primer. These primer sets  30 ,  32  allow a single template strand to be amplified and clustered across both primer sets, and also enable the generation of forward and reverse strands on the adjacent functionalized layers due to the cleavage groups being present on the opposite primers of the sets. Examples of these primer sets  30 ,  32  will be discussed in reference to  FIG.  3 A  through  FIG.  3 D . 
       FIG.  3 A  through  FIG.  3 D  depict different configurations of the primer sets  30 A,  32 A,  30 B,  32 B,  30 C,  32 C, and  30 D,  32 D attached to the functionalized layers  24 ,  26 . 
     Each of the first primer sets  30 A,  30 B,  30 C, and  30 D includes an un-cleavable first primer  34  or  34 ′ and a cleavable second primer  36  or  36 ′; and each of the second primer sets  32 A,  32 B,  32 C, and  32 D includes a cleavable first primer  38  or  38 ′ and an un-cleavable second primer  40  or  40 ′. 
     The un-cleavable first primer  34  or  34 ′ and the cleavable second primer  36  or  36 ′ are oligonucleotide pairs, e.g., where the un-cleavable first primer  34  or  34 ′ is a forward amplification primer and the cleavable second primer  36  or  36 ′ is a reverse amplification primer or where the cleavable second primer  36  or  36 ′ is the forward amplification primer and the un-cleavable first primer  34  or  34 ′ is the reverse amplification primer. In each example of the first primer set  30 A,  30 B,  30 C, and  30 D the cleavable second primer  36  or  36 ′ includes a cleavage site  42 , while the un-cleavable first primer  34  or  34 ′ does not include a cleavage site  42 . 
     The cleavable first primer  38  or  38 ′ and the un-cleavable second primer  40  or  40 ′ are also oligonucleotide pairs, e.g., where the cleavable first primer  38  or  38 ′ is a forward amplification primer and the un-cleavable second primer  40  or  40 ′ is a reverse amplification primer or where the un-cleavable second primer  40  or  40 ′ is the forward amplification primer and the cleavable first primer  38  or  38 ′ is the reverse amplification primer. In each example of the second primer set  32 A,  32 B,  32 C, and  32 D, the cleavable first primer  38  or  38 ′ includes a cleavage site  42 ′ or  44 , while the un-cleavable second primer  40  or  40 ′ does not include a cleavage site  42 ′ or  44 . 
     It is to be understood that the un-cleavable first primer  34  or  34 ′ of the first primer set  30 A,  30 B,  30 C, and  30 D and the cleavable first primer  38  or  38 ′ of the second primer set  32 A,  32 B,  32 C, and  32 D have the same nucleotide sequence (e.g., both are forward amplification primers), except that the cleavable first primer  38  or  38 ′ includes the cleavage site  42 ′ or  44  integrated into the nucleotide sequence or into a linker  46 ′ attached to the nucleotide sequence. Similarly, the cleavable second primer  36  or  36 ′ of the first primer set  30 A,  30 B,  30 C, and  30 D and the un-cleavable second primer  40  or  40 ′ of the second primer set  32 A,  32 B,  32 C, and  32 D have the same nucleotide sequence (e.g., both are reverse amplification primers), except that the cleavable second primer  36  or  36 ′ includes the cleavage site  42  integrated into the nucleotide sequence or into a linker  46  attached to the nucleotide sequence. 
     It is to be understood that when the first primers  34  and  38  or  34 ′ and  38 ′ are forward amplification primers, the second primers  36  and  40  or  36 ′ and  40 ′ are reverse primers, and vice versa. 
     The un-cleavable primers  34 ,  40  or  34 ′,  40 ′ may be any primers with a universal sequence for capture and/or amplification purposes, such as P5 and P7 primers, or any combination of PA, PB, PC, and PD primers (e.g., PA and PB or PA and PD, etc.). 
     Examples of the P5 and P7 primers are used on the surface of commercial flow cells sold by Illumina Inc. for sequencing, for example, on HISEQ™′ HISEQX™, MISEQ™, MISEQDX™, MINISEQ™, NEXTSEQ™, NEXTSEQDX™, NOVASEQ™, ISEQ™, GENOME ANALYZER™, and other instrument platforms. The P5 primer is: 
                            P5: 5’→3’           (SEQ. ID. NO. 1)           AATGATACGGCGACCACCGAGACTACAC            
The P7 primer may be any of the following:
 
                            P7 #1: 5’→3’           (SEQ. ID. NO. 2)           CAAGCAGAAGACGGCATACGAAT                         P7 #2: 5’→3’           (SEQ. ID. NO. 3)           CAAGCAGAAGACGGCATACAGAT            
The other primers (PA-PD) mentioned above include:
 
     
       
         
           
               
               
            
               
                   
                 PA 5’→3’ 
               
               
                   
                 (SEQ. ID. NO. 4) 
               
               
                   
                 GCTGGCACGTCCGAACGCTTCGTTAATCCGTTGAG 
               
               
                   
                   
               
               
                   
                 cPA (PA’) 5’→3’ 
               
               
                   
                 (SEQ. ID. NO. 5) 
               
               
                   
                 CTCAACGGATTAACGAAGCGTTCGGACGTGCCAGC 
               
               
                   
                   
               
               
                   
                 PB 5’→3’ 
               
               
                   
                 (SEQ. ID. NO. 6) 
               
               
                   
                 CGTCGTCTGCCATGGCGCTTCGGTGGATATGAACT 
               
               
                   
                   
               
               
                   
                 cPB (PB’) 5’→3’ 
               
               
                   
                 (SEQ. ID. NO. 7) 
               
               
                   
                 AGTTCATATCCACCGAAGCGCCATGGCAGACGACG 
               
               
                   
                   
               
               
                   
                 PC 5’→3’ 
               
               
                   
                 (SEQ. ID. NO. 8) 
               
               
                   
                 ACGGCCGCTAATATCAACGCGTCGAATCCGCAACT 
               
               
                   
                   
               
               
                   
                 cPC (PC’) 5’→3’ 
               
               
                   
                 (SEQ. ID. NO. 9) 
               
               
                   
                 AGTTGCGGATTCGACGCGTTGATATTAGCGGCCGT 
               
               
                   
                   
               
               
                   
                 PD 5’→3’ 
               
               
                   
                 (SEQ. ID. NO. 10) 
               
               
                   
                 GCCGCGTTACGTTAGCCGGACTATTCGATGCAGC 
               
               
                   
                   
               
               
                   
                 cPD (PD’) 5’→3’ 
               
               
                   
                 (SEQ. ID. NO. 11) 
               
               
                   
                 GCTGCATCGAATAGTCCGGCTAACGTAACGCGGC 
               
            
           
         
       
     
     These primers are un-cleavable primers  34 ,  40  or  34 ′,  40 ′ because they do not include a cleavage site  42 ,  42 ′,  44 . It is to be understood that any suitable universal sequence can be used as the un-cleavable primers  34 ,  40  or  34 ′,  40 ′. 
     Examples of cleavable primers  36 ,  38  or  36 ′,  38 ′ include the P5 and P7 primers or other universal sequence primers (e.g., the PA, PB, PC, PD primers) with the respective cleavage sites  42 ,  42 ′,  44  incorporated into the respective nucleic acid sequences (e.g.,  FIG.  3 A  and  FIG.  3 C ), or into a linker  46 ′,  46  that attaches the cleavable primers  36 ,  38  or  36 ′,  38 ′ to the respective functionalized layers  24 ,  26  ( FIG.  3 B  and  FIG.  3 D ). Examples of suitable cleavage sites  42 ,  42 ′,  44  include enzymatically cleavable nucleobases or chemically cleavable nucleobases, modified nucleobases, or linkers (e.g., between nucleobases), as described herein. Some specific examples of the cleavage sites  42 ,  42 ′,  44  include uracil, 8-oxoguanine, allyl-T. The cleavage sites  42 ,  42 ′,  44  may be incorporated at any point in the strand. 
     Some specific examples of the cleavable primers  36 ,  38  or  36 ′,  38 ′ are shown below, where the cleavage site  42 ,  42 ′,  44  is shown as “U” or at “n”: 
                            P5: 5’→3’           (SEQ. ID. NO. 12)           AATGATACGGCGACCACCGAGAnCTACAC            
wherein “n” is uracil or allyl T.
 
The P7 primer may be any of the following:
 
                            P7 #1: 5’→3’           (SEQ. ID. NO. 13)           CAAGCAGAAGACGGCATACGAnAT                       P7 #2: 5’→3’           (SEQ. ID. NO. 14)           CAAGCAGAAGACGGCATACnAGAT            
where “n” is 8-oxoguanine in each of the sequences.
 
     Each primer set  30 A and  32 A or  30 B and  32 B or  30 C and  32 C or  30 D and  32 D is attached to a respective functionalized layer  24 ,  26 . As described herein, the functionalized layers  24 ,  26  include different functional groups that can selectively react with the respective primers  34 ,  36  or  34 ′,  36 ′ or  38 ,  40  or  38 ′,  40 ′. 
     While not shown in  FIG.  3 A  through  FIG.  3 D , it is to be understood that one or both of the primer sets  30 A,  30 B,  30 C,  30 D or  32 A,  32 B,  32 C or  32 D may also include a PX primer for capturing a library template seeding molecule. As one example, PX may be included with the primer set  30 A,  30 B,  30 C,  30 D, but not with primer set  32 A,  32 B,  32 C or  32 D. As another example, PX may be included with the primer set  30 A,  30 B,  30 C,  30 D and with the primer set  32 A,  32 B,  32 C or  32 D. The density of the PX motifs should be relatively low in order to minimize polyclonality within each depression  20 ,  20 ′. The PX capture primers may be: 
     
       
         
           
               
               
            
               
                   
                 PX 5’→3’ 
               
               
                   
                 (SEQ. ID. NO. 15) 
               
               
                   
                 AGGAGGAGGAGGAGGAGGAGGAGG 
               
               
                   
                   
               
               
                   
                 cPX (PX’) 5’→3’ 
               
               
                   
                 (SEQ. ID. NO. 16) 
               
               
                   
                 CCTCCTCCTCCTCCTCCTCCTCCT 
               
            
           
         
       
     
       FIG.  3 A  through  FIG.  3 D  depict different configurations of the primer sets  30 A,  32 A,  30 B,  32 B,  30 C,  32 C, and  30 D,  32 D attached to the functionalized layers  24 ,  26 . More specifically,  FIG.  3 A  through  FIG.  3 D  depict different configurations of the primers  34 ,  36  or  34 ′,  36 ′ and  38 ,  40  or  38 ′,  40 ′ that may be used. 
     In the example shown in  FIG.  3 A , the primers  34 ,  36  and  38 ,  40  of the primer sets  30 A and  32 A are directly attached to the functionalized layers  24 ,  26 , for example, without a linker  46 ,  46 ′. The functionalized layer  24  has surface functional groups that can immobilize the terminal groups at the 5′ end of the primers  34 ,  36 . Similarly, the functionalized layer  26  has surface functional groups that can immobilize the terminal groups at the 5′ end of the primers  38 ,  40 . As described, the immobilization chemistry between the functionalized layer  24  and the primers  34 ,  36  and the immobilization chemistry between the functionalized layer  26  and the primers  38 ,  40  is different so that the primers  34 ,  36  or  38 ,  40  selectively attach to the desirable functionalized layer  24 ,  26 . The immobilization chemistry between the functionalized layer  24  and the primers  34 ,  36  and the immobilization chemistry between the functionalized layer  26  and the primers  38 ,  40  may be different so that the primers  34 ,  36  or  38 ,  40  selectively attach to the desirable functionalized layer  24 ,  26 . Alternatively, the primers  34 ,  36  or  38 ,  40  may be pre-grafted or sequentially applied via some of the methods disclosed herein. 
     Also, in the example shown in  FIG.  3 A , the cleavage site  42 ,  42 ′ of each of the cleavable primers  36 ,  38  is incorporated into the sequence of the primer. In this example, the same type of cleavage site  42 ,  42 ′ is used in the cleavable primers  36 ,  38  of the respective primer sets  30 A,  32 A. As an example, the cleavage sites  42 ,  42 ′ are uracil bases, and the cleavable primers  36 ,  38  are P5U and P7U. The uracil bases or other cleavage sites may also be incorporated into any of the PA, PB, PC, and PD primers to generate the cleavable primers  36 ,  38 . In this example, the un-cleavable primer  34  of the oligonucleotide pair  34 ,  36  may be P7, and the un-cleavable primer  40  of the oligonucleotide pair  38 ,  40  may be P5. Thus, in this example, the first primer set  30 A includes P7, P5U and the second primer set  32 A includes P5, P7U. The primer sets  30 A,  32 A have opposite linearization chemistries, which, after amplification, cluster generation, and linearization, allows forward template strands to be formed on one functionalized layer  24  and reverse strands to be formed on the other functionalized layer  26 . 
     In the example shown in  FIG.  3 B , the primers  34 ′,  36 ′ and  38 ′,  40 ′ of the primer sets  30 B and  32 B are attached to the functionalized layers  24 ,  26 , for example, through linkers  46 ,  46 ′. The functionalized layers  24 ,  26  include respective functional groups of the functional group pairs disclosed herein, and the terminal ends of the respective linkers  46 ,  46 ′ are capable of covalently attaching to the respective functional groups. As such, the functionalized layer  24  may have surface functional groups that can immobilize the linker  46  at the 5′ end of the primers  34 ′,  36 ′. Similarly, the functionalized layer  26  may have surface functional groups that can immobilize the linker  46 ′ at the 5′ end of the primers  38 ′,  40 ′. The immobilization chemistry for the functionalized layer  24  and the linkers  46  and the immobilization chemistry for the functionalized layer  26  and the linkers  46 ′ is different so that the primers  34 ′,  36 ′ or  38 ′,  40 ′ selectively graft to the desirable functionalized layer  24 ,  26 . Alternatively, the primers  34 ,  36  or  38 ,  40  may be pre-grafted or sequentially applied via some of the methods disclosed herein. 
     Examples of suitable linkers  46 ,  46 ′ may include nucleic acid linkers (e.g., 10 nucleotides or less) or non-nucleic acid linkers, such as a polyethylene glycol chain, an alkyl group or a carbon chain, an aliphatic linker with vicinal diols, a peptide linker, etc. An example of a nucleic acid linker is a polyT spacer, although other nucleotides can also be used. In one example, the spacer is a 6T to 10T spacer. The following are some examples of nucleotides including non-nucleic acid linkers with terminal alkyne groups (where B is the nucleobase and “oligo” is the primer): 
     
       
         
         
             
             
         
       
     
     In the example shown in  FIG.  3 B , the primers  34 ′,  38 ′ have the same sequence (e.g., P5). The primer  34 ′ is un-cleavable, whereas the primer  38 ′ includes the cleavage site  42 ′ incorporated into the linker  46 ′. Also in this example, the primers  36 ′,  40 ′ have the same sequence (e.g., P7). The primer  40 ′ in un-cleavable, and the primer  36 ′ includes the cleavage site  42  incorporated into the linker  46 . The same type of cleavage site  42 ,  42 ′ is used in the linker  46 ,  46 ′ of each of the cleavable primers  36 ′,  38 ′. As an example, the cleavage sites  42 ,  42 ′ may be uracil bases that are incorporated into nucleic acid linkers  46 ,  46 ′. The primer sets  30 B,  32 B have opposite linearization chemistries, which, after amplification, cluster generation, and linearization, allows forward template strands to be formed on one functionalized layer  24  and reverse strands to be formed on the other functionalized layer  26 . 
     The example shown in  FIG.  3 C  is similar to the example shown in  FIG.  3 A , except that different types of cleavage sites  42 ,  44  are used in the cleavable primers  36 ,  38  of the respective primer sets  30 C,  32 C. As examples, two different enzymatic cleavage sites may be used, two different chemical cleavage sites may be used, or one enzymatic cleavage site and one chemical cleavage site may be used. Examples of different cleavage sites  42 ,  44  that may be used in the respective cleavable primers  36 ,  38  include any combination of the following: vicinal diol, uracil, allyl ether, disulfide, restriction enzyme site, and 8-oxoguanine. 
     The example shown in  FIG.  3 D  is similar to the example shown in  FIG.  3 B , except that different types of cleavage sites  42 ,  44  are used in the linkers  46 ,  46 ′ attached to the cleavable primers  36 ′,  38 ′ of the respective primer sets  30 D,  32 D. Examples of different cleavage sites  42 ,  44  that may be used in the respective linkers  46 ,  46 ′ attached to the cleavable primers  36 ′,  38 ′ include any combination of the following: vicinal diol, uracil, allyl ether, disulfide, restriction enzyme site, and 8-oxoguanine. 
     In any of the examples shown in  FIG.  2    and  FIG.  3 A  through  FIG.  3 D , the attachment of the primers  34 ,  36  and  38 ,  40  or  34 ′,  36 ′ and  38 ′,  40 ′ to the functionalized layers  24 ,  26  leaves a template-specific portion of the primers  34 ,  36  and  38 ,  40  or  34 ′,  36 ′ and  38 ′,  40 ′ free to anneal to its cognate template and the 3′ hydroxyl group free for primer extension. 
     As will be described in more detail below, the primers  34 ,  36  and  38 ,  40  or  34 ′,  36 ′ and  38 ′,  40 ′ may be attached to the respective functionalized layer  24 ,  26  prior to its application to a flow cell substrate, and thus the functionalized layer  24 ,  26  may be pre-grafted. In other examples, the primers  34 ,  36  and  38 ,  40  or  34 ′,  36 ′ and  38 ′,  40 ′ may be attached to the respective functionalized layer  24 ,  26  after its application to the flow cell substrate. 
     As shown in  FIG.  2 B  through  FIG.  2 E , the functionalized layers  24 ,  26  and primer sets  30 ,  32  are positioned in particular positions in the different architectures. Different methods may be used to generate these flow cell architectures (including the positioning of the functionalized layers  24 ,  26  and primer sets  30 ,  32 ), and these methods will now be described. 
     Methods for Making Flow Cells 
     The architecture within the flow cell  10  may be obtained through a variety of methods. 
     Methods with Timed Dry Etching 
     Some examples of the method utilize a time dry etching process of a sacrificial layer (e.g., a photoresist) in order to pattern one or more layers. These methods are shown in  FIG.  4 A  through  FIG.  4 D  in combination with any of i)  FIG.  5 A  through  FIG.  5 E , ii)  FIG.  6 A  through  FIG.  6 F , iii)  FIG.  7 A  through  FIG.  7 F , or  FIG.  7 A  through  FIG.  7 D  and  FIG.  7 G , or  FIG.  7 A  through  FIG.  7 D  and  FIG.  7 H  through  FIG.  7 L , or iv)  FIG.  8 A  through  FIG.  8 G .  FIG.  9 A  through  FIG.  9 H  depict another example method. 
     The beginning of examples of the method that utilize the time dry etching process are shown in  FIG.  4 A  through  FIG.  4 D . 
     As shown in  FIG.  4 A , the multi-depth depression  20 ′ is defined in either the single layer base support  14  or the resin layer  18  of the multi-layered structure  16  as described herein. In these example methods, the single layer base support  14  is one example of the resins set forth herein, and thus is also referred to as the resin layer  14 . As such, the term “resin layer” is referred to as “resin layer  14 ,  18 ” throughout the description of these methods. With the underlying base support  17  being shown in phantom, both the multi-layered structure  16  and the single layer base support  14  are represented in  FIG.  4 A  through  FIG.  4 D . 
     In the examples when the resin layer  14 ,  18  is the single layer base support  14 , the resin layer  14 ,  18  may be any of the resins described herein. 
     In the examples when the resin layer  14 ,  18  is the resin layer  18  of the multi-layered structure  16 , the resin layer  18  may be any of the resins described herein. The base support  17  may be any of the substrates described herein. 
     The multi-depth depression  20 ′ may be etched, imprinted, or defined in the resin layer  14 ,  18  using any suitable technique. In one example, nanoimprint lithography is used. In this example, a working stamp is pressed into the resin layer  14 ,  18  while the material is soft, which creates an imprint (negative replica) of the working stamp features in the resin layer  14 ,  18 . The resin layer  14 ,  18  may then be cured with the working stamp in place. 
     Curing may be accomplished by exposure to actinic radiation, such as visible light radiation or ultraviolet (UV) radiation, when a radiation-curable resin material is used; or by exposure to heat when a thermal-curable resin material is used. Curing may promote polymerization and/or cross-linking. As an example, curing may include multiple stages, including a softbake (e.g., to drive off any liquid carrier that may be used to deposit the resin) and a hardbake. The softbake may take place at a lower temperature, ranging from about 50° C. to about 150° C., for greater than 0 seconds to about 3 minutes. The duration of the hardbake may last from about 5 seconds to about 10 minutes at a temperature ranging from about 100° C. to about 300° C. Examples of devices that can be used for softbaking and/or hard baking include a hot plate, oven, etc. 
     After curing, the working stamp is released. This creates topographic features in the resin layer  14 ,  18 . In this example, the topographic features of the multi-depth depression  20 ′ include the shallow portion  50 , the deep portion  48 , the internal wall  29 , I separating the deep portion  48  and the shallow portion  50 , and the perimeter sidewall  29 , P, each of which is shown in  FIG.  4 A . 
     While one multi-depth depression  20 ′ is shown in  FIG.  4 A , it is to be understood that the method may be performed to generate an array of multi-depth depressions  20 ′ including respective deep portions  48  and shallow portions  50 , separated by interstitial regions  22 , across the surface of the resin layer  14 ,  18 . 
     If the resin layer  14 ,  18  does not include surface groups to covalently attach to the functionalized layers  24 ,  26 , the resin layer  14 ,  18  may first be activated, e.g., through silanization or plasma ashing. If the resin layer  14 ,  18  does include surface groups to covalently attach to the functionalized layers  24 ,  26 , the activation process is not performed. As examples, the resin layer  14 ,  18  is Ta 2 O 5  which can be silanized to generate surface groups to react with the functionalized layers  24 ,  26 , or the resin layer  14 ,  18  is a polyhedral oligomeric silsesquioxane based resin which can be plasma ashed or silanized to generate surface groups to react with the functionalized layers  24 ,  26 . 
     Some examples of the methods disclosed herein include depositing a first functionalized layer  24  over the resin layer  14 ,  18  including a plurality of multi-depth depressions  20 ′ separated by interstitial regions  22 , each multi-depth depression  20 ′ including a deep portion  48  and a shallow portion  50  adjacent to the deep portion  48  (as shown in  FIG.  4 B ); patterning the first functionalized layer  24 , whereby a portion of the first functionalized layer  24  in the deep portion  48  is covered by a region  53  of a sacrificial layer  52  and portions of the first functionalized layer  24  in the shallow portion  50  and over the interstitial regions  22  are removed (as shown in  FIGS.  4 C and  4 D ); and utilizing at least one additional sacrificial layer to define a second functionalized layer adjacent to the portion of the first functionalized layer  24  in the deep portion  48 . Different ways of utilizing the additional sacrificial layer to define the second functionalized layer are described in i)  FIG.  5 A  through  FIG.  5 E , ii)  FIG.  6 A  through  FIG.  6 F , iii)  FIG.  7 A  through  FIG.  7 F , or  FIG.  7 A  through  FIG.  7 D  and  FIG.  7 G , or  FIG.  7 A  through  FIG.  7 D  and  FIG.  7 H  through  FIG.  7 L , and iv)  FIG.  8 A  through  FIG.  8 G . 
     Referring specifically to  FIG.  4 B , the functionalized layer  24  is deposited over the resin layer  14 ,  18 . As depicted, the functionalized layer  24  is positioned over the exposed surfaces of the resin layer  14 ,  18 , including a surface  64  of the resin layer  14 ,  18  at the deep portion  48 , a surface  66  of the resin layer  14 ,  18  at the shallow portion  50 , the interstitial regions  22 , and the sidewalls  29 , P, I. The functionalized layer  24  may be any of the gel materials described herein and may be applied using any suitable deposition technique. A curing process may be performed after deposition. The functionalized layer  24  covalently attaches to the resin layer  14 ,  18  in the depression  20 ′. Covalent linking is helpful for maintaining the primer set(s)  30 ,  32  in the desired regions throughout the lifetime of the flow cell  10  during a variety of uses. 
     The first functionalized layer  24  is then patterned, which is shown and described in reference to  FIG.  4 C  and  FIG.  4 D . Patterning the first functionalized layer  24  involves applying the sacrificial layer  52  over the first functionalized layer  24  ( FIG.  4 C ); and dry etching the sacrificial layer  52  and portions of the first functionalized layer  24  in the shallow portion  50  and over the interstitial regions  22 . 
     Referring specifically to  FIG.  4 C , the sacrificial layer  52  is deposited over the first functionalized layer  24 . In this example, the sacrificial layer  52  may be any material that is susceptible to plasma etching conditions and is soluble in an organic solvent. As examples, the sacrificial material  52  is a negative photoresist, a positive photoresist, poly(methyl methacrylate), or the like. The sacrificial material  52  may be applied using any suitable deposition technique disclosed herein (e.g., spin coating, etc.) and may be cured (e.g., using heating). 
     An example of a suitable negative photoresist includes the NR® series photoresist (available from Futurrex). Other suitable negative photoresists include the SU-8 Series and the KMPR® Series (both of which are available from Kayaku Advanced Materials, Inc.), or the UVN™ Series (available from DuPont). 
     Examples of suitable positive photoresists include the MICROPOSIT® S1800 series or the AZ® 1500 series, both of which are available from Kayaku Advanced Materials, Inc. Another example of a suitable positive photoresist is SPR™-220 (from DuPont). 
     Referring now to  FIG.  4 D , the sacrificial layer  52  and the first functionalized layer  24  are dry etched to expose the surface  66  in the shallow portion  50  and the interstitial regions  22 . This dry etching process is performed for a measured amount of time to expose the desired surfaces/regions  66 ,  22 . As shown in  FIG.  4 D , the timed dry etching is stopped so that the region  53  of the sacrificial layer  52  and the underlying portion  25  of the functionalized layer  24  remain in the portion of the deep portion  48  that is next to the interior wall  29 , I. As such, the remaining sacrificial layer  52  is at least substantially co-planar with the surface  66  at the shallow portion  50 . In one example, the timed dry etch may involve a reactive ion etch (e.g., with 10% CF 4  and 90% O 2 ) where the sacrificial layer  52  and functionalized layer  24  are etched at a rate of about 17 nm/min. In another example, the timed dry etch may involve a 100% O 2  plasma etch where the sacrificial layer  52  and functionalized layer  24  are etched at a rate of about 98 nm/min. 
     One example of the method continues from  FIG.  4 D  to  FIG.  5 A  through  FIG.  5 E .  FIG.  5 A  through  FIG.  5 E  together depict one example of utilizing the at least one additional sacrificial layer  68  ( FIG.  5 C  and  FIG.  5 D ) to define the second functionalized layer  26  adjacent to the portion  25  of the first functionalized layer  24  in the deep portion  48 . This example method also includes utilizing the at least one additional sacrificial layer  68  to keep the interstitial regions  22  free of the second functionalized layer  26  and to remove the portion of the second functionalized layer  26  from the perimeter  29 , P of the multi-depth depression  20 ′. As will be discussed further in reference to  FIG.  5 D , the presence of the additional sacrificial layer  68  within the multi-depth depression  20 ′ allows the second functionalized layer  26  to be removed from the interstitial regions  22  and from a portion of the sidewall  29 , P. This reduces the padlock like conformation within the multi-depth depression  20 ′. 
     As such, in this example method, utilizing the at least one additional sacrificial layer  68  to define the second functionalized layer  26 , to keep the interstitial regions  22  free of the second functionalized layer  26 , and to remove the portion of the second functionalized layer  26  from the perimeter  29 , P of the multi-depth depression  20 ′ involves depositing the second functionalized layer  26  in the shallow portion  50  and over the region  53  of the sacrificial layer  52  and the interstitial regions  22  (as shown in  FIG.  5 A ); lifting off the region  53  of the sacrificial layer  52 , thereby exposing the portion  25  of the first functionalized layer  24  (as shown in  FIG.  5 B ); applying the at least one additional sacrificial layer  68  over the second functionalized layer  26  and over the portion  25  of the first functionalized layer  24  (as shown in  FIG.  5 C ); dry etching the at least one additional sacrificial layer  68  and the second functionalized layer  26  until the second functionalized layer  26  is removed from the interstitial regions  22  and remains in the shallow portion  50  (as shown in  FIG.  5 D ); and lifting off the at least one additional sacrificial layer  68  (shown in  FIG.  5 E ). 
     Referring specifically to  FIG.  5 A , the second functionalized layer  26  is deposited in the shallow portion  50 , and over the region  53  of the sacrificial layer  52  and over the interstitial regions  22 . By “in the shallow portion,” it is meant that the second functionalized layer  26  is deposited over portions of the resin layer  14 ,  18  that are exposed in the shallow portion  50 , e.g., the surface  66  and the perimeter sidewall  29 , P. It is to be understood that the second functionalized layer  26  may also be deposited over other exposed portions of the perimeter sidewall  29 , P as well as the internal wall  29 , I. 
     The second functionalized layer  26  may be any of the gel materials described herein and may be applied using any suitable deposition technique. A curing process, as described herein, may be performed after deposition. The second functionalized layer  26  covalently attaches to the resin layer  14 ,  18  in the depression  20 ′. Covalent linking is helpful for maintaining the primer set(s)  30 ,  32  in the desired regions throughout the lifetime of the flow cell  10  during a variety of uses. 
     Referring specifically to  FIG.  5 B , the sacrificial layer  52  is removed in a lift-off process. The lift-off process may involve an organic solvent that is capable of dissolving or otherwise lifting off the sacrificial layer  52 . A cured positive photoresist may be lifted off with removers such as dimethylsulfoxide (DMSO) with sonication, an acetone wash, a propylene glycol monomethyl ether acetate wash, or an NMP (N-methyl-2-pyrrolidone) based stripper wash. A cured negative photoresist may be lifted off with removers such as dimethylsulfoxide (DMSO) with sonication, an acetone wash, or an NMP (N-methyl-2-pyrrolidone) based stripper wash. Cured poly(methyl methacrylate) may be lifted off with dimethylsulfoxide (DMSO) using sonication, or in acetone, or with an NMP (N-methyl-2-pyrrolidone) based stripper. The sacrificial layer  52  is soluble (at least 99% soluble) in the organic solvent used in the lift-off process. The lift-off process removes i) at least 99% of the region  53  of the sacrificial layer  52  and ii) the functionalized layer  26  positioned thereon. The lift-off process does not remove the portion  25  of the functionalized layer  24  that had been in contact with the region  53  of the sacrificial layer  52 ′. Thus, the lift-off process exposes the functionalized layer  24  at the surface  64  of the resin layer  14 ,  18  at the deep portion  48 , as depicted in  FIG.  5 B . 
     Referring now to  FIG.  5 C , an additional sacrificial layer  68  is applied over the second functionalized layer  26  and over the exposed portion  25  of the first functionalized layer  24 . In this example, the additional sacrificial layer  68  may be a negative or positive photoresist or poly(methyl methacrylate) and may be applied and cured so that all of the additional sacrificial layer  68  remains over the functionalized layers  24 ,  26 . 
     The additional sacrificial layer  68  is then timed dry etched, using any of the timed dry etching techniques described herein. The result of time dry etching is depicted in  FIG.  5 D . This dry etching process is performed for a measured amount of time to expose the interstitial regions  22  and a portion of the perimeter sidewall  29 , P. As shown in  FIG.  5 D , the timed dry etching is stopped so that the functionalized layer  26  remains on the surface  66  in the shallow portion  50 . Timed dry etching does remove some of the functionalized layer  26  from portions of the perimeter sidewall  29 , P near the opening of the multi-depth depression  20 ′. This reduces the padlock like conformation  33 . 
     Because the functionalized layer  24  is positioned over the lower surface  64  in the deep portion  48 , along the interior wall  29 , I (which is lower than the surface  66 ), and along a portion of the perimeter sidewall  29 , P that is lower than the surface  66 , the timed dry etching does not affect the functionalized layer  24 . Thus, the timed dry etching is stopped so that at least some of the additional sacrificial layer  68  and the underlying functionalized layer  24  remain in an area of the deep portion  48  that is next to the interior wall  29 , I. As a result of timed dry etching, the portion  25  of the functionalized layer  24  and the functionalized layer  26  over the surface  66  remain intact. 
     In some instances, timed dry etching is stopped so that some of the additional sacrificial layer  68  remains over the functionalized layer  26  on the surface  66  and adjacent to the functionalized layer  26  along the perimeter sidewall  29 , P. In these instances, the functionalized layer  26  along the perimeter sidewall  29 , P and the additional sacrificial layer  68  are substantially co-planar in the multi-depth depression  20 ′. As noted above, however, dry etching does remove some of the functionalized layer  26  from portions of the perimeter sidewall  29 , P near the opening of the multi-depth depression  20 ′ to reduce the padlock like conformation  33 . As shown in  FIG.  5 E , the portions  35  of the functionalized layer  26  that remain along the perimeter sidewall  29 , P after timed dry etching may still form a minimal padlock like conformation  33 . By “minimal padlock like conformation,” it is meant that signal interference from the padlock like conformation  33  is 50% or less. In other words, the signals from the nascent strands that are attached to the functionalized layer  24  make up 50% or more of the of the signals that are imaged in the area corresponding to the functionalized layer  24 . 
     Referring specifically to  FIG.  5 E , the additional insoluble sacrificial layer  68  is removed in a lift-off process. The lift-off process may be performed using any of the suitable organic solvents described herein, which depends on the type of sacrificial layer  68  used. 
     While not shown, the method shown in  FIG.  4 A  through  FIG.  4 D  and  FIG.  5 A  through  FIG.  5 E  also includes attaching respective primer sets  30 ,  32  to the functionalized layers  24 ,  26 . In some examples, the primers  34 ,  36  or  34 ′,  36 ′ (not shown in  FIG.  4 A  through  FIG.  4 D  or  FIG.  5 A  through  FIG.  5 E ) may be pre-grafted to the functionalized layer  24 . Similarly, the primers  38 ,  40  or  38 ′,  40 ′ (not shown in Fig.  FIG.  4 A  through  FIG.  4 D  or  FIG.  5 A  through  FIG.  5 E ) may be pre-grafted to the functionalized layer  26 . In these examples, additional primer grafting is not performed. 
     In other examples, the primers  34 ,  36  or  34 ′,  36 ′ are not pre-grafted to the functionalized layer  24 . In these examples, the primers  34 ,  36  or  34 ′,  36 ′ may be grafted after the functionalized layer  24  is applied (e.g., at  FIG.  4 B ). In these examples, the primers  38 ,  40  or  38 ′,  40 ′ may be pre-grafted to the second functionalized layer  26 . Alternatively, in these examples, the primers  38 ,  40  or  38 ′,  40 ′ may not be pre-grafted to the second functionalized layer  26 . Rather, the primers  38 ,  40  or  38 ′,  40 ′ may be grafted immediately after the second functionalized layer  26  is applied (e.g., at  FIG.  5 A ); or after the region  53  is removed ( FIG.  5 B ) as long as i) the functionalized layer  26  has different functional groups (than functionalized layer  24 ) for attaching the primers  38 ,  40  or  38 ′,  40 ′ or ii) any unreacted functional groups of the functionalized layer  24  have been quenched, e.g., using the Staudinger reduction to amines or additional click reaction with a passive molecule such as hexynoic acid. 
     When grafting is performed during the method, grafting may be accomplished using any of the grafting techniques described herein. 
     While a single set of the functionalized layers  24 ,  26  is shown in  FIG.  5 E , it is to be understood that the method described in reference to  FIG.  4 A  through  FIG.  4 D  and  FIG.  5 A  through  FIG.  5 E  may be performed to generate an array of depressions  20 ′ (each having functionalized layers  24 ,  26  therein) separated by interstitial regions  22  across the surface of the support  14  or resin layer  18  of the multi-layer structure  16 . 
     Still another method is shown in  FIG.  4 A  through  FIG.  4 D  and continues at  FIG.  6 A  through  FIG.  6 F .  FIG.  6 A  through  FIG.  6 F  together depict one example of utilizing the at least one additional sacrificial layer  68  ( FIG.  6 C  and  FIG.  6 D ) to define the second functionalized layer  26  adjacent to the portion  25  of the first functionalized layer  24  in the deep portion  48 . This example method also includes utilizing the at least one additional sacrificial layer  68  to keep the interstitial regions  22  free of the second functionalized layer  26 , and to remove the portion of the second functionalized layer  26  from the perimeter  29 , P of the multi-depth depression  20 ′. As will be discussed further in reference to  FIG.  6 D , the presence of the additional sacrificial layer  68  within the multi-depth depression  20 ′ allows the second functionalized layer  26  to be removed from the interstitial regions  22  and from a portion of the sidewall  29 , P. This reduces the padlock like conformation  33  within the multi-depth depression  20 ′. 
     As such, in this example method, utilizing the at least one additional sacrificial layer  68  to define the second functionalized layer  26 , to keep the interstitial regions  22  free of the second functionalized layer  26 , and to remove the portion of the second functionalized layer  26  from the perimeter  29 , P of the multi-depth depression  20 ′ involves depositing the second functionalized layer  26  in the shallow portion  50  and over the region  53  of the sacrificial layer  52  and the interstitial regions  22  (as shown in  FIG.  6 A ); lifting off the region  53  of the sacrificial layer  52 , thereby exposing the portion  25  of the first functionalized layer  24  (as shown in  FIG.  6 B ); applying the at least one additional sacrificial layer  68  over the second functionalized layer  26  and over the portion  25  of the first functionalized layer  24  (as shown in  FIG.  6 C ); dry etching the at least one additional sacrificial layer  68  and the second functionalized layer  26  until the second functionalized layer  26  is removed from the interstitial regions  22  and remains in the shallow portion  50  (as shown in  FIG.  6 D ); dry etching the resin layer  14 ,  18  at the interstitial regions  22  until the interstitial regions (shown at  22 ′) are substantially co-planar with the second functionalized layer  26  in the shallow portion  50  (as shown in  FIG.  6 E ); and lifting off the at least one additional sacrificial layer  68  (as shown in  FIG.  6 F ). 
     Referring specifically to  FIG.  6 A , the second functionalized layer  26  is deposited in the shallow portion  50 , and over the region  53  of the sacrificial layer  52  and over the interstitial regions  22 . As depicted, the second functionalized layer  26  is deposited over portions of the resin layer  14 ,  18  that are exposed in the shallow portion  50 , e.g., the surface  66  and the perimeter sidewall  29 , P. It is to be understood that the second functionalized layer  26  may also be deposited over other exposed portions of the perimeter sidewall  29 , P as well as the internal wall  29 , I. 
     The second functionalized layer  26  may be any of the gel materials described herein and may be applied using any suitable deposition technique. A curing process, as described herein, may be performed after deposition. The second functionalized layer  26  covalently attaches to the resin layer  14 ,  18  in the depression  20 ′, including the sidewall  29 , P. 
     Referring specifically to  FIG.  6 B , the sacrificial layer  52  is removed in a lift-off process. The lift-off process may be performed using any of the suitable organic solvent described herein, which depends, in part, on the type of sacrificial layer  52  used. The lift-off process exposes the portion  25  of the functionalized layer  24  in the deep portion  48 . 
     Referring now to  FIG.  6 C , the additional sacrificial layer  68  is applied over the second functionalized layer  26  and over the exposed portion  25  of the first functionalized layer  24 . In this example, the additional sacrificial layer  68  may be a negative or positive photoresist or poly(methyl methacrylate), and may be applied and cured as described in reference to  FIG.  4 C  so that all of the sacrificial layer  68  remains over the functionalized layers  24 ,  26 . 
     The additional sacrificial layer  68  is then timed dry etched, using any of the timed dry etching techniques described herein. The result of timed dry etching is depicted in  FIG.  6 D . This dry etching process is performed for a measured amount of time to expose the interstitial regions  22  and a portion of the perimeter sidewall  29 , P. As shown in  FIG.  6 D , the timed dry etching is stopped so that the functionalized layer  26  remains on the surface  66  in the shallow portion  50 . Timed dry etching does remove some of the functionalized layer  26  from portions of the perimeter sidewall  29 , P near the opening of the multi-depth depression  20 ′. This reduces the padlock like conformation  33 . 
     Because the functionalized layer  24  is positioned over the lower surface  64  in the deep portion  48 , along the interior wall  29 , I (which is lower than the surface  66 ), and along a portion of the perimeter sidewall  29 , P that is lower than the surface  66 , the timed dry etching does not affect the functionalized layer  24 . Thus, the timed dry etching is stopped so that at least some of the additional sacrificial layer  68  and the underlying functionalized layer  24  remain in an area of the deep portion  48  that is next to the interior wall  29 , I. As a result of timed dry etching, the portion  25  of the functionalized layer  24  and the functionalized layer  26  over the surface  66  remain intact. 
     In some instances, timed dry etching is stopped so that some of the additional sacrificial layer  68  remains over the functionalized layer  26  on the surface  66  and adjacent to the functionalized layer  26  along the perimeter sidewall  29 , P. In these instances, the functionalized layer  26  along the perimeter sidewall  29 , P and the additional sacrificial layer  68  are substantially co-planar in the multi-depth depression  20 ′. As noted above, however, dry etching does remove some of the functionalized layer  26  from portions of the perimeter sidewall  29 , P near the opening of the multi-depth depression  20 ′ to reduce the padlock like conformation  33 . The portions  35  ( FIG.  6 D ) of the functionalized layer  26  that remain along the perimeter sidewall  29 , P after timed dry etching may still form the minimal padlock like conformation  33 , which exhibit reduced signal interference relative to the padlock like conformation  33 . In this example, the minimal padlock like conformation  33  is even further reduced in subsequent processing. 
     Referring now specifically to  FIG.  6 E , the resin layer  14 ,  18  at the interstitial regions  22  is then timed dry etched to form new interstitial regions  22 ′ that are substantially co-planar with the second functionalized layer  26  in (what had been) the shallow portion  50 , i.e., the portion of the second functionalized layer  26  on the surface  66 . Etching of the resin layer  14 ,  18  may involve a dry etching process, such as an anisotropic oxygen plasma, a CF 4  plasma, or a mixture of 90% CF 4  and 10% O 2  plasma. It is to be understood that the dry etching of the resin layer  14 ,  18  shown in  FIG.  6 E  may use the same ions as the dry etching of the sacrificial layer  68  shown in  FIG.  6 D  at a different ratio. The dry etching process shown in  FIG.  6 E  removes the perimeter sidewall  29 , P, and thus, may also remove at least some of the portions  35  of the functionalized layer  26  that remained along the perimeter sidewall  29 , P after the timed dry etching process of  FIG.  6 D . The portions  35  of the functionalized layer  26  on the perimeter sidewall  29 , P are susceptible to the dry etching process of  FIG.  6 E , and thus at least some of the portions  35  are removed with the resin layer  14 ,  18 . The removal of the at least some of the portions  35  further reduces the minimal padlock like conformation  33 . 
     Referring specifically to  FIG.  6 F , the additional sacrificial layer  68  is removed in a lift-off process. The lift-off process may be performed using any of the suitable organic solvents described herein, which depends on the type of sacrificial layer  68  used. 
     While not shown, the method shown in  FIG.  4 A  through  FIG.  4 D  and  FIG.  6 A  through  FIG.  6 F  also includes attaching respective primer sets  30 ,  32  to the functionalized layers  24 ,  26 . In some examples, the primers  34 ,  36  or  34 ′,  36 ′ (not shown in  FIG.  4 A  through  FIG.  4 D  or  FIG.  6 A  through  FIG.  6 F ) may be pre-grafted to the functionalized layer  24 . Similarly, the primers  38 ,  40  or  38 ′,  40 ′ (not shown in  FIG.  4 A  through  FIG.  4 D  or  FIG.  6 A  through  FIG.  6 F ) may be pre-grafted to the functionalized layer  26 . In these examples, additional primer grafting is not performed. 
     In other examples, the primers  34 ,  36  or  34 ′,  36 ′ are not pre-grafted to the functionalized layer  24 . In these examples, the primers  34 ,  36  or  34 ′,  36 ′ may be grafted after the functionalized layer  24  is applied (e.g., at  FIG.  4 B ). In these examples, the primers  38 ,  40  or  38 ′,  40 ′ may be pre-grafted to the second functionalized layer  26 . Alternatively, in these examples, the primers  38 ,  40  or  38 ′,  40 ′ may not be pre-grafted to the second functionalized layer  26 . Rather, the primers  38 ,  40  or  38 ′,  40 ′ may be grafted immediately after the second functionalized layer  26  is applied (e.g., at  FIG.  6 A ); or after the region  53  is removed ( FIG.  6 B ) as long as i) the functionalized layer  26  has different functional groups (than functionalized layer  24 ) for attaching the primers  38 ,  40  or  38 ′,  40 ′ or ii) any unreacted functional groups of the functionalized layer  24  have been quenched, e.g., using the Staudinger reduction to amines or additional click reaction with a passive molecule such as hexynoic acid. 
     When grafting is performed during the method, grafting may be accomplished using any of the grafting techniques described herein. 
     While a single set of the functionalized layers  24 ,  26  is shown in  FIG.  6 F , it is to be understood that the method described in reference to  FIG.  4 A  through  FIG.  4 D  and  FIG.  6 A  through  FIG.  6 F  may be performed to generate an array of depressions  20 ′ (each having functionalized layers  24 ,  26  therein) separated by interstitial regions  22 ′ across the surface of the resin layer  14 ,  18 . 
     Still further examples of the method described in  FIG.  4 A  through  FIG.  4 D  continue from  FIG.  4 D  to  FIG.  7 A  through  FIG.  7 D . Different examples of these methods then continue from  FIG.  7 D  to  FIG.  7 E  through  FIG.  7 F , or from  FIG.  7 D  to FIG.  7 G, or from  FIG.  7 D  to  FIG.  7 H  through  FIG.  7 L . In all of these examples of the method, the resin layer  18  is positioned on a base support  17  (as shown in phantom in  FIG.  4 A ). It is to be understood that in these examples of the method, the multi-layer structure  16  is used (i.e., the resin layer  18  positioned on the base support  17 ), even though the base support  17  is shown in phantom in  FIG.  4 A . In these examples of the method, patterning the first functionalized layer  24  involves applying a sacrificial layer  52  over the first functionalized layer (as shown in  FIG.  4 C ); and dry etching the sacrificial layer  52  and the portions of the first functionalized layer  24  in the shallow portion  50  and over the interstitial regions  22  (as shown in  FIG.  4 D ). It is to be understood that in the methods shown, in part, in  FIG.  7 E  through  FIG.  7 F  or  FIG.  7 G , the resin layer  18  is positioned on the base support  17  which does not include surface groups to covalently attach the second functionalized layer  26 . In contrast, in the method shown, in part, in  FIG.  7 H  through  FIG.  7 L , the base support  17  may or may not include surface groups that are capable of covalently attaching to the second functionalized layer  26 , as the base support  17  is covered by a third photoresist during the application of the second functionalized layer  26 . 
       FIG.  7 A  through  FIG.  7 D  together with either  FIG.  7 E  and  FIG.  7 F  or  FIG.  7 G  depict utilizing the at least one additional sacrificial layer  68  (as shown in  FIG.  7 B ) to define the second functionalized layer  26  adjacent to the portion  25  of the first functionalized layer  24  in the deep portion  48  (shown in  FIG.  7 A ). The utilizing of the at least one additional sacrificial layer  68  involves lifting off the region  53  of the sacrificial layer  52 , thereby exposing the portion  25  of the first functionalized layer  24  (shown in  FIG.  7 A ); applying the at least one additional sacrificial layer  68  over the portion  25  of the first functionalized layer  24  and the resin layer  18  (shown in  FIG.  7 B ); dry etching the at least one additional sacrificial layer  68  to expose the interstitial regions  22  and to remove at least some of the at least one additional sacrificial layer  68  from each multi-depth depression  20 ′ (shown in  FIG.  7 C ); sequentially dry etching the resin layer  18  and the at least one additional sacrificial layer  68  to respectively expose a surface  70  of the base support  17  underlying the interstitial regions  22  and a surface  66  of the resin layer  18  at the shallow portion  50  (shown in  FIG.  7 D ); and depositing the second functionalized layer  26  over the exposed surface  66  of the resin layer  18 , whereby the second functionalized layer  26  does not adhere to the exposed surface  70  of the base support  17  (which will be described in further detail in reference to either  FIG.  7 E  and  FIG.  7 F , or  FIG.  7 G ). 
     Referring now specifically to  FIG.  7 A , the sacrificial layer  52  (shown in  FIG.  4 D ) is removed in a lift-off process. The lift-off process may be performed using any of the suitable organic solvents described herein, which depends on the type of sacrificial layer  52  used. The lift-off process exposes the portion  25  of the first functionalized layer  24 . 
     Referring now specifically to  FIG.  7 B , the at least one additional sacrificial layer  68  is applied over the exposed portion  25  of the first functionalized layer  24  and the resin layer  18 . In this example, the additional sacrificial layer  68  may be a negative or positive photoresist or poly(methyl methacrylate), and may be applied and cure as described in reference to  FIG.  4 C . 
     Referring now to  FIG.  7 C , the additional sacrificial layer  68  is then timed dry etched, using any of the timed dry etching techniques described herein for the sacrificial layer  52  or  68 . The result of time dry etching is depicted in  FIG.  7 C . This dry etching process is performed for a measured amount of time to expose the interstitial regions  22  and a portion of the perimeter sidewall  29 , P. As shown in  FIG.  7 C , the timed dry etching is stopped so that some of the additional sacrificial layer  68  remains over the surface  66  of the resin layer  18 . This will protect the surface  66  and the underlying resin layer  18  when other portions of the resin layer  18 , e.g., at the interstitial regions  22 , are removed. 
       FIG.  7 D  illustrates a protrusion  28  that is formed by sequentially dry etching the resin layer  18  and the at least one additional sacrificial layer  68 . In performing the sequential dry etching processes, first the resin layer  18  at the interstitial regions  22  is dry etched until the interstitial regions  22  are removed to expose a surface  70  of the base support  17 . In this example, the base support  17  acts as an etch stop. The dry etch of these portions of the resin layer  18  eliminates the multi-depth depression  20 ′ and forms the protrusion  28 . Without the portions of the resin layer  18  that define the perimeter sidewall  29 , P, the padlock like conformation  33  within the multi-depth depression  20 ′ cannot be generated. The protrusion  28  includes a top surface  27  (which may correspond with the surface  66  of the multi-depth depression  20 ′ located at the shallow portion  50 ), a lower surface  27 ′ (which may correspond with the surface  64  of the multi-depth depression  20 ′ located at the deep portion  48 ), and a sidewall  29 ′ (which may correspond with the interior wall  29 , I of the multi-depth depression  20 ′) separating the surfaces  27 ,  27 ′. 
     The resin layer  18  may be dry etched using any of the examples set forth herein specifically for the resin layer  14 ,  18 . 
     After the resin layer  18  is etched away to expose the base support surface  70 , the at least one additional sacrificial layer  68  is then timed dry etched using any of the timed dry etching techniques described herein. This dry etching process is performed for a measured amount of time to expose the surface  66 , which was in/at the shallow portion  50  when the multi-depth depression  20 ′ was present. This surface  66  or an area of the resin layer  18  directly underlying this surface  66  becomes the top (or outermost) surface  27  of the protrusion  28 . 
     From  FIG.  7 D , one example of the method continues to  FIG.  7 E  through  FIG.  7 F . In the example method depicted in  FIG.  7 E  and  FIG.  7 F , depositing the second functionalized layer  26  over the exposed surface  66  of the resin layer  18  (i.e., surface  27  of the protrusion  28 ) also deposits the second functionalized layer  26  over the at least one additional sacrificial layer  68  (shown in  FIG.  7 E ); and the method further comprises lifting off the at least one additional sacrificial layer  68  to expose the portion  25  of the first functionalized layer  24  (shown in  FIG.  7 F ). 
     In this example, the second functionalized layer  26  may be any of the gel materials described herein and may be applied using any suitable deposition technique. A curing process, as described herein, may be performed after deposition. As shown in  FIG.  7 E , the second functionalized layer  26  is applied over the resin layer  18  at the top surface  27  of the protrusion  28 , and over the at least one additional sacrificial layer  68 , but is not applied over the surface  70  of the base support  17 . The second functionalized layer  26  covalently attaches to the resin layer  18  at the top surface  27  of the protrusion  28 , and can also covalently attach to other exposed surfaces  71  (e.g., exterior sidewalls) of the resin layer  18 /protrusion  28 . The second functionalized layer  26  may or may not covalently attach to the at least one sacrificial layer  68 . The functionalized layer  26  does not covalently attach to the exposed surface  70  of the base support  17 , as the base support  17  in this example does not have surface groups for the functionalized layer  26  to attach to. Because of the different interactions at the surface(s)  27 ,  71  of the resin layer  18  and at the surface  70  of the base support  17 , the functionalized layer  26  remains over the surface(s)  27 ,  71  (e.g., along exterior sidewalls), and can be easily removed (e.g., via sonication, washing, wiping, etc.) from the surface  70 . This reduces the padlock like conformation  33  such that signal interference from the second functionalized layer  26  adjacent to the surface  27 ′ is expected to be less than 10%. 
     Referring specifically now to  FIG.  7 F , the at least one additional sacrificial layer  68  is lifted off to expose the portion  25  of the first functionalized layer  24 . The lift-off process may be performed using any of the suitable organic solvents described herein, which depends on the type of sacrificial layer  68  used. This lift-off process removes the additional sacrificial layer  68  as well as the second functionalized layer  26  that may be positioned on the additional sacrificial layer  68 . 
     As depicted in  FIG.  7 F , a sidewall  29 ′ is positioned between the top surface  27  and the lower surface  27 ′ of the protrusion  28 . This sidewall  29 ′ corresponds with at least a portion of the interior wall  29 , I of the multi-depth depression  20 ′ and may have the first functionalized  24  covalently attached thereto. 
     While not shown, the method shown in  FIG.  4 A  through  FIG.  4 D  and  FIG.  7 A  through  FIG.  7 F  also includes attaching respective primer sets  30 ,  32  to the functionalized layers  24 ,  26 . In some examples, the primers  34 ,  36  or  34 ′,  36 ′ (not shown in  FIG.  4 A  through  FIG.  4 D  or  FIG.  7 A  through  FIG.  7 F ) may be pre-grafted to the functionalized layer  24 . Similarly, the primers  38 ,  40  or  38 ′,  40 ′ (not shown in  FIG.  4 A  through  FIG.  4 D  or  FIG.  7 A  through  FIG.  7 F ) may be pre-grafted to the functionalized layer  26 . In these examples, additional primer grafting is not performed. 
     In other examples, the primers  34 ,  36  or  34 ′,  36 ′ are not pre-grafted to the functionalized layer  24 . In these examples, the primers  34 ,  36  or  34 ′,  36 ′ may be grafted after the functionalized layer  24  is applied (e.g., at  FIG.  4 B ). In these examples, the primers  38 ,  40  or  38 ′,  40 ′ may be pre-grafted to the second functionalized layer  26 . Alternatively, in these examples, the primers  38 ,  40  or  38 ′,  40 ′ may not be pre-grafted to the second functionalized layer  26 . Rather, the primers  38 ,  40  or  38 ′,  40 ′ may be grafted immediately after the second functionalized layer  26  is applied (e.g., at  FIG.  7 E ); or after the at least one additional sacrificial layer  68  is lifted off ( FIG.  7 F ) as long as i) the functionalized layer  26  has different functional groups (than functionalized layer  24 ) for attaching the primers  38 ,  40  or  38 ′,  40 ′ or ii) any unreacted functional groups of the functionalized layer  24  have been quenched, e.g., using the Staudinger reduction to amines or additional click reaction with a passive molecule such as hexynoic acid. 
     When grafting is performed during the method, grafting may be accomplished using any of the grafting techniques described herein. 
     While a single set of the functionalized layers  24 ,  26  is shown in  FIG.  7 F , it is to be understood that the method described in reference to  FIG.  4 A  through  FIG.  4 D  and  FIG.  7 A  through  FIG.  7 F  may be performed to generate an array of protrusions  28  (each having functionalized layers  24 ,  26  thereon) across the base support  17 , where the protrusions  28  are separated by the exposed surface  70  of the base support  17 . 
     Referring back to  FIG.  7 D , another example of the method continues to  FIG.  7 G . In this example, prior to depositing the second functionalized layer  26 , the method further comprises lifting off the at least one additional sacrificial layer  68  to expose the portion  25  of the first functionalized layer  24 ; and wherein depositing the second functionalized layer  26  over the exposed surface  66  of the resin layer  18  involves a selective deposition process. 
     In this example method, the at least one additional sacrificial layer  68  is lifted off to expose the portion  25  of the first functionalized layer  24  ( FIG.  7 G ). The lift-off process may be performed using any of the suitable organic solvents described herein, which depends on the type of sacrificial layer  68  used. As depicted in  FIG.  7 G , the first functionalized layer  24  is positioned over the lower surface  27 ′ as well as the sidewall  29 ′ of the protrusion  28 . 
     In this example, after the additional sacrificial layer  68  is removed, the second functionalized layer  26  is selectively deposited. The second functionalized layer  26  may be any of the gel materials described herein and may be applied using any suitable deposition technique under high ionic strength conditions (e.g., in the presence of 10×PBS, NaCl, KCl, etc.). A curing process, as described herein, may be performed after deposition. 
     When the deposition of the gel material of the functionalized layer  26  is performed under high ionic strength, the second functionalized layer  26  does not deposit on or adhere to the first functionalized layer  24 . As such, the second functionalized layer  26  does not contaminate the first functionalized layer  24 . 
     The second functionalized layer  26  does attach to the exposed surfaces (e.g.,  27 ,  71 ) of the resin layer  18 , which has surface groups capable of attaching to the second functionalized layer  26 . 
     The second functionalized layer  26  does not attach to the exposed surface  70  of the base support  17 , as the base support  17  does not have surface groups for the functionalized layer  26  to attach to, as described above. 
     While not shown, the method shown in  FIG.  4 A  through  FIG.  4 D ,  FIG.  7 A  through  FIG.  7 D  and  FIG.  7 G  also includes attaching respective primer sets  30 ,  32  to the functionalized layers  24 ,  26 . In some examples, the primers  34 ,  36  or  34 ′,  36 ′ (not shown in  FIG.  4 A  through  FIG.  4 D ,  FIG.  7 A  through  FIG.  7 D  and  FIG.  7 G ) may be pre-grafted to the functionalized layer  24 . Similarly, the primers  38 ,  40  or  38 ′,  40 ′ (not shown in  FIG.  4 A  through  FIG.  4 D ,  FIG.  7 A  through  FIG.  7 D  and  FIG.  7 G ) may be pre-grafted to the functionalized layer  26 . In these examples, additional primer grafting is not performed. 
     In other examples, the primers  34 ,  36  or  34 ′,  36 ′ are not pre-grafted to the functionalized layer  24 . In these examples, the primers  34 ,  36  or  34 ′,  36 ′ may be grafted after the functionalized layer  24  is applied (e.g., at  FIG.  4 B ). In these examples, the primers  38 ,  40  or  38 ′,  40 ′ may be pre-grafted to the second functionalized layer  26 . Alternatively, in these examples, the primers  38 ,  40  or  38 ′,  40 ′ may not be pre-grafted to the second functionalized layer  26 . Rather, the primers  38 ,  40  or  38 ′,  40 ′ may be grafted immediately after the second functionalized layer  26  is applied (e.g., at  FIG.  7 G ) as long as i) the functionalized layer  26  has different functional groups (than functionalized layer  24 ) for attaching the primers  38 ,  40  or  38 ′,  40 ′ or ii) any unreacted functional groups of the functionalized layer  24  have been quenched, e.g., using the Staudinger reduction to amines or additional click reaction with a passive molecule such as hexynoic acid. 
     When grafting is performed during the method, grafting may be accomplished using any of the grafting techniques described herein. 
     While a single set of the functionalized layers  24 ,  26  is shown in  FIG.  7 G , it is to be understood that the method described in reference to  FIG.  4 A  through  FIG.  4 D ,  FIG.  7 A  through  FIG.  7 D  and  FIG.  7 G  may be performed to generate an array of protrusions  28  (each having functionalized layers  24 ,  26  thereon) across the base support  17 , where the protrusions  28  are separated by the exposed surface  70  of the base support  17 . 
     The method shown in  FIG.  4 A  through  FIG.  4 D ,  FIG.  7 A  through  FIG.  7 D , and  FIG.  7 H  through  FIG.  7 L  utilizes two additional sacrificial layers  68  (as shown in  FIG.  7 B ) and  72 ,  60 ′ (as shown in  FIG.  7 J ). In this example, the additional sacrificial layer  72 ,  60 ′ is a negative photoresist. In this example of the method, the base support  17 ′ and the resin layer  18 ′ are utilized. As described herein, the base support  17 ′ is a UV transmitting material, and thick and thin portions of the resin layer  18 ′ are adjusted to achieve the desired UV absorption (at thicker portions) and UV transmittance (at thinner portions). The UV transmitting materials may be any of the UV transmitting materials described herein, providing the base support  17 ′ allows a dosage of ultraviolet light to be transmitted through the material and the thickness of the resin layer  18 ′ can be adjusted to be transparent or absorbing. Additionally, in these examples, the base support  17 ′ may or may not include surface groups to covalently attach to the functionalized layers  24 ,  26 , as the base support  17 ′ is not exposed during the deposition of either the first functionalized layer  24  (see  FIG.  4 B ) or the second functionalized layer  26  (see  FIG.  7 K ). 
     The portion of the method depicted in  FIG.  4 A  through  FIG.  4 D  may be performed as described herein. The method continues at  FIG.  7 A  through  FIG.  7 D  and then at  FIG.  7 G  through  FIG.  7 L . In this example, utilizing the at least one additional sacrificial layer  68  to define the second functionalized layer  26  involves lifting off the region  53  of the sacrificial layer  52 , thereby exposing the portion  25  of the first functionalized layer  24  (shown in  FIG.  7 A ); applying a first of the at least one additional sacrificial layer  68  over the portion  25  of the first functionalized layer  24  and the resin layer  18  (shown in  FIG.  7 B ); dry etching the first of the at least one additional sacrificial layer  68  to expose the interstitial regions  22  and to remove at least some of the first of the at least one additional sacrificial layer  68  from each multi-depth depression  20 ′ (shown at  FIG.  7 C ); sequentially dry etching the resin layer  18  and the first of the at least one additional sacrificial layer  68  to respectively expose a surface  70  of the base support  17 ′ underlying the interstitial regions  22  and a surface  66  of the resin layer  18  at the shallow portion  50  (shown in  FIG.  7 D ); lifting off the at least one additional sacrificial layer  68  to expose the portion  25  of the first functionalized layer  24  (shown in  FIG.  7 H ); applying a second of the at least one additional sacrificial layer  72  over the portion  25  of the first functionalized layer  24 , the exposed surface  70  of the base support  17 ′, and the exposed surface  66  of the resin layer  18 ′ wherein the second of the at least one additional sacrificial layer  72  is a negative photoresist (shown in  FIG.  7 I ); directing, through the base support  17 ′, an ultraviolet light dosage, thereby forming an insoluble negative photoresist  60 ′ over the portion  25  of the first functionalized layer  24  and the exposed surface  70  of the base support  17 ′ and a soluble negative photoresist  60 ″ over the exposed surface  66  of the resin layer  18 ′ at the shallow portion  50  (also shown in  FIG.  7 I ); removing the soluble negative photoresist  60 ″ such that the exposed surface  66  of the resin layer  18 ′ (e.g., top surface  27  of the protrusion  28 ) remains exposed (shown in  FIG.  7 J ); depositing the second functionalized layer  26  over the exposed surface  66  of the resin layer  18 ′ and the insoluble negative photoresist  60 ′ (shown in  FIG.  7 K ); and lifting off the insoluble negative photoresist  60 ′ (shown in  FIG.  7 L ). 
     The portions of this example of the method depicted in  FIG.  7 A  through  FIG.  7 D  may be performed as described herein. 
     Referring now to  FIG.  7 H , the additional sacrificial layer  68  is lifted off to expose the portion  25  of the first functionalized layer  24 . The lift-off process may be performed using any of the suitable organic solvents described herein, which depends on the type of sacrificial layer  68  used. 
       FIG.  7 I  depicts the deposition of the second of the at least one additional sacrificial layer  72  over the portion  25  of the first functionalized layer  24 , the exposed surface  66  of the resin layer  18 ′, and the surface  70  of the base support  17 ′. The second of the at least one additional sacrificial layer  72  is a negative photoresist, and can be any of the negative photoresists described herein. The negative photoresist may be applied using any suitable technique. To develop the negative photoresist, an ultraviolet light dosage is directed through the base support  17 ′ and the resin layer  18 ′. The thicker resin portion(s) (e.g., the portion that defines the surfaces  66 ,  27 ) blocks at least 75% of light that is transmitted through the base support  17 ′ and the resin layer  18 ′ from reaching the third (negative) photoresist  72  that is positioned directly in line with the thicker resin portions. As such, these portions become the soluble negative photoresist  60 ″. The soluble portions are removed, e.g., with the developer, to re-expose the surface  66 ,  27 . In contrast, the UV light is able to transmit through the base support  17 ′ and the thinner resin portions (to which the first functionalized layer  24  is attached). Thus, portions of the negative photoresist (third additional sacrificial layer  72 ) in direct contact with the surface  70  and over the first functionalized layer  24  become insoluble.  FIG.  7 J  depicts the negative insoluble photoresist  60 ′ that is formed over the portion  25  of the first functionalized layer  24  as well as the surface  70  of the base support  17 ′ after development of the third photoresist  72 . 
     Referring now to  FIG.  7 K , the second functionalized layer  26  is deposited over the exposed surface  66  of the resin layer  18 ′ and the negative insoluble photoresist  60 ′. The second functionalized layer  26  may be any of the gel materials described herein and may be applied using any suitable technique described herein. A curing process, as described herein, may be performed after deposition. The second functionalized layer  26  covalently attaches to the resin layer  18 ′ at the surface  66  (surface  27  of the protrusion  28 ). 
       FIG.  7 K  depicts the lift-off of the negative insoluble photoresist  60 ′. The lift-off process may be performed using any suitable removers for the negative photoresists described herein. 
     The lift-off process removes i) at least 99% of the negative insoluble photoresist  60 ′ and ii) the functionalized layer  26  positioned thereon. The negative insoluble photoresist  60 ′ is lifted off to expose the portion  25  of the first functionalized layer  24 , shown in  FIG.  7 K . 
     The resulting protrusion  28  includes the functionalized layers  24 ,  26  on the surfaces  27 ′,  27 . As depicted in  FIG.  7 K , a sidewall  29 ′ is positioned between the top surface  27  and the lower surface  27 ′ of the protrusion  28 . This sidewall  29 ′ corresponds with at least a portion of the interior wall  29 , I of the multi-depth depression  20 ′ and may have the first functionalized  24  covalently attached thereto. 
     While not shown, the method shown in  FIG.  4 A  through  FIG.  4 D ,  FIG.  7 A  through  FIG.  7 D  and  FIG.  7 H  through  FIG.  7 L  also includes attaching respective primer sets  30 ,  32  to the functionalized layers  24 ,  26 . In some examples, the primers  34 ,  36  or  34 ′,  36 ′ (not shown in  FIG.  4 A  through  FIG.  4 D ,  FIG.  7 A  through  FIG.  7 D  and  FIG.  7 H  through  FIG.  7 L ) may be pre-grafted to the functionalized layer  24 . Similarly, the primers  38 ,  40  or  38 ′,  40 ′ (not shown in  FIG.  4 A  through  FIG.  4 D ,  FIG.  7 A  through  FIG.  7 D  and  FIG.  7 H  through  FIG.  7 L ) may be pre-grafted to the functionalized layer  26 . In these examples, additional primer grafting is not performed. 
     In other examples, the primers  34 ,  36  or  34 ′,  36 ′ are not pre-grafted to the functionalized layer  24 . In these examples, the primers  34 ,  36  or  34 ′,  36 ′ may be grafted after the functionalized layer  24  is applied (e.g., at  FIG.  4 B ). In these examples, the primers  38 ,  40  or  38 ′,  40 ′ may be pre-grafted to the second functionalized layer  26 . Alternatively, in these examples, the primers  38 ,  40  or  38 ′,  40 ′ may not be pre-grafted to the second functionalized layer  26 . Rather, the primers  38 ,  40  or  38 ′,  40 ′ may be grafted immediately after the second functionalized layer  26  is applied (e.g., at  FIG.  7 K ); or after the negative insoluble photoresist  60 ′ is removed as long as i) the functionalized layer  26  has different functional groups (than functionalized layer  24 ) for attaching the primers  38 ,  40  or  38 ′,  40 ′ or ii) any unreacted functional groups of the functionalized layer  24  have been quenched, e.g., using the Staudinger reduction to amines or additional click reaction with a passive molecule such as hexynoic acid. 
     When grafting is performed during the method, grafting may be accomplished using any of the grafting techniques described herein. 
     While a single set of the functionalized layers  24 ,  26  is shown in  FIG.  7 L , it is to be understood that the method described in reference to  FIG.  4 A  through  FIG.  4 D ,  FIG.  7 A  through  FIG.  7 D  and  FIG.  7 H  through  FIG.  7 L  may be performed to generate an array of protrusions  28  (each having functionalized layers  24 ,  26  thereon) across the base support  17 ′, where the protrusions  28  are separated by the exposed surface  70  of the base support  17 ′. 
     Another example of the method continues from  FIG.  4 A  through  FIG.  4 D  at  FIG.  8 A  to  FIG.  8 G . As discussed herein, the portion of the method described in reference to  FIG.  4 A  through  FIG.  4 D  generates the portion  25  of the functionalized layer  24  in the deep portion  48 , which is covered by the region  53  of the sacrificial layer  52 . This example method continues at  FIG.  8 A  and includes utilizing the at least one additional insoluble sacrificial layer  68  to define the second functionalized layer  26 , which involves: lifting off the region  53  of the sacrificial layer  52 , thereby exposing the portion  25  of the first functionalized layer  24  ( FIG.  8 A ); applying the at least one additional sacrificial layer  68  over the portion  25  of the first functionalized layer  24  and the resin layer  14 ,  14 ′,  18 ,  18 ′ ( FIG.  8 B ); dry etching the at least one additional sacrificial layer  68  to expose the interstitial regions  22  and to remove at least some of the at least one additional sacrificial layer  68  from each multi-depth depression  20 ′ ( FIG.  8 C ); depositing a metal film  62  over the interstitial regions  22  and the at least one additional sacrificial layer  68  ( FIG.  8 D ); lifting off the at least one additional sacrificial layer  68 , thereby exposing the portion  25  of the first functionalized layer  24  and the resin layer  14 ,  14 ′,  18 ,  18 ′ at the shallow portion  50 , and whereby the metal film  62  remains intact over the interstitial regions  22  and on at least a portion  37  of the sidewall  29 , P of each multi-depth depression  20 ′ ( FIG.  8 E ); depositing the second functionalized layer  26  over the metal film  62  and the resin layer  14 ,  14 ′,  18 ,  18 ′ at the shallow portion  50  ( FIG.  8 F ); and etching the metal film  62  from the interstitial regions  22  and the portion  37  of the sidewall  29 , P of each multi-depth depression  20 ′ (as shown in  FIG.  8 G ). 
     Referring specifically to  FIG.  8 A , the region  53  of the sacrificial layer  52  is lifted off to expose the portion  25  of the first functionalized layer  24 . The first sacrificial layer  52  may be lifted off using any suitable technique described herein, and any suitable remover. The lift-off process and remover will depend, in part, on what type of material is used as the sacrificial layer  52 . 
     Referring specifically to  FIG.  8 B , the at least one additional sacrificial layer  68  is then applied over the portion  25  of the first functionalized layer  24  and the resin layer  14 ,  14 ′,  18 ,  18 ′. In this example, the sacrificial layer  68  may be any example of the negative or positive photoresist disclosed herein or poly(methyl methacrylate). The additional sacrificial layer  68  may be applied using any suitable deposition technique disclosed and exposed to curing. 
       FIG.  8 C  depicts the at least one additional sacrificial layer  68  after it has been dry etched to expose the interstitial regions  22  and to remove at least some of the at least one additional sacrificial layer  68  from the multi-depth depression  20 ′. The dry etching process may be performed as described herein, for example, in reference to  FIG.  7 B . The dry etching process exposes a portion  37  of the perimeter of the sidewall  29 , P, but does not expose the surface  66  or the portion  25  of the functionalized layer  24 . As such, the additional sacrificial layer  68  that remains after dry etching covers both the surface  66  and the portion  25  of the functionalized layer  24 . Because dry etching is substantially uniform and the surface  66  is not exposed, the additional sacrificial layer  68  that remains will also cover some of the perimeter sidewall  29 , P (e.g., portion  39  shown in  FIG.  8 D ). The portion  37  of the perimeter of the sidewall  29 , P that is exposed is between the interstitial region  22  and the top of the remaining additional sacrificial layer  68 , and ultimately defines the region of the sidewall  29 , P where a metal film  62  will be formed to prevent the second functionalized layer  26  from being deposited, which will reduce the padlock like conformation  33 . As such, dry etching is controlled (e.g., via time) so that the length of the portion  37  is maximized without exposing the surface  66 . 
       FIG.  8 D  depicts when the metal film  62  is deposited over the interstitial regions  22  and the at least one additional sacrificial layer  68 . Examples of suitable materials for the metal film  62  include semi-metals, such as silicon, or metals, such as aluminum, copper, titanium, gold, silver, etc. In some examples, the semi-metal or metal may be at least substantially pure (&lt;99% pure). In other examples, molecules or compounds of the listed elements may be used. For example, oxides of any of the listed semi-metals (e.g., silicon dioxide) or metals (e.g., aluminum oxide) may be used, alone or in combination with the listed semi-metal or metal. These materials may be deposited using any suitable technique disclosed herein that results in a substantially uniform film, such as chemical vapor deposition (CVD), spray coating (e.g., ultrasonic spray coating), spin coating, etc. A directional coating method may be used, such as sputtering or thermally evaporating, which generates a metal film  62  with varying thicknesses. In these instances, the portion of the metal film  62  on the interstitial regions  22  is thicker than on the portion of the metal film  62  on the sidewalls  29  and on the additional sacrificial layer  68 . 
     Referring now to  FIG.  8 E , the at least one additional sacrificial layer  68  is removed to expose the resin layer  14 ,  14 ′,  18 ,  18 ′ at the shallow portion  50 . In one example, the at least one additional sacrificial layer  68  may be lifted off using any suitable technique described herein, depending, in part, on what type of sacrificial material is used for the at least one additional sacrificial layer  68 . Lift-off may be used when the portion of the metal layer  62  overlying the additional sacrificial layer  68  is thin. A combination of the organic solvent and agitation may attack the thinner portions of the metal  62 , enabling lift-off of both the additional sacrificial layer  68  and the portion of the metal layer  62  thereon. In another example, the portion of the metal layer  62  overlying the additional sacrificial layer  68  and the additional sacrificial layer  68  may be removed sequentially. In this example, the portion of the metal layer  62  overlying the additional sacrificial layer  68  may be anisotropically etched, and then the organic solvent may be used to lift-off the additional sacrificial layer  68 . As shown in  FIG.  8 E , the removal exposes the surface  66 , the functionalized layer  24 , and other portions  39  of the perimeter sidewall  29 , P that are not covered by the metal film  62  and that had been covered by the additional sacrificial layer  68 . In contrast, another portion of the metal film  62  remains intact over the interstitial regions  22  and may remain intact on the portion  37  of the perimeter sidewall  29 , P of each multi-depth depression  20 ′. 
     As shown at  FIG.  8 E , the removal of the additional sacrificial layer  68  also exposes the portion  25  of the functionalized layer  24 . 
     Referring specifically to  FIG.  8 F , the second functionalized layer  26  is then deposited over the metal film  62  and the resin layer  14 ,  14 ′,  18 ,  18 ′ at the shallow portion  50 . As depicted in  FIG.  8 F , the exposed portions of the resin layer  14 ,  14 ′,  18 ,  18 ′, including surface  66  at the shallow portion  50  and portions  39  of the perimeter sidewall  29 , P, have the second functionalized layer  26  deposited thereon. The metal film  62  prevents the functionalized layer  26  from adhering to the resin layer  14 ,  14 ′,  18 ,  18 ′ at the portion  37  of the perimeter sidewall  29 , P. This reduces the padlock like conformation  33 . 
     The second functionalized layer  26  may be any of the gel materials described herein, and may be deposited using any suitable technique under high ionic strength conditions (e.g., in the presence of 10×PBS, NaCl, KCl, etc.) as described herein. When the deposition of the gel material of the functionalized layer  26  is performed under high ionic strength, the second functionalized layer  26  does not deposit on or adhere to the first functionalized layer  24 . As such, the second functionalized layer  26  does not contaminate the first functionalized layer  24 , as shown in  FIG.  8 F . A curing process, as described herein, may be performed after deposition. 
     Referring specifically to  FIG.  8 G , the metal film  62  is then etched, and thus removed from the interstitial regions  22  and the portion  37  of the perimeter sidewall  29 , P of each multi-depth depression  20 ′. The metal film  62  may be dry or wet etched. The dry etching is performed as described herein, e.g., using reactive ion etching with BCl 3 +Cl 2 . As examples of wet etching, an aluminum metal film  62  can be removed in acidic (e.g., nitric acid based) or basic (e.g., KOH based) conditions, a copper metal film  62  can be removed using FeCl 3 , a copper, gold or silver metal film  62  can be removed in an iodine and iodide solution, and a silicon metal film  62  can be removed in basic (pH) conditions. The resin layer  14 ,  14 ′,  18 ,  18 ′ is not susceptible to the etching process, and thus the resin layer  14 ,  14 ′,  18 ,  18 ′ at the interstitial regions  22  and at the portion  37  of the perimeter sidewall  29 , P is exposed by the wet etching process and remains intact. The first and second functionalized layers  24 ,  26  are covalently attached to the resin layer  14 ,  14 ′,  18 ,  18 ′, and thus are not affected by the etching process. 
     The resulting multi-depth depression  20 ′ includes the functionalized layers  24 ,  26  therein. As depicted in  FIG.  8 G , the portion  39  of the perimeter sidewall  29 , P of the multi-depth depression  20 ′ may have the second functionalized layer  26  covalently attached thereto after metal film  62  removal, which creates the padlock like conformation  33 . However, the second functionalized layer  26  on the portion  39  of the perimeter sidewall  29 , P is minimized by the metal mask  62  and how much of the sacrificial layer  68  is removed in the timed dry etch. 
     While not shown, the method shown in  FIG.  4 A  through  FIG.  4 D  and  FIG.  8 A  through  FIG.  8 G  also includes attaching respective primer sets  30 ,  32  to the functionalized layers  24 ,  26 . In some examples, the primers  34 ,  36  or  34 ′,  36 ′ (not shown in  FIG.  4 A  through  FIG.  4 D  and  FIG.  8 A  through  FIG.  8 G ) may be pre-grafted to the functionalized layer  24 . Similarly, the primers  38 ,  40  or  38 ′,  40 ′ (not shown in  FIG.  4 A  through  FIG.  4 D  and  FIG.  8 A  through  FIG.  8 G ) may be pre-grafted to the functionalized layer  26 . In these examples, additional primer grafting is not performed. 
     In other examples, the primers  34 ,  36  or  34 ′,  36 ′ are not pre-grafted to the functionalized layer  24 . In these examples, the primers  34 ,  36  or  34 ′,  36 ′ may be grafted after the functionalized layer  24  is applied (e.g., at  FIG.  4 B ). In these examples, the primers  38 ,  40  or  38 ′,  40 ′ may be pre-grafted to the second functionalized layer  26 . Alternatively, in these examples, the primers  38 ,  40  or  38 ′,  40 ′ may not be pre-grafted to the second functionalized layer  26 . Rather, the primers  38 ,  40  or  38 ′,  40 ′ may be grafted immediately after the second functionalized layer  26  is applied (e.g., at  FIG.  8 F ) or after the metal mask  62  is removed (e.g., at  FIG.  8 G ) as long as i) the functionalized layer  26  has different functional groups (than functionalized layer  24 ) for attaching the primers  38 ,  40  or  38 ′,  40 ′ or ii) any unreacted functional groups of the functionalized layer  24  have been quenched, e.g., using the Staudinger reduction to amines or additional click reaction with a passive molecule such as hexynoic acid. 
     When grafting is performed during the method, grafting may be accomplished using any of the grafting techniques described herein. 
     While a single set of the functionalized layers  24 ,  26  is shown in  FIG.  8 G , it is to be understood that the method described in reference to  FIG.  4 A  through  FIG.  4 D  and  FIG.  8 A  through  FIG.  8 G  may be performed to generate an array of depressions  20 ′ (each having functionalized layers  24 ,  26  therein) across the surface of the resin layer  14 ,  14 ′,  18 ,  18 ′. 
     Another example method is shown in  FIG.  9 A  through  FIG.  9 H , and this method generally includes: forming a metal film  62  on at least a portion of a sidewall  29 , P of each of a plurality of multi-depth depressions  20 ′ defined in a resin layer  14 ,  14 ′,  18 ,  18 ′ and separated by interstitial regions  22  ( FIG.  9 A ), wherein each multi-depth depression  20 ′ includes a deep portion  48  and a shallow portion  50  adjacent to the deep portion  48  and wherein at least some of a bottom surface  64 ,  66  of each multi-depth depression  20 ′ is free of the metal film  62 ; depositing a first functionalized layer  24  over each of the multi-depth depressions  20 ′ and the interstitial regions  22  ( FIG.  9 B ); patterning the first functionalized layer  24 , whereby a portion  25  of the first functionalized layer  24  in the deep portion  48  is covered by a region  53  of a sacrificial layer  52  and portions of the first functionalized layer  24  in the shallow portion  50 , over the metal film  62 , and over the interstitial regions  22  are removed ( FIG.  9 D ); depositing a second functionalized layer  26  over the interstitial regions  22 , over the metal film  62 , over the region  53  of the sacrificial layer  52 , and in the shallow portion  50  ( FIG.  9 E ); lifting off the region  53  of the sacrificial layer  52 , thereby exposing the portion  25  of the first functionalized layer  24  ( FIG.  9 F ); wet etching the metal film  62 , thereby removing the second functionalized layer  26  positioned over the metal film  62  ( FIG.  9 G ); and polishing the interstitial regions  22 , whereby the portion  25  of the first functionalized layer  24  in the deep portion  48  and the second functionalized layer  26  in the shallow portion  50  remain intact. 
       FIG.  9 A  depicts the application of a metal film  62  to at least a portion of the perimeter sidewall  29 , P of the multi-depth depression  20 ′. In this example, the portion of the perimeter sidewall  29 , P that is covered by the metal film  62  is between the interstitial region  22  and the surface  66 . Around the perimeter sidewall  29 , P, the length of the metal film  62  is the same as the depth of the shallow portion  50 . It is to be understood that the bottom surface  64  of the depression  20 ′ is free of the metal film  62 , and the bottom surface  66  of the depression  20 ′ is free of the metal film  62  except at the intersection of the surface  66  and the perimeter sidewall  29 , P. 
     While not shown, the metal film  62  shown in  FIG.  9 A  may be formed using yet another sacrificial layer. In this example, the sacrificial layer is deposited on the resin layer  14 ,  18  and cured. The sacrificial layer may then be etched back so that the sacrificial layer remains in the deep portion  48  adjacent to the step feature  80 , and so that the surface  66  has a thin layer of the sacrificial layer thereon. The metal film  62  may then be applied using a directional coating method. This would result in a metal film  62  on the interstitial regions  22 , on the sidewalls  29 , and on the sacrificial layer. Anisotropic etching may then be used to remove the metal film  62  from the interstitial regions  22  and from the sacrificial layer, and then the sacrificial layer may be removed using a suitable organic solvent. The metal film  62  remains on the sidewalls  29  as shown in  FIG.  9 A . Alternatively, the metal film  62  could be conformally coated using sputtering, and anisotropic etching could be used to remove the metal film  62  from desired areas. 
       FIG.  9 B  depicts the deposition of a first functionalized layer  24  over the multi-depth depression  20 ′ and the interstitial regions  22 . The functionalized layer  24  may be any of the gel materials described herein and may be applied using any suitable deposition technique. A curing process may be performed after deposition. The functionalized layer  24  covalently attaches to the exposed surfaces of the resin layer  14 ,  14 ′ or to the exposed surfaces of the resin layer  18 ,  18 ′. When the resin layer  14 ,  14 ′ is used, the applied functionalized layer  24  is positioned over the metal film  62  and over exposed surfaces of the resin layer  14 ,  14 ′, including over the surface  64  of the deep portion  48 , the surface  66  of the shallow portion  50 , and the interstitial regions  22 . When the multi-layer structure  16  is used, the applied functionalized layer  24  is positioned over the metal film  62  and over the exposed surfaces of the resin layer  18 ,  18 ′, including over the exposed surface  64  of the deep portion  48 , the surface  66  of the shallow portion  50 , and the interstitial regions  22 . 
     The first functionalized layer  24  is then patterned. In an example, patterning the first functionalized layer  24  involves: applying a sacrificial layer  52  over the first functionalized layer  24 ; and dry etching the sacrificial layer  52  and the portions of the first functionalized layer  24  in the shallow portion  50  and over the interstitial regions  22 . 
       FIG.  9 C  depicts the deposition of the sacrificial layer  52 . The sacrificial layer  52  may be any of the photoresists described herein, i.e., a positive photoresist  56  or a negative photoresist  60 , or poly(methyl methacrylate). The sacrificial layer  52  may be applied using any suitable deposition technique disclosed herein and then may be exposed to curing. 
       FIG.  9 D  depicts the remaining region  53  of the sacrificial layer  52  and the portion  25  of the functionalized layer  24  after a dry etch process, which exposes the metal film  62 , the surface  66  at the shallow portion  50 , and the interstitial regions  22 . This dry etching may be performed by any suitable technique described herein (e.g., in reference to  FIG.  4 D ). The dry etching process removes the sacrificial layer  52  from the multi-depth depression  20 ′ at the shallow portion  50 , and also removes the sacrificial layer  52  from the interstitial regions  22 . The metal film  62  remains intact over the portion of the sidewall  29 , P. The remaining sacrificial layer  52  forms the region  53  that remains in the deep portion  48 , as shown in  FIG.  9 D . The region  53  of the sacrificial layer  52  is directly over, i.e. covers, the portion  25  of the first functionalized layer  24  in the deep portion  48 . 
     This dry etching process may be performed for a measured amount of time to expose the surface  66 . In these instances, the region  53  of the sacrificial layer  52  and underlying functionalized layer  24  remain in the portion of the deep portion  48  that is next to the interior wall  29 , I. As such, the remaining sacrificial layer  52 ,  53  is at least substantially co-planar with the surface  66  at the shallow portion  50 , and the perimeter sidewall  29 , P adjacent to the deep portion  48  is not exposed. Alternatively, this dry etching process may be performed to extend deeper than the surface  66 . In these instances, some of the region  53  of the sacrificial layer  52  and underlying functionalized layer  24  are removed such that some of the perimeter sidewall  29 , P adjacent to the deep portion  48  and some of the interior sidewall  29 , I are exposed. This exposure is shown in  FIG.  9 D . 
       FIG.  9 E  depicts the second functionalized layer  26  deposited over the interstitial regions  22 , over the metal film  62 , over the region  53  of the sacrificial layer  52 , and on the surface  66  in the shallow portion  50 . The second functionalized layer  26  (e.g., the gel material that forms the second functionalized layer  26 ) may be applied using any suitable deposition technique. When the perimeter sidewall  29 , P adjacent to the deep portion  48  is not exposed as a result of dry etching, the second functionalized layer  26  does not deposit over the perimeter sidewall  29 , P adjacent to the deep portion  48 . In this example, the padlock like conformation  33  is eliminated. Alternatively, when the perimeter sidewall  29 , P adjacent to the deep portion  48  is partially exposed as a result of dry etching, the second functionalized layer  26  may deposit over the perimeter sidewall  29 , P adjacent to the deep portion  48  and the interior sidewall  29 , I due to covalent attachment to the exposed resin layer  14 ,  14 ′,  18 ,  18 ′. In this example, the padlock like conformation  33  is reduced. 
     The second functionalized layer  26  does not contaminate the portion of first functionalized layer  24 , which is covered by the region  53  of the sacrificial layer  52 . 
       FIG.  9 F  depicts the removal of the region  53  of the sacrificial layer  52 . The region  53  of the sacrificial layer  52  is removed through a lift-off process. The lift-off process may be any suitable lift-off process described herein that involves any suitable organic solvent, which depends, in part, on the type of sacrificial layer  52  used. The lift-off process removes i) at least 99% of the sacrificial layer  52  and ii) the functionalized layer  26  positioned thereon. The sacrificial layer  52  is lifted off to expose the portion  25  of the first functionalized layer  24 , shown in  FIG.  9 F . 
       FIG.  9 G  depicts the removal of the metal film  62 . In an example, the removal of the metal film  62  may involve a wet etching or lift-off process, which depends upon the material of the metal film  62 . As examples, an aluminum metal film  62  can be removed in acidic or basic conditions, a copper metal film  62 ′ can be removed using FeCl 3 , a copper, gold or silver metal film  62  can be removed in an iodine and iodide solution, and a silicon metal film  62  can be removed in basic (pH) conditions. The removal of the metal film  62  also removed the second functionalized layer  26  thereon and exposes the sidewall  29 , P of the multi-depth depression  20 ′. 
     In  FIG.  9 H , the functionalized layer  26  that is positioned over the interstitial regions  22  is removed, e.g., using a polishing process. The polishing process may be performed with a chemical slurry (including, e.g., an abrasive, a buffer, a chelating agent, a surfactant, and/or a dispersant) which can remove the functionalized layer  26  from the interstitial regions  22  without deleteriously affecting the underlying resin layer  14 ,  14 ′,  18 ,  18 ′ at those regions  22 . Alternatively, polishing may be performed with a solution that does not include the abrasive particles. 
     The chemical slurry may be used in a chemical mechanical polishing system to polish the surface of the interstitial regions  22 . The polishing head(s)/pad(s) or other polishing tool(s) is/are capable of polishing the functionalized layer  26  that may be present over the interstitial regions  22  while leaving the functionalized layers  24 ,  26  in the depression(s)  20 ′ at least substantially intact. As an example, the polishing head may be a Strasbaugh ViPRR II polishing head. 
     Cleaning and drying processes may be performed after polishing. The cleaning process may utilize a water bath and sonication. The water bath may be maintained at a relatively low temperature ranging from about 22° C. to about 30° C. The drying process may involve spin drying, or drying via another suitable technique. 
     As depicted in  FIG.  9 H , the functionalized layer  24  is positioned in the deep portion  48  of the multi-depth depression  20 ′ and the functionalized layer  26  is positioned on the surface  66  in the shallow portion  50  and the adjacent portion of the perimeter  29 , P along the deep portion  48 . As such, the padlock like conformation  33  is reduced, or in some instances eliminated, depending, in part, on how much of the portion of the sidewall  29 , P is covered by the metal film  62  and whether dry etching is extended deeper than the surface  66 . 
     While not shown, the method of  FIG.  9 A  through  FIG.  9 H  also includes attaching respective primer sets  30 ,  32  to the functionalized layers  24 ,  26 . In some examples, the primers  34 ,  36  or  34 ′,  36 ′ (not shown in  FIG.  9 A  through  FIG.  9 H ) may be pre-grafted to the functionalized layer  24 . Similarly, the primers  38 ,  40  or  38 ′,  40 ′ (not shown in  FIG.  9 A  through  FIG.  9 H ) may be pre-grafted to the functionalized layer  26 . In these examples, additional primer grafting is not performed. 
     In other examples, the primers  34 ,  36  or  34 ′,  36 ′ are not pre-grafted to the functionalized layer  24 . In these examples, the primers  34 ,  36  or  34 ′,  36 ′ may be grafted after the functionalized layer  24  is applied (e.g., at  FIG.  9 B ). In these examples, the primers  38 ,  40  or  38 ′,  40 ′ may be pre-grafted to the second functionalized layer  26 . Alternatively, in these examples, the primers  38 ,  40  or  38 ′,  40 ′ may not be pre-grafted to the second functionalized layer  26 . Rather, in some instances, the primers  38 ,  40  or  38 ′,  40 ′ may be grafted immediately after the second functionalized layer  26  is applied (e.g., at  FIG.  9 E ). In other instances, the primers  38 ,  40  or  38 ′,  40 ′ may be grafted after the portion  53  of the sacrificial layer  52  is removed (e.g., at  FIG.  9 F ) or after the metal film  62  has been removed (e.g., at  FIG.  9 G ) or after the interstitial regions  22  have been polished (e.g., at  FIG.  9 H ) as long as i) the functionalized layer  26  has different functional groups (than functionalized layer  24 ) for attaching the primers  38 ,  40  or  38 ′,  40 ′ or ii) any unreacted functional groups of the functionalized layer  24  have been quenched, e.g., using the Staudinger reduction to amines or additional click reaction with a passive molecule such as hexynoic acid. 
     When grafting is performed during the method, grafting may be accomplished using any of the grafting techniques described herein. 
     While a single set of the functionalized layers  24 ,  26  is shown in  FIG.  9 H , it is to be understood that the method described in reference to  FIG.  9 A  through  FIG.  9 H  may be performed to generate an array of depressions  20 ′ (each having functionalized layers  24 ,  26  therein) across the resin layer  14 ,  14 ′,  18 ,  18 ′. 
     Methods with a Metal Film 
     Some examples of the method disclosed herein use a metal film to pattern one or more layers. In these examples, the metal film is a sacrificial layer that protects the underlying resin layer  18 ,  18 ′ (of the multi-layer structure  16 ,  16 ′) or base support  17 ,  17 ′ during processing, but is readily removable at a desirable time. These methods are shown in  FIG.  10 A  through  FIG.  10 C  or  FIG.  10 A ,  FIG.  10 D ,  FIG.  10 B , and  FIG.  10 C  in combination with any of i)  FIG.  11 A  through  FIG.  11 E , ii)  FIG.  12 A  through  FIG.  12 G , iii)  FIG.  12 A  through  FIG.  12 D  and  FIG.  12 H  through  FIG.  12 J , iv)  FIG.  13 A  through  FIG.  13 I , or v)  FIG.  14 A  through  FIG.  14 I , or vi)  FIG.  15 A  through  FIG.  15 F . In some of these examples, the metal film also functions as a mask for photoresist development. 
     The examples of the method shown in each of these series of figures generally include forming a metal film  62  over a resin layer  14 ,  14 ′,  18 , or  18 ′ including the plurality of multi-depth depressions  20 ′ separated by interstitial regions  22 , each multi-depth depression  20 ′ including a deep portion  48  and a shallow portion  50  adjacent to the deep portion  48  ( FIG.  10 B ); forming a sacrificial layer  52  over the metal film  62  ( FIG.  10 B ); and sequentially dry etching the sacrificial layer  52  and the metal film  62  to expose a surface  66  of the resin layer  14 ,  14 ′,  18 , or  18 ′ at the shallow portion  50  and the interstitial regions  22  ( FIG.  10 C ). As such, the beginning of each of the example methods that utilize the metal film  62  as a sacrificial layer are shown in  FIG.  10 A  through  FIG.  10 C  or  FIG.  10 A ,  FIG.  10 D ,  FIG.  10 B , and  FIG.  10 C . 
     As shown in  FIG.  10 A , the multi-depth depression  20 ′ is defined in either the single layer base support  14 ,  14 ′ or the resin layer  18 ,  18 ′ of the multi-layered structure  16 ,  16 ′ as described herein. As such, the term “resin layer” may be referred to as “resin layer  14 ,  14 ′,  18 , or  18 ” throughout the description of these methods. In examples where the “resin layer  14 ,  14 ” is specifically mentioned, it is meant that the resin layer is the single layer base support  14 ,  14 ′. In other examples where “the resin layer  18 ,  18 ” is mentioned, it is meant that the resin layer is the resin layer  18 ,  16 ′ of the multi-layer structure  16 ,  16 ′. The underlying base support  17 ,  17 ′ is shown in phantom, which indicates that both the multi-layered structure  16  and the single layer base support  14  are represented in  FIG.  10 A  through  FIG.  10 D , as well as each of the series of figures with which they can be combined. 
     The resin layer  14  may be any of the examples of the resin set forth herein for the single layer base support  14 , the resin layer  14 ′ may be any of the examples of the resin set forth herein for the single layer base support  14 ′, the resin layer  18  may be any of the resins described herein for the resin layer  18  of the multi-layer structure  16 , and the resin layer  18 ′ may be any of the resins described herein for the resin layer  18 ′ of the multi-layer structure  16 ′. When included, the base support  17  or  17 ′ may be any of the respective examples described herein. 
     The multi-depth depression  20 ′ may be etched, imprinted, or defined in the resin layer  14 ,  14 ′,  18 , or  18 ′ using any suitable technique, such as the nanoimprint lithography process described in reference to  FIG.  4 A . While one multi-depth depression  20 ′ is shown in  FIG.  10 A , it is to be understood that the method may be performed to generate an array of multi-depth depressions  20 ′ including respective deep portions  48  and shallow portions  50 , separated by interstitial regions  22 , across the surface of the resin layer  14 ,  14 ′,  18 , or  18 ′. 
     The method shown in  FIG.  10 A  through  FIG.  10 C  includes the resin layer  14 ,  14 ′ i.e., the single layer base support  14 ,  14 ′. After the multi-depth depression  20 ′ is formed in the resin layer  14 ,  14 ′, the resin layer  14 ,  14 ′ may be exposed to activation, e.g., through silanization or plasma ashing, before the metal film  62  is deposited. If the resin layer  14 ,  14 ′ includes surface groups to covalently attach to the functionalized layers  24 ,  26 , the activation process is not performed. 
     As shown in  FIG.  10 B , the metal film  62  is deposited over the resin layer  14 ,  14 ′. Examples of suitable materials for the metal film  62  include semi-metals, such as silicon, or metals, such as aluminum, copper, titanium, gold, silver, etc. In some examples, the semi-metal or metal may be at least substantially pure (&lt;99% pure). In other examples, molecules or compounds of the listed elements may be used. When the method uses backside exposure for development of a photoresist the metal film  62  is selected to be opaque (non-transparent or having transmittance less than 0.25) to the light energy used for photoresist development. For example, oxides of any of the listed semi-metals (e.g., silicon dioxide) or metals (e.g., aluminum oxide) may be used, alone or in combination with the listed semi-metal or metal. These materials may be deposited using any suitable technique disclosed herein. 
     Also as shown in  FIG.  10 B , the sacrificial layer  52  is deposited over the metal film  62  and cured. In this example, the sacrificial layer  52  may be a negative or positive photoresist or poly(methyl methacrylate). 
     Referring now to  FIG.  10 C , the sacrificial layer  52  and the metal film  62  are sequentially dry etched to expose the surface  66  of the resin layer  14 ,  14 ′ in the shallow portion  50  and to expose the interstitial regions  22 . The first dry etching process removes some of the sacrificial layer  52 , and is performed for a measured amount of time to expose the metal film  62  that overlies the surface  66 . Examples of the first dry etching process include a reactive ion etch (e.g., with 10% CF 4  and 90% O 2 ) or a 100% O 2  plasma etch. The second dry etching process removes some of the metal film  62 , and is performed for a measured amount of time to expose the resin layer  14 ,  14 ′ that overlies the surface  66 . Examples of the second dry etching process include a reactive ion etch, e.g., with BCl 3 +Cl 2 . As shown in  FIG.  10 C , the dry etching processes are stopped so that the region  53  of the sacrificial layer  52  and the underlying metal film  62 ′ remain in the portion of the deep portion  48  that is next to the interior wall  29 , I. 
     The method shown in  FIG.  10 A ,  FIG.  10 D ,  FIG.  10 B , and  FIG.  10 C  includes the resin layer  18 ,  18 ′ positioned on the base support  17 ,  17 ′. After the multi-depth depression  20 ′ is formed in the resin layer  18 ,  18 ′ ( FIG.  10 A ), the method includes additional processing to expose a surface  74  of the underlying base support  17 ,  17 ′ as shown in  FIG.  10 D . In this example method, the resin layer  18 ,  18 ′ is positioned over the base support  17 ,  17 ′, and prior to forming the metal film  62 , the method further comprises dry etching the resin layer  18 ,  18 ′ at the deep portion  48  to expose a first region  74  of a surface of the base support  17 ,  17 ′, wherein the first region  74  of the surface is the surface at the deep portion  48  and may be referred to herein as “surface  74 .” In this example, the resin layer  18 ,  18 ′ may be etched using a dry etching process, such as an anisotropic oxygen plasma, a CF 4  plasma, or a mixture of 90% CF 4  and 10% O 2  plasma, and the underlying base support  17 ,  17 ′ acts as an etch stop. 
     In this example method, if the base support  17 ,  17 ′ (including first region  74 ) includes surface groups to covalently attach to the functionalized layers  24 ,  26  and the resin layer  18 ,  18 ′ does not include surface groups to covalently attach to the functionalized layers  24 ,  26 , the resin layer  18 ,  18 ′ can be exposed to activation, e.g., through silanization or plasma ashing, after the depression  20 ′ is formed and before the first region  74  is exposed. As examples, tantalum pentoxide and fused silica include surface groups that can attach to PAZAM and may be suitable for the base support  17 ,  17 ′. Alternatively, if the resin layer  18 ,  18 ′ and the base support  17 ,  17 ′ (including first region  74 ) include surface groups to covalently attach to the functionalized layers  24 ,  26 , the activation process is not performed. In still other examples, if neither of the resin layer  18 ,  18 ′ nor the base support  17 ,  17 ′ includes surface groups to covalently attach to the functionalized layers  24 ,  26 , the resin layer  18 ,  18 ′ and the first region  74  may be exposed to activation after the first region  74  is exposed and before the metal film  62  is deposited. 
     As shown in  FIG.  10 B , the metal film  62  is deposited over the resin layer  18 ,  18 ′ and the first region  74 , and the sacrificial layer  52  is deposited and cured over the metal film  62 . Each of these processes may be performed as described herein. In this example, when the metal film  62  is applied, a portion of it is in direct contact with the first region  74  of the base support  17 ,  17 ′. 
     In this example method, the sacrificial layer  52  and the metal film  62  are sequentially dry etched to expose the surface  66  of the resin layer  18 ,  18 ′ in the shallow portion  50  and to expose the interstitial regions  22 , as described herein in reference to  FIG.  10 C . In this example, the region  53  of the sacrificial layer  52  and the underlying metal film  62 ′ remain in the portion of the deep portion  48  that is next to the interior wall  29 , I, and the metal film  62 ′ is in direct contact with the first region  74  of the base support  17 ,  17 ′. 
     As mentioned, the methods shown in  FIG.  10 A  through  FIG.  10 C  or  FIG.  10 A ,  FIG.  10 D ,  FIG.  10 B , and  FIG.  10 C  may continue at any of i)  FIG.  11 A  through  FIG.  11 E , ii)  FIG.  12 A  through  FIG.  12 G , iii)  FIG.  12 A  through  FIG.  12 D  and  FIG.  12 H  through  FIG.  12 J , iv)  FIG.  13 A  through  FIG.  13 I , or v)  FIG.  14 A  through  FIG.  14 I , or vi)  FIG.  15 A  through  FIG.  15 F . Each of these methods will now be described. 
     In addition to the processes described in reference to either  FIG.  10 A  through  FIG.  10 C  or  FIG.  10 A ,  FIG.  10 D ,  FIG.  10 B , and  FIG.  10 C , the method shown in  FIG.  11 A  through  FIG.  11 E  generally includes: removing portions of the resin layer  14 ,  14 ′,  18 ,  18 ′ i) at the shallow portion  50  of the multi-depth depression  20 ′ to form a depression region  76  having a surface  78 ,  78 ′ that is directly adjacent to a surface  64  or  74  at the deep portion  48  and ii) at the interstitial regions  22  to form new interstitial regions  22 ′ surrounding the deep portion  48  and the depression region  76  ( FIG.  11 A ); depositing a first functionalized layer  24  over the metal film  62 ′, the depression region  76 , and the new interstitial regions  22 ′ ( FIG.  11 B ); removing the metal film  62 ′ from the deep portion  48  ( FIG.  11 C ); depositing a second functionalized layer  26  over the surface  64  or  74  at the deep portion  48  ( FIG.  11 D ); and polishing the new interstitial regions  22 ′ ( FIG.  11 E ). 
     The removal of the portions of the resin layer  14 ,  14 ′,  18 ,  18 ′ to form the depression region  76  and the new interstitial regions  22 ′ is shown in  FIG.  11 A . The resin layer  14 ,  14 ′,  18 ,  18 ′ may be dry etched using any of the examples set forth herein, e.g., an anisotropic oxygen plasma, a CF 4  plasma, or a mixture of 90% CF 4  and 10% O 2  plasma. In this example, dry etching removes exposed portions of the resin layer  14 ,  14 ′,  18 ,  18 ′, e.g., at the interstitial regions  22  and at the step feature  80  that defines the surface  66  and the shallow portion  50  (see  FIG.  10 C ). When the resin layer  14 ,  14 ′ is used, this dry etching process may be a timed dry etch that is performed for a measured amount of time to create the surface  78  which is substantially co-planar with the surface  64  that had been at the deep portion  48  (see  FIG.  11 A ). In this particular example, the surface  78  is the surface of the depression region  76 . Alternatively, when the resin layer  18 ,  18 ′ is used, this dry etching process may be performed until the surface  78 ′ is reached, which acts as an etch stop. The surface  78 ′ is co-planar with the surface  74  (see  FIG.  11 A ). In this example, the removal of portions (e.g., step feature  80 ) of the resin layer  18 ,  18 ′ at the shallow portion  50  of the multi-depth depression  20 ′ exposes a second region of the surface of the base support  17 ,  17 ′, wherein second region of the surface of the base support  17 ,  17 ′ is the surface  78 ′ of the depression region  76 . As shown in  FIG.  11 A , this dry etching process removes the step feature  80  of the resin layer  14 ,  14 ′,  18 ,  18 ′ (which had defined the shallow portion  50 ) in order to create the depression region  76 . This dry etching process also removes a portion of the perimeter sidewall  29 , P. The resulting structure is the single depth depression  20  shown in  FIG.  11 A . 
     As shown in  FIG.  11 A , the metal film  62 ′ remains intact after the resin layer  14 ,  14 ′,  18 ,  18 ′ is dry etched. 
     Referring now to  FIG.  11 B , the method then includes depositing the functionalized layer  24 . When the resin layer  14 ,  14 ′ is used, the applied functionalized layer  24  is positioned over the metal film  62 ′ and over exposed surfaces of the resin layer  14 ,  14 ′ (including over surface  78  and new interstitial regions  22 ′). When the multi-layer structure  16 ,  16 ′ is used, the applied functionalized layer  24  is positioned over the metal film  62 ′, over exposed surfaces of the resin layer  18 ,  18 ′, and over the exposed surface  78 ′ of the base support  17 ,  17 ′. The functionalized layer  24  may be any of the gel materials described herein and may be applied using any suitable deposition technique. A curing process may be performed after deposition. The functionalized layer  24  covalently attaches to the exposed surfaces of the resin layer  14 ,  14 ′ or to the exposed surfaces of the resin layer  18 ,  18 ′ and the base support  17 ,  17 ′ (including surface  78 ′). 
     The metal film  62 ′ is then removed from what had been, prior to resin layer  14 ,  14 ′,  18 ,  18 ′ etching, the deep portion  48 . The metal film  62 ′ may be removed by a wet etching or lift-off process, which depends upon the material of the metal film  62 ′. As examples, an aluminum metal film  62 ′ can be removed in acidic or basic conditions, a copper metal film  62 ′ can be removed using FeCl 3 , a copper, gold or silver metal film  62 ′ can be removed in an iodine and iodide solution, and a silicon metal film  62 ′ can be removed in basic (pH) conditions. The underlying surface  64 ,  74  may be inert to the wet etching or lift-off process. 
     As shown in  FIG.  11 C , the wet etching or lift-off process removes i) at least 99% of the metal film  62 ′ and ii) the first functionalized layer  24  thereon. This process exposes the surface  64  of the resin layer  14 ,  14 ′ or the surface  74  of the base support  17 ,  17 ′. 
     As shown in  FIG.  11 D , the second functionalized layer  26  may then be applied over the surface  64  or  74 . The second functionalized layer  26  (e.g., the gel material that forms the second functionalized layer  26 ) may be applied using any suitable deposition technique. In this example, when deposition of the gel material is performed under high ionic strength (e.g., in the presence of 10×PBS, NaCl, KCl, etc.), the second functionalized layer  26  does not deposit on or adhere to the first functionalized layer  24 . As such, the second functionalized layer  26  does not contaminate the first functionalized layer  24 . 
     In  FIG.  11 E , the functionalized layer  24  that is positioned over the new interstitial regions  22 ′ is removed, e.g., using a polishing process as described in reference to  FIG.  9 H . 
     Cleaning and drying processes may be performed after polishing. The cleaning process may utilize a water bath and sonication. The water bath may be maintained at a relatively low temperature ranging from about 22° C. to about 30° C. The drying process may involve spin drying, or drying via another suitable technique. 
     While not shown, the methods of  FIG.  10 A  through  FIG.  10 C  or  FIG.  10 A ,  FIG.  10 D ,  FIG.  10 B , and  FIG.  10 C  in combination with  FIG.  11 A  through  FIG.  11 E  also include attaching respective primer sets  30 ,  32  to the functionalized layers  24 ,  26 . In some examples, the primers  34 ,  36  or  34 ′,  36 ′ (not shown in  FIG.  10 A  through  FIG.  10 D  or in  FIG.  11 A  through  FIG.  11 E ) may be pre-grafted to the functionalized layer  24 . Similarly, the primers  38 ,  40  or  38 ′,  40 ′ (not shown in  FIG.  10 A  through  FIG.  10 D  or in  FIG.  11 A  through  FIG.  11 E ) may be pre-grafted to the functionalized layer  26 . In these examples, additional primer grafting is not performed. 
     In other examples, the primers  34 ,  36  or  34 ′,  36 ′ are not pre-grafted to the functionalized layer  24 . In these examples, the primers  34 ,  36  or  34 ′,  36 ′ may be grafted after the functionalized layer  24  is applied (e.g., at  FIG.  11 B ). In these examples, the primers  38 ,  40  or  38 ′,  40 ′ may be pre-grafted to the second functionalized layer  26 . Alternatively, in these examples, the primers  38 ,  40  or  38 ′,  40 ′ may not be pre-grafted to the second functionalized layer  26 . Rather, the primers  38 ,  40  or  38 ′,  40 ′ may be grafted after the second functionalized layer  26  is applied (e.g., at  FIG.  11 D  or  FIG.  11 E ) as long as i) the functionalized layer  26  has different functional groups (than functionalized layer  24 ) for attaching the primers  38 ,  40  or  38 ′,  40 ′ or ii) any unreacted functional groups of the functionalized layer  24  have been quenched, e.g., using the Staudinger reduction to amines or additional click reaction with a passive molecule such as hexynoic acid. 
     When grafting is performed during the method, grafting may be accomplished using any of the grafting techniques described herein. 
     While a single set of the functionalized layers  24 ,  26  is shown in  FIG.  11 E , it is to be understood that the method described in reference to  FIG.  10 A  through  FIG.  10 C  or  FIG.  10 A ,  FIG.  10 D ,  FIG.  10 B , and  FIG.  10 C  in combination with  FIG.  11 A  through  FIG.  11 E  may be performed to generate an array of depressions  20  (each having functionalized layers  24 ,  26  therein) across the resin layer  14 ,  14 ′,  18 ,  18 ′. 
     In addition to the processes described in reference to either  FIG.  10 A  through  FIG.  10 C  or  FIG.  10 A ,  FIG.  10 D ,  FIG.  10 B , and  FIG.  10 C  (which generates the metal film  62 ′), the method shown in  FIG.  12 A  through  FIG.  12 G  generally includes: removing portions of the resin layer  14 ′,  18 ′ i) at the shallow portion  50  of the multi-depth depression  20 ′ to form a depression region  76  having a surface  78 ,  78 ′ that is directly adjacent to a surface  64  or  74  at the deep portion  48  and ii) at the interstitial regions  22  to form new interstitial regions  22 ′ surrounding the deep portion  48  and the depression region  76  ( FIG.  12 A ); depositing a first functionalized layer  24  over the metal film  62 ′, the depression region  76 , and the new interstitial regions  22 ′ ( FIG.  12 B ); prior to the removal of the metal film  62 ′ from the deep portion  48 : depositing a negative photoresist  60  over the first functionalized layer  24  ( FIG.  12 B ); directing, through the resin layer  14 ′, or alternatively through the base support  17 ′, an ultraviolet light dosage, thereby forming an insoluble negative photoresist  60 ′ over the surface  78 ,  78 ′ of the depression region  76  and the new interstitial regions  22 ′ and a soluble negative photoresist  60 ″ over the first functionalized layer  24  over the metal film  62 ′ ( FIG.  12 B ); removing the soluble negative photoresist  60 ″ ( FIG.  12 C ); and ashing the first functionalized layer  24  from over the metal film  62 ′ ( FIG.  12 D ); wherein removing the metal film  62 ′ from the deep portion  48  involves etching the metal film  62 ′ ( FIG.  12 D ); and the method further comprises removing the insoluble negative photoresist  60 ′ before depositing the second functionalized layer  26  ( FIG.  12 E ). This example method also includes depositing the second functionalized layer  26  over the surface  64  or  74  at the deep portion  48  ( FIG.  12 F ); and polishing the new interstitial regions  22 ′ ( FIG.  12 G ). 
     The removal of the portions of the resin layer  14 ′,  18 ′ to form the depression region  76  and the new interstitial regions  22 ′ is shown in  FIG.  12 A . The resin layer  14 ′,  18 ′ may be dry etched using any of the examples set forth herein, e.g., an anisotropic oxygen plasma, a CF 4  plasma, or a mixture of 90% CF 4  and 10% O 2  plasma. In this example, dry etching removes exposed portions of the resin layer  14 ′,  18 ′, e.g., at the interstitial regions  22  and at the step feature  80  that defines the surface  66  and the shallow portion  50  (see  FIG.  10 C ). When the resin layer  14 ′ is used, this dry etching process may be a timed dry etch that is performed for a measured amount of time to create the surface  78  which is substantially co-planar with the surface  64  that had been at the deep portion  48  (see  FIG.  12 A ). In this particular example, the surface  78  is the surface of the depression region  76 . Alternatively, when the resin layer  18 ′ is used, this dry etching process may be performed until the surface  78 ′ of the base support  17 ′ is reached, which acts as an etch stop. In this example, the removal of portions (e.g., step feature  80 ) of the resin layer  18 ′ at the shallow portion  50  of the multi-depth depression  20 ′ exposes a second region of the surface of the base support  17 ′, wherein the second region of the surface of the base support  17 ′ is the surface  78 ′ of the depression region  76 . In these examples, the surface  78  or  78 ′ is at least substantially co-planar with the surface  64  or  74  (see  FIG.  12 A ). The dry etching process also removes a portion of the perimeter sidewall  29 , P. The resulting structure is the single depth depression  20  shown in  FIG.  12 A . 
     As shown in  FIG.  12 A , the metal film  62 ′ remains intact after the resin layer  14 ′,  18 ′ is dry etched. 
     Referring now to  FIG.  12 B , the method then includes depositing the functionalized layer  24 . When the resin layer  14 ′ is used, the applied functionalized layer  24  is positioned over the metal film  62 ′ and over exposed surfaces of the resin layer  14 ′ (including over surface  78  and new interstitial regions  22 ′). When the multi-layer structure  16 ′ is used, the applied functionalized layer  24  is positioned over the metal film  62 ′, over exposed surfaces of the resin layer  18 ′, and over the exposed surface  78 ′ of the base support  17 ′. The functionalized layer  24  may be any of the gel materials described herein and may be applied using any suitable deposition technique. A curing process may be performed after deposition. The functionalized layer  24  covalently attaches to the exposed surfaces of the resin layer  14 ′ (including surface  78 ) or to the exposed surfaces of the resin layer  18 ′ and the base support  17 ′ (including surface  78 ′). 
       FIG.  12 B  also depicts depositing a negative photoresist  60  over the first functionalized layer  24 . The negative photoresist  60  may be any of the negative photoresists described herein. The negative photoresist  60  is then exposed to an ultraviolet light dosage through the resin layer  14 ′ or, alternatively, through the base support  17 ′, which forms an insoluble negative photoresist  60 ′ over the surface  78 ,  78 ′ of the depression region  76  and over the new interstitial regions  22 ′, and a soluble negative photoresist  60 ″ over the first functionalized layer  24  that is positioned over the metal film  62 ′. The metal film  62 ′ blocks the light from reaching the negative photoresist  60  overlying the metal film  62 ′, and thus this portion becomes soluble. The remainder of the negative photoresist  60  is exposed to the light and thus becomes insoluble. 
       FIG.  12 C  depicts when the soluble negative photoresist  60 ″ is removed from (what had been) the deep portion  48 . The soluble negative photoresist  60 ″ is removed using any suitable developer. Examples of suitable developers for the negative photoresist include aqueous-alkaline solutions, such as diluted sodium hydroxide, diluted potassium hydroxide, or an aqueous solution of the metal ion free organic TMAH (tetramethylammoniumhydroxide). 
     After developer exposure, the insoluble negative photoresist  60 ′ remains over the first functionalized layer  24  at what had been, prior to resin layer  14 ′,  18 ′ etching, the shallow portion  50 , and the new interstitial regions  22 ′. 
     Referring now to  FIG.  12 D , a portion of the first functionalized layer  24  and the metal film  62 ′ are sequentially removed. The portion of the first functionalized layer  24  that is positioned over the metal film  62 ′ may be removed via ashing. The ashing process that is used to remove the functionalized layer  24  may be performed with plasma, such as 100% O 2  plasma, air plasma, argon plasma, etc. This process may also be used to remove the metal film  62 ′. Alternatively, this process may be stopped to leave the metal film  62 ′ intact. In these instances, the metal film  62 ′ is then removed from what had been, prior to resin layer  14 ′,  18 ′ etching, the deep portion  48 . The metal film  62 ′ may be removed by a dry etching process or by wet etching or lift-off process, which depends upon the material of the metal film  62 ′. The dry etching process that is used to remove the metal film  62 ′ may be reactive ion etching with BCl 3 +Cl 2 . As examples of the wet etch or lift-off process, an aluminum metal film  62 ′ can be removed in acidic or basic conditions, a copper metal film  62 ′ can be removed using FeCl 3 , a copper, gold or silver metal film  62 ′ can be removed in an iodine and iodide solution, and a silicon metal film  62 ′ can be removed in basic (pH) conditions. The removal of the metal film  62 ′ exposes the surface  64  or  74 . When the resin layer  14 ′ has been used, the surface exposed is the resin layer surface  64 . When the multi-layer structure  16 ′ has been used, the surface exposed is the base support surface  74 . The underlying surface  64 ,  74  may be inert to the wet etching or lift-off process. The underlying surface  64 ,  74  may or may not be inert to the dry etching process. If not, the etch rate of the underlying surface  64 ,  74  is much slower than that of the metal film  62 ′, and thus effectively acts as an etch stop. 
     At  FIG.  12 E , the insoluble negative photoresist  60 ′ is removed before depositing the second functionalized layer  26 . The insoluble negative photoresist  60 ′ may be removed by any suitable remover, which depends, in part, on the type of negative photoresist  60  used. 
     At  FIG.  12 F , the second functionalized layer  26  may then be applied over the surface  64  or  74 . The second functionalized layer  26  (e.g., the gel material that forms the second functionalized layer  26 ) may be applied using any suitable deposition technique. In this example, when deposition of the gel material is performed under high ionic strength (e.g., in the presence of 10×PBS, NaCl, KCl, etc.), the second functionalized layer  26  does not deposit on or adhere to the first functionalized layer  24 . As such, the second functionalized layer  26  does not contaminate the first functionalized layer  24 . 
     In  FIG.  12 G , the functionalized layer  24  that is positioned over the new interstitial regions  22 ′ is removed, e.g., using a polishing process. The polishing process may be performed as described herein, e.g., in reference to  FIG.  9 H . 
     Cleaning and drying processes may be performed after polishing. The cleaning process may utilize a water bath and sonication. The water bath may be maintained at a relatively low temperature ranging from about 22° C. to about 30° C. The drying process may involve spin drying, or drying via another suitable technique. 
     While not shown, the methods of  FIG.  10 A  through  FIG.  10 C  or  FIG.  10 A ,  FIG.  10 D ,  FIG.  10 B , and  FIG.  10 C  in combination with  FIG.  12 A  through  FIG.  12 G  also include attaching respective primer sets  30 ,  32  to the functionalized layers  24 ,  26 . In some examples, the primers  34 ,  36  or  34 ′,  36 ′ (not shown in  FIG.  10 A  through  FIG.  10 D  or in  FIG.  12 A  through  FIG.  12 G ) may be pre-grafted to the functionalized layer  24 . Similarly, the primers  38 ,  40  or  38 ′,  40 ′ (not shown in  FIG.  10 A  through  FIG.  10 D  or in  FIG.  12 A  through  FIG.  12 G ) may be pre-grafted to the functionalized layer  26 . In these examples, additional primer grafting is not performed. 
     In other examples, the primers  34 ,  36  or  34 ′,  36 ′ are not pre-grafted to the functionalized layer  24 . In these examples, the primers  34 ,  36  or  34 ′,  36 ′ may be grafted after the functionalized layer  24  is applied (e.g., at  FIG.  12 B ). In these examples, the primers  38 ,  40  or  38 ′,  40 ′ may be pre-grafted to the second functionalized layer  26 . Alternatively, in these examples, the primers  38 ,  40  or  38 ′,  40 ′ may not be pre-grafted to the second functionalized layer  26 . Rather, the primers  38 ,  40  or  38 ′,  40 ′ may be grafted after the second functionalized layer  26  is applied (e.g., at  FIG.  12 F ) as long as i) the functionalized layer  26  has different functional groups (than functionalized layer  24 ) for attaching the primers  38 ,  40  or  38 ′,  40 ′ or ii) any unreacted functional groups of the functionalized layer  24  have been quenched, e.g., using the Staudinger reduction to amines or additional click reaction with a passive molecule such as hexynoic acid. 
     When grafting is performed during the method, grafting may be accomplished using any of the grafting techniques described herein. 
     While a single set of the functionalized layers  24 ,  26  is shown in  FIG.  12 G , it is to be understood that the method described in reference to  FIG.  10 A  through  FIG.  10 C  or  FIG.  10 A ,  FIG.  10 D ,  FIG.  10 B , and  FIG.  10 C  in combination with  FIG.  12 A  through  FIG.  12 G  may be performed to generate an array of depressions  20  (each having functionalized layers  24 ,  26  therein) across the resin layer  14 ′,  18 ′. 
     In addition to the processes described in reference to either  FIG.  10 A  through  FIG.  10 C  or  FIG.  10 A ,  FIG.  10 D ,  FIG.  10 B , and  FIG.  10 C , the method shown in  FIG.  12 A  through  FIG.  12 D  and continuing at  FIG.  12 H  through  FIG.  12 J  generally includes: removing portions of the resin layer  14 ′,  18 ′ i) at the shallow portion  50  of the multi-depth depression  20 ′ to form a depression region  76  having a surface  78 ,  78 ′ that is directly adjacent to a surface  64  or  74  at the deep portion  48  and ii) at the interstitial regions  22  to form new interstitial regions  22 ′ surrounding the deep portion  48  and the depression region  76  ( FIG.  12 A ); depositing a first functionalized layer  24  over the metal film  62 ′, the depression region  76 , and the new interstitial regions  22 ′ ( FIG.  12 B ); prior to the removal of the metal film  62 ′ from the deep portion  48 : depositing a negative photoresist  60  over the first functionalized layer  24  ( FIG.  12 B ); directing, through the resin layer  14 ′, or alternatively through the base support  17 ′, an ultraviolet light dosage, thereby forming an insoluble negative photoresist  60 ′ over the surface  78  or  78 ′ of the depression region  76  and the new interstitial regions  22 ′ and a soluble negative photoresist  60 ″ over the first functionalized layer  24  over the metal film  62 ′ (also  FIG.  12 B ); removing the soluble negative photoresist  60 ″ ( FIG.  12 C ); and ashing the first functionalized layer from over the metal film  62 ′ ( FIG.  12 D ); wherein removing the metal film  62 ′ from the deep portion  48  involves etching the metal film  62 ′ ( FIG.  12 D ); wherein the second functionalized layer  26  is also deposited over the insoluble negative photoresist  60 ′ ( FIG.  12 H ); and the method further comprises removing the insoluble negative photoresist  60 ′ ( FIG.  12 I ). This example of the method also involves polishing the new interstitial regions  22 ′ ( FIG.  12 J ). 
     In this example method, the processes shown in  FIG.  12 A  through  FIG.  12 D  may be performed as described herein. 
     The method then continues from  FIG.  12 A  through  FIG.  12 D  to  FIG.  12 H . At  FIG.  12 H , the second functionalized layer  26  may then be applied over the surface  64  or  74  and the insoluble negative photoresist  60 ′. The second functionalized layer  26  (e.g., the gel material that forms the second functionalized layer  26 ) may be applied using any suitable deposition technique. The second functionalized layer  26  does not contaminate the first functionalized layer  24 , which is covered by the insoluble negative photoresist  60 ′. 
     At  FIG.  12 I , the insoluble negative photoresist  60 ′ is removed. The insoluble negative photoresist  60 ′ may be removed by any suitable remover, which depends, in part, on the type of negative photoresist  60  used. As shown in  FIG.  12 I , the removal process removes i) at least 99% of the insoluble negative photoresist  60 ′ and ii) the second functionalized layer  26  thereon. This removal process leaves the second functionalized layer  26  that is positioned on the surface  64  or  74 , and also leaves the first functionalized layer  24  intact. These portions of the functionalized layers  24 ,  26  remain intact, in part because they are covalently attached to the resin layer  14 ′ or  18 ′ and/or base support  17 ′. 
     In  FIG.  12 J , the functionalized layer  24  that is positioned over the new interstitial regions  22 ′ is removed, e.g., using a polishing process as described, for example, in reference to  FIG.  9 H . 
     Cleaning and drying processes may be performed after polishing. The cleaning process may utilize a water bath and sonication. The water bath may be maintained at a relatively low temperature ranging from about 22° C. to about 30° C. The drying process may involve spin drying, or drying via another suitable technique. 
     While not shown, the methods of  FIG.  10 A  through  FIG.  10 C  or  FIG.  10 A ,  FIG.  10 D ,  FIG.  10 B , and  FIG.  10 C  in combination with  FIG.  12 A  through  FIG.  12 D  and continued at  FIG.  12 H  through  FIG.  12 J  also include attaching respective primer sets  30 ,  32  to the functionalized layers  24 ,  26 . In some examples, the primers  34 ,  36  or  34 ′,  36 ′ (not shown in  FIG.  10 A  through  FIG.  10 D  or in  FIG.  12 A  through  FIG.  12 D  and continued at  FIG.  12 H  through  FIG.  12 J ) may be pre-grafted to the functionalized layer  24 . Similarly, the primers  38 ,  40  or  38 ′,  40 ′ (not shown in  FIG.  10 A  through  FIG.  10 D  or in  FIG.  12 A  through  FIG.  12 D  and continued at  FIG.  12 H  through  FIG.  12 J ) may be pre-grafted to the functionalized layer  26 . In these examples, additional primer grafting is not performed. 
     In other examples, the primers  34 ,  36  or  34 ′,  36 ′ are not pre-grafted to the functionalized layer  24 . In these examples, the primers  34 ,  36  or  34 ′,  36 ′ may be grafted after the functionalized layer  24  is applied (e.g., at  FIG.  12 B ). In these examples, the primers  38 ,  40  or  38 ′,  40 ′ may be pre-grafted to the second functionalized layer  26 . Alternatively, in these examples, the primers  38 ,  40  or  38 ′,  40 ′ may not be pre-grafted to the second functionalized layer  26 . Rather, the primers  38 ,  40  or  38 ′,  40 ′ may be grafted after the second functionalized layer  26  is applied and prior to additional processing (e.g., at  FIG.  12 H ); or after the second functionalized layer  26  is applied and exposed to additional processing (e.g., at  FIG.  12 I  or at  12 J) as long as i) the functionalized layer  26  has different functional groups (than functionalized layer  24 ) for attaching the primers  38 ,  40  or  38 ′,  40 ′ or ii) any unreacted functional groups of the functionalized layer  24  have been quenched, e.g., using the Staudinger reduction to amines or additional click reaction with a passive molecule such as hexynoic acid. 
     When grafting is performed during the method, grafting may be accomplished using any of the grafting techniques described herein. 
     While a single set of the functionalized layers  24 ,  26  is shown in  FIG.  12 J , it is to be understood that the method described in reference to  FIG.  10 A  through  FIG.  10 C  or  FIG.  10 A ,  FIG.  10 D ,  FIG.  10 B , and  FIG.  10 C  in combination with  FIG.  12 A  through  FIG.  12 D  and continued in  FIG.  12 H  through  FIG.  12 J  may be performed to generate an array of depressions  20  (each having functionalized layers  24 ,  26  therein) across the resin layer  14 ′,  18 ′. 
     In addition to the processes described in reference to either  FIG.  10 A  through  FIG.  10 C  or  FIG.  10 A ,  FIG.  10 D ,  FIG.  10 B , and  FIG.  10 C  (which generates the metal film  62 ′), the method shown in  FIG.  13 A  through  FIG.  13 I  generally includes: removing portions of the resin layer  14 ′,  18 ′ i) at the shallow portion  50  of the multi-depth depression  20 ′ to form a depression region  76  having a surface  78 ,  78 ′ that is directly adjacent to a surface  64  or  74  at the deep portion  48  and ii) at the interstitial regions  22  to form new interstitial regions  22 ′ surrounding the deep portion  48  and the depression region  76  ( FIG.  13 A ); depositing a first functionalized layer  24  over the metal film  62 ′, the depression region  76 , and the new interstitial regions  22 ′ ( FIG.  13 B ); prior to the removal of the metal film  62 ′ from the deep portion  48 : depositing a positive photoresist  56  over the first functionalized layer  24  ( FIG.  13 B ); directing, through the resin layer  14 ′, or alternatively through the base support  17 ′, an ultraviolet light dosage, thereby forming an insoluble positive photoresist  56 ′ over the first functionalized layer  24  over the metal film  62 ′ and a soluble positive photoresist  56 ″ over the surface  78 ,  78 ′ of the depression region  76  and the new interstitial regions  22 ′ ( FIG.  13 B ); removing the soluble positive photoresist  56 ″ ( FIG.  13 C ); ashing the first functionalized layer  24  from the surface  78 ,  78 ′ of the depression region  76  and the new interstitial regions  22 ′ ( FIG.  13 D ); wherein the deposition of the second functionalized layer  26  over the surface  64 ,  74  at the deep portion  48  involves depositing the second functionalized layer  26  over the insoluble positive photoresist  56 ′, the surface  78 ,  78 ′ of the depression region  76 , and the new interstitial regions  22 ′ ( FIG.  13 E ); and removing the insoluble positive photoresist  56 ′ ( FIG.  13 F ); and after the removal of the metal film  62 ′ from the deep portion  48 , the method further comprises increasing adhesion between the first functionalized layer  24  and the surface  64  at the deep portion  48 , or between the first functionalized layer  24  and the first region  74  of the surface of the base support  17 ′ ( FIG.  13 G ). 
     The removal of the portions of the resin layer  14 ′,  18 ′ to form the depression region  76  and the new interstitial regions  22 ′ is shown in  FIG.  13 A . The resin layer  14 ′,  18 ′ may be dry etched using any of the examples set forth herein, e.g., an anisotropic oxygen plasma, a CF 4  plasma, or a mixture of 90% CF 4  and 10% O 2  plasma. In this example, dry etching removes exposed portions of the resin layer  14 ′,  18 ′, e.g., at the interstitial regions  22  and at the step feature  80  that defines the surface  66  and the shallow portion  50  (see  FIG.  10 C ). When the resin layer  14 ′ is used, this dry etching process may be a timed dry etch that is performed for a measured amount of time to create the surface  78  which is substantially co-planar with the surface  64  or  74  that had been at the deep portion  48  (see  FIG.  13 A ). In this particular example, the surface  78  is the surface of the depression region  76 . Alternatively, when the resin layer  18 ′ is used, this dry etching process may be performed until the surface  78 ′ of the base support  17 ′ is reached, which acts as an etch stop. The surface  78 ′ is co-planar with the surface  74  (see  FIG.  13 A ). In this example, the removal of portions (e.g., step feature  80 ) of the resin layer  18 ′ at the shallow portion  50  of the multi-depth depression  20 ′ exposes a second region of the surface of the base support  17 ′, wherein the second region of the surface of the base support  17 ′ is the surface  78 ′ of the depression region  76 . This dry etching process also removes a portion of the perimeter sidewall  29 , P. The resulting structure is the single depth depression  20  shown in  FIG.  13 A . 
     As shown in  FIG.  13 A , the metal film  62 ′ remains intact after the resin layer  14 ′,  18 ′ is dry etched. 
     Referring now to  FIG.  13 B , the method then includes depositing the functionalized layer  24 . When the resin layer  14 ′ is used, the applied functionalized layer  24  is positioned over the metal film  62 ′ and over exposed surfaces of the resin layer  14 ′ (including over surface  78  and new interstitial regions  22 ′). When the multi-layer structure  16 ′ is used, the applied functionalized layer  24  is positioned over the metal film  62 ′, over exposed surfaces of the resin layer  18 ′, and over the exposed surface  78 ′ of the base support  17 ′. The functionalized layer  24  may be any of the gel materials described herein and may be applied using any suitable deposition technique. A curing process may be performed after deposition. The functionalized layer  24  covalently attaches to the exposed surfaces of the resin layer  14 ′ or to the exposed surfaces of the resin layer  18 ′ and the base support  17 ′ (including surface  78 ′). 
       FIG.  13 B  also depicts depositing a positive photoresist  56  over the first functionalized layer  24 . The positive photoresist  56  may be any of the positive photoresists described herein. The positive photoresist  56  is then exposed to an ultraviolet light dosage through the resin layer  14 ′ or, alternatively, the base support  17 ′, which forms an insoluble positive photoresist  56 ′ over the metal film  62 ′ and the first functionalized layer  24  at the deep portion  48 , and a soluble positive photoresist  56 ″ over the surface  78 ,  78 ′ of the depression region  76  and the new interstitial regions  22 ′. The metal film  62 ′ blocks the light from reaching the positive photoresist  56  overlying the metal film  62 ′, and thus this portion becomes insoluble. The remainder of the positive photoresist  56  is exposed to the light and thus becomes soluble. 
       FIG.  13 C  depicts when the soluble positive photoresist  56 ″ is removed from over the surface  78 ,  78 ′ of the depression region  76  and from over the new interstitial regions  22 ′. The soluble positive photoresist  56 ″ is removed using any suitable developer. Examples of suitable developers for the positive photoresist  56  include aqueous-alkaline solutions, such as diluted sodium hydroxide, diluted potassium hydroxide, or an aqueous solution of the metal ion free organic TMAH (tetramethylammonium hydroxide). 
     After developer exposure, the insoluble positive photoresist  56 ′ remains over the metal film  62 ′ positioned over the first functionalized layer  24  at what had been, prior to resin layer  14 ′,  18 ′ etching, the deep portion  48 . 
       FIG.  13 D  depicts ashing the first functionalized layer  24  from the surface  78 ,  78 ′ of the depression region  76  and the new interstitial regions  22 ′. The ashing may be performed as described herein, and removes the first functionalized layer  24  to expose the surface  78 ,  78 ′ of the depression region  76  and the new interstitial regions  22 ′. The insoluble positive photoresist  56 ′ is not susceptible to the ashing process, and thus the insoluble photoresist  56 ′, the first functionalized layer  24 , and the metal film  62 ′ remain in what had been the deep portion  48  after the ashing process. 
     Referring now to  FIG.  13 E , the second functionalized layer  26  may then be applied over the surface  78 ,  78 ′, the new interstitial regions  22 ′, and the insoluble positive photoresist  56 ′. The second functionalized layer  26  (e.g., the gel material that forms the second functionalized layer  26 ) may be applied using any suitable deposition technique. The second functionalized layer  26  does not contaminate the first functionalized layer  24 , which is covered by the insoluble positive photoresist  56 ′. 
     Referring now to  FIG.  13 F , the insoluble positive photoresist  56 ′ is removed. The insoluble positive photoresist  56 ′ may be removed by any suitable remover, which depends, in part, on the type of positive photoresist  56  used. As shown in  FIG.  13 F , the removal process removes i) at least 99% of the insoluble positive photoresist  56 ′ and ii) the second functionalized layer  26  thereon. This removal process leaves the second functionalized layer  26  that is positioned over the surface  78 ,  78 ′ and the new interstitial regions  22 ′. These portions of the functionalized layer  26  remain intact, in part because they are covalently attached to the resin layer  14 ′ or  18 ′ and/or base support  17 ′. 
       FIG.  13 G  depicts when the metal film  62 ′ is removed from what had been, prior to resin layer  14 ′,  18 ′ etching, the deep portion  48 . The metal film  62 ′ may be removed by a wet etching process, which depends upon the material of the metal film  62 ′. In an example, the metal film  62 ′ (e.g., aluminum having about 30 nm thickness) can be etched by exposure to a 1-2% KOH solution or a sodium carbonate buffer (pH˜10) for about 3 to 5 minutes, without mechanical stress including agitation or sonication. The etching process can be slowed by diluting the etchant and increasing the duration of the process, which may improve the retention of the functionalized layer  24 . The removal of the metal film  62 ′ does not remove the first functionalized layer  24  deposited over the metal film  62 ′ at what had been the deep portion  48 , but does expose the surface  64 ,  74 . The underlying surface  64 ,  74  may also be inert to the wet etching process. 
     As depicted in  FIG.  13 G , the metal film  62 ′ removal creates a gap between the surface  64 ,  74  and the first functionalized layer  24 . A variety of methods may be performed for increasing adhesion between the first functionalized layer  24  and either the surface  64  of the resin layer  14 ′ at the deep portion  48  or the first region  74  of the surface of the base support  17 ′. These methods may also improve the adhesion between the first functionalized layer  24  and the remaining portion of the perimeter  29 , P. 
     The following are examples of methods that may be used to increase adhesion between the first functionalized layer  24  and the surface  64  of the resin layer  14 ′. 
     In one example, increasing the adhesion between the first functionalized layer  24  and the surface  64  at the deep portion  48  involves heating the first functionalized layer  24  and the surface  64  at the deep portion  48 . Heating can speed up covalent bonding between the first functionalized layer  24  and the underlying surface  64 . In an example, heating may be performed at a temperature ranging from about 55° C. to about 65° C. for a time ranging from about 25 minutes to about 35 minutes. In another example, heating may be performed at a temperature of about 60° C. for a time of about 30 minutes. 
     In another example, increasing the adhesion between the first functionalized layer  24  and the surface  64  at the deep portion  48  involves applying a protective coating (not shown) over the first and the second functionalized layers  24 ,  26 ; heating the first functionalized layer  24  and the surface  64  at the deep portion  48 ; and removing the protective coating. The protective coating may be generated using an aqueous solution that includes up to about 15% (mass to volume) of a water soluble material selected from the group consisting of a polyvinyl alcohol/polyethylene glycol graft copolymer (one example of which includes KOLLICOAT® IR, available from BASF Corp.), sucrose, polyacrylamide, dextran (e.g., molecular weight of 200,000 Da), polyacrylamide (e.g., molecular weight of 40,000 Da, 200,000 Da, etc.), polyethylene glycol, ethylenediaminetetraacetic acid sodium salt (i.e., EDTA), tris(hydroxymethyl)aminomethane with ethylenediaminetetraacetic acid, (tris(2-carboxyethyl)phosphine), tris(3-hydroxypropyltriazolylmethyl)amine, bathophenanthrolinedisulfonic acid disodium salt, hydroxyl functional polymers, glycerol, or saline sodium citrate. Any suitable deposition technique may be used to apply the aqueous solution. After the aqueous solution is applied, it may be heated to evaporate the water and form the protective coating. The protective coating may then be removed by exposure to water. 
     In still another example, increasing the adhesion between the first functionalized layer  24  and the surface  64  at the deep portion  48  involves selectively silanizing the surface  64  at the deep portion  48 . For selective silanization, a silane may be used that includes functional groups that can attach to functional groups of the first functionalized layer  24  and functional groups that can attach to the surface  64 . Examples of suitable silanes include an amino silane, an alkynyl silane, and a norbornene silane. The amino silane or the alkynyl silane can attach to an azide functional group of the functionalized layer  24 . The norbornene silane can respectively attach to an azide functional group or a tetrazine of the functionalized layer. An example of the amino silane may include (3-aminopropyl)trimethoxysilane) (APTMS), (3-aminopropyl)triethoxysilane) (APTES), N-(6-aminohexyl)am inomethyltriethoxysilane (AHAMTES), N-(2-aminoethyl)-3-aminopropyltriethoxysilane (AEAPTES), and N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAPTMS), each of which is available from Gelest. The alkynyl silane may include a cycloalkyne unsaturated moiety, such as O-propargyl)-N-(triethoxysilylpropyl)carbamate, cyclooctyne, a cyclooctyne derivative, or bicyclononynes (e.g., bicyclo[6.1.0]non-4-yne or derivatives thereof, bicyclo[6.1.0]non-2-yne, or bicyclo[6.1.0]non-3-yne). The norbornene silane may be a norbornene derivative, e.g., a (hetero)norbornene including an oxygen or nitrogen in place of one of the carbon atoms. An example of the norbornene silane includes [(5-bicyclo[2.2.1]hept-2-enyl)ethyl]trimethoxysilane. 
     The silane is introduced into an aqueous solution that the functionalized layer  24  can take up (e.g., absorb), and the appropriate reactions take place between the silane and the respective functional groups. The aqueous silane solution may be applied using any suitable technique, e.g., vapor deposition (e.g., a YES method), spin coating, or other deposition method disclosed herein. 
     The following are examples of methods that may be used to increase adhesion between the first functionalized layer  24  and the first region  74  of the surface of the base support  17 ′. 
     One example of the method of increasing the adhesion between the first functionalized layer  24  and the first region  74  of the surface of the base support  17 ′ involves heating the first functionalized layer  24  and the first region  74  of the surface of the base support  17 ′. This process may be performed as described herein. 
     Another example of the method of increasing the adhesion between the first functionalized layer  24  and the first region  74  of the surface of the base support  17 ′ involves: applying a protective coating over the first and the second functionalized layers; heating the first functionalized layer  24  and the first region  74  of the surface of the base support  17 ′; and removing the protective coating. This process may be performed as described herein. 
     Still another example of the method of increasing the adhesion between the first functionalized layer  24  and the first region  74  of the surface of the base support  17 ′ involves selectively silanizing the first region  74  of the surface of the base support  17 ′. This process may be performed as described herein. 
     In any of the examples of the method of increasing the adhesion between the first functionalized layer  24  and the surface  64  or the first region  74  of the surface of the base support  17 ′, the first functionalized layer  24  is brought into direct contact with the surface  64  or the first region  74 . The method may also covalently attach the functionalized layer  24  to the surface  64  or the first region  74 . The resulting structure is shown schematically in  FIG.  13 H .  FIG.  13 H  depicts the depression  20  with the first functionalized layer  24  in direct contact with the surface  64  or the first region  74  of the base support  17 ′, and the second functionalized layer  26  adjacent to the first functionalized layer  26 . 
     In  FIG.  13 I , the functionalized layer  26  that is positioned over the new interstitial regions  22 ′ is removed, e.g., using a polishing process as described, for example, in reference to  FIG.  9 H . 
     Cleaning and drying processes may be performed after polishing. The cleaning process may utilize a water bath and sonication. The water bath may be maintained at a relatively low temperature ranging from about 22° C. to about 30° C. The drying process may involve spin drying, or drying via another suitable technique. 
     While not shown, the methods of  FIG.  10 A  through  FIG.  10 C  or  FIG.  10 A ,  FIG.  10 D ,  FIG.  10 B , and  FIG.  10 C  in combination with  FIG.  13 A  through  FIG.  13 I  and also include attaching respective primer sets  30 ,  32  to the functionalized layers  24 ,  26 . In some examples, the primers  34 ,  36  or  34 ′,  36 ′ (not shown in  FIG.  10 A  through  FIG.  10 D  or in  FIG.  13 A  through  FIG.  13 I ) may be pre-grafted to the functionalized layer  24 . Similarly, the primers  38 ,  40  or  38 ′,  40 ′ (not shown in  FIG.  10 A  through  FIG.  10 D  or in  FIG.  13 A  through  FIG.  13 I ) may be pre-grafted to the functionalized layer  26 . In these examples, additional primer grafting is not performed. 
     In other examples, the primers  34 ,  36  or  34 ′,  36 ′ are not pre-grafted to the functionalized layer  24 . In these examples, the primers  34 ,  36  or  34 ′,  36 ′ may be grafted after the functionalized layer  24  is applied (e.g., at  FIG.  13 B ). In these examples, the primers  38 ,  40  or  38 ′,  40 ′ may be pre-grafted to the second functionalized layer  26 . Alternatively, in these examples, the primers  38 ,  40  or  38 ′,  40 ′ may not be pre-grafted to the second functionalized layer  26 . Rather, the primers  38 ,  40  or  38 ′,  40 ′ may be grafted after the second functionalized layer  26  is applied and prior to subsequent processing (e.g., at  FIG.  13 E ), or after the second functionalized layer  26  is applied and processed (e.g., at  FIG.  13 F ,  FIG.  13 H , or  FIG.  13 I ) as long as i) the functionalized layer  26  has different functional groups (than functionalized layer  24 ) for attaching the primers  38 ,  40  or  38 ′,  40 ′ or ii) any unreacted functional groups of the functionalized layer  24  have been quenched, e.g., using the Staudinger reduction to amines or additional click reaction with a passive molecule such as hexynoic acid. 
     When grafting is performed during the method, grafting may be accomplished using any of the grafting techniques described herein. 
     While a single set of the functionalized layers  24 ,  26  is shown in  FIG.  13 I , it is to be understood that the method described in reference to  FIG.  10 A  through  FIG.  10 C  or  FIG.  10 A ,  FIG.  10 D ,  FIG.  10 B , and  FIG.  10 C  in combination with  FIG.  13 A  through  FIG.  13 I  may be performed to generate an array of depressions  20  (each having functionalized layers  24 ,  26  therein) across the resin layer  14 ′,  18 ′. 
     In addition to the processes described in reference to either  FIG.  10 A  through  FIG.  10 C  or  FIG.  10 A ,  FIG.  10 D ,  FIG.  10 B , and  FIG.  10 C  (which generates the metal film  62 ′), the method shown in  FIG.  14 A  through  FIG.  14 I  generally includes: removing portions of the resin layer i) at the shallow portion  50  of the multi-depth depression  20 ′ to form a depression region  76  having a surface  78 ,  78 ′ that is directly adjacent to a surface  64 ,  74  at the deep portion  48  and ii) at the interstitial regions  22  to form new interstitial regions  22 ′ surrounding the deep portion  48  and the depression region  76  ( FIG.  14 A ); depositing a first functionalized layer  24  over the metal film  62 ′, the depression region  76 , and the new interstitial regions  22 ′ ( FIG.  14 B ); depositing a positive photoresist  56  over the first functionalized layer  24  ( FIG.  14 B ); directing, through the resin layer  14 ′ or, alternatively, through the base support  17 ′ and the resin layer  18 ′, an ultraviolet light dosage, thereby forming an insoluble positive photoresist  56 ′ over the first functionalized layer  24  over the metal film  62 ′ and a soluble positive photoresist  56 ″ over the surface  78 ,  78 ′ of the depression region  76  and the new interstitial regions  22 ′; removing the soluble positive photoresist  56 ″ ( FIG.  14 C ); ashing the first functionalized layer  24  from the surface  78 ,  78 ′ of the depression region  76  and the new interstitial regions  22 ′ ( FIG.  14 D ); removing the insoluble positive photoresist  56 ′ ( FIG.  14 E ); wet etching the metal film  62 ′ from the deep portion  48 , whereby the first functionalized layer  24  over the metal film  62 ′ remains intact ( FIG.  14 F ); increasing adhesion between the first functionalized layer  24  and the surface  64 ,  74  at the deep portion  48  ( FIG.  14 G ); depositing a second functionalized layer  26  over the surface  78 ,  78 ′ of the depression region  76  and the new interstitial regions  22 ′ ( FIG.  14 H ); and polishing the new interstitial regions  22 ′ ( FIG.  14 I ). 
     The removal of the portions of the resin layer  14 ′,  18 ′ to form the depression region  76  and the new interstitial regions  22 ′ is shown in  FIG.  14 A . The resin layer  14 ′,  18 ′ may be dry etched using any of the examples set forth herein, e.g., an anisotropic oxygen plasma, a CF 4  plasma, or a mixture of 90% CF 4  and 10% O 2  plasma. In this example, dry etching removes exposed portions of the resin layer  14 ′,  18 ′, e.g., at the interstitial regions  22  and at the step feature  80  that defines the surface  66  and the shallow portion  50  (see  FIG.  10 C ). When the resin layer  14 ′ is used, this dry etching process may be a timed dry etch that is performed for a measured amount of time to create the surface  78  which is substantially co-planar with the surface  64  that had been at the deep portion  48  (see  FIG.  14 A ). In this particular example, the surface is the surface  78  of the depression region  76 . Alternatively, when the resin layer  18 ′ is used, this dry etching process may be performed until the surface  78 ′ of the base support  17 ′ is reached, which acts as an etch stop. The surface  78 ′ is co-planar with the surface  74  (see  FIG.  14 A ). In this example, the removal of portions (e.g., step feature  80 ) of the resin layer  18 ′ at the shallow portion  50  of the multi-depth depression  20 ′ exposes a second region of the surface of the base support  17 ′, wherein the second region of the surface of the base support  17 ′ is the surface  78 ′ of the depression region  76 . As shown in  FIG.  14 A , this dry etching process removes the step feature  80  of the resin layer  14 ′,  18 ′ (which had defined the shallow portion  50 ) in order to create the depression region  76 . This dry etching process also removes a portion of the perimeter sidewall  29 , P. The resulting structure is the single depth depression  20  shown in  FIG.  14 A . 
     As shown in  FIG.  14 A , the metal film  62 ′ remains intact after the resin layer  14 ′,  18 ′ is dry etched. 
     Referring now to  FIG.  14 B , the method then includes depositing the functionalized layer  24 . When the resin layer  14 ′ is used, the applied functionalized layer  24  is positioned over the metal film  62 ′ and over exposed surfaces of the resin layer  14 ′ (including over surface  78  and new interstitial regions  22 ′). When the multi-layer structure  16 ′ is used, the applied functionalized layer  24  is positioned over the metal film  62 ′, over exposed surfaces of the resin layer  18 ′, and over the exposed surface  78 ′ of the base support  17 ′. The functionalized layer  24  may be any of the gel materials described herein and may be applied using any suitable deposition technique. A curing process may be performed after deposition. The functionalized layer  24  covalently attaches to the exposed surfaces of the resin layer  14 ′ or to the exposed surfaces of the resin layer  18 ′ and the base support  17 ′ (including surface  78 ′). 
       FIG.  14 B  also depicts depositing a positive photoresist  56  over the first functionalized layer  24 . The positive photoresist  56  may be any of the positive photoresists described herein. The positive photoresist  56  is then exposed to an ultraviolet light dosage through the resin layer  14 ′ or the base support  17 ′ and the resin layer  18 ′, which forms an insoluble positive photoresist  56 ′ over the metal film  62 ′ and the first functionalized layer  24  at (what had been) the deep portion  48 , and a soluble positive photoresist  56 ″ over the surface  78 ,  78 ′ of the depression region  76  and the new interstitial regions  22 ′. The metal film  62 ′ blocks the light from reaching the positive photoresist  56  overlying the metal film  62 ′, and thus this portion becomes insoluble. The insoluble positive photoresist  56 ′ is shown in  FIG.  14 C . The remainder of the positive photoresist  56  is exposed to the light and thus becomes soluble.  FIG.  14 C  also depicts when the soluble positive photoresist  56 ″ is removed from the surface  78 ,  78 ′ of the depression region  76  and the new interstitial regions  22 ′. The soluble positive photoresist  56 ″ is removed using any suitable developer described herein for positive photoresists  56 . 
     After developer exposure, the insoluble positive photoresist  56 ′ remains over the metal film  62 ′ positioned over the first functionalized layer  24  at what had been, prior to resin layer  14 ′,  18 ′ etching, the deep portion  48 . 
       FIG.  14 D  depicts ashing the first functionalized layer  24  from the surface  78 ,  78 ′ of the depression region  76  and the new interstitial regions  22 ′. The ashing may be performed as described herein, and removes the first functionalized layer  24  to expose the surface  78 ,  78 ′ of the depression region  76  and the new interstitial regions  22 ′. The insoluble positive photoresist  56 ′ is not susceptible to the ashing process, and thus the insoluble positive photoresist  56 ′, the first functionalized layer  24 , and the metal film  62 ′ remain in what had been the deep portion  48  after the ashing process. 
     Referring now to  FIG.  14 E , the insoluble positive photoresist  56 ′ is removed, e.g., with a lift-off process. The lift-off process may be performed with any suitable remover, which depends, in part, on the type of positive photoresist  56  used. The first functionalized layer  24  and the metal film  62 ′ remain intact, and are not removed with the insoluble positive photoresist  56 ′ as the layer and film  24 ,  62 ′ are inert to the remover. 
       FIG.  14 F  depicts when the metal film  62 ′ is removed from what had been, prior to resin layer  14 ′,  18 ′ etching, the deep portion  48 . The metal film  62 ′ may be removed by a wet etching process, which depends upon the material of the metal film  62 ′. In an example, the metal film  62 ′ (e.g., aluminum having about 30 nm thickness) can be etched by exposure to a 1-2% KOH solution or a sodium carbonate buffer (pH˜10) for about 3 to 5 minutes, without mechanical stress including agitation or sonication. The etching process can be slowed by diluting the etchant and increasing the duration of the process, which may improve the retention of the functionalized layer  24 . The removal of the metal film  62 ′ does not remove the first functionalized layer  24  deposited over the metal film  62 ′ at what had been the deep portion  48 , but does expose the surface  64 ,  74 . The underlying surface  64 ,  74  may be inert to the wet etching process. 
     As depicted in  FIG.  14 F , the metal film  62 ′ removal creates a gap between the surface  64 ,  74  and the first functionalized layer  24 . A variety of methods may be performed for increasing adhesion between the first functionalized layer  24  and either the surface  64  of the resin layer  14 ′ at the deep portion  48  or the first region  74  of the surface of the base support  17 ′. These methods may also improve the adhesion between the first functionalized layer  24  and the remaining portion of the perimeter  29 , P. Any of the methods for increasing adhesion described in reference to the  FIG.  13    series of figures may be used. 
     In any of the examples of the method of increasing the adhesion between the first functionalized layer  24  and the surface  64  or the first region  74  of the surface of the base support  17 ′, the first functionalized layer  24  is brought into direct contact with the surface  64  or the first region  74 . The method may also covalently attach the functionalized layer  24  to the surface  64  or the first region  74 . The resulting structure is shown schematically in  FIG.  14 G .  FIG.  14 G  depicts the depression  20  with the first functionalized layer  24  in direct contact with the surface  64  or the first region  74  of the base support  17 ′. 
     Referring now to  FIG.  14 H , the second functionalized layer  26  may then be applied over the surface  78 ,  78 ′ and the new interstitial regions  22 ′. The second functionalized layer  26  (e.g., the gel material that forms the second functionalized layer  26 ) may be applied using any suitable deposition technique. In this example, the second functionalized layer  26  is applied under high ionic strength as described herein, and thus does not contaminate the first functionalized layer  24 . 
     In  FIG.  14 I , the functionalized layer  26  that is positioned over the new interstitial regions  22 ′ is removed, e.g., using a polishing process as described, for example, in reference to  FIG.  9 H . 
     Cleaning and drying processes may be performed after polishing. The cleaning process may utilize a water bath and sonication. The water bath may be maintained at a relatively low temperature ranging from about 22° C. to about 30° C. The drying process may involve spin drying, or drying via another suitable technique. 
     While not shown, the methods of  FIG.  10 A  through  FIG.  10 C  or  FIG.  10 A ,  FIG.  10 D ,  FIG.  10 B , and  FIG.  10 C  in combination with  FIG.  14 A  through  FIG.  14 I  and also include attaching respective primer sets  30 ,  32  to the functionalized layers  24 ,  26 . In some examples, the primers  34 ,  36  or  34 ′,  36 ′ (not shown in  FIG.  10 A  through  FIG.  10 D  or in  FIG.  14 A  through  FIG.  14 I ) may be pre-grafted to the functionalized layer  24 . Similarly, the primers  38 ,  40  or  38 ′,  40 ′ (not shown in  FIG.  10 A  through  FIG.  10 D  or in  FIG.  14 A  through  FIG.  14 I ) may be pre-grafted to the functionalized layer  26 . In these examples, additional primer grafting is not performed. 
     In other examples, the primers  34 ,  36  or  34 ′,  36 ′ are not pre-grafted to the functionalized layer  24 . In these examples, the primers  34 ,  36  or  34 ′,  36 ′ may be grafted after the functionalized layer  24  is applied (e.g., at  FIG.  14 B ). In these examples, the primers  38 ,  40  or  38 ′,  40 ′ may be pre-grafted to the second functionalized layer  26 . Alternatively, in these examples, the primers  38 ,  40  or  38 ′,  40 ′ may not be pre-grafted to the second functionalized layer  26 . Rather, the primers  38 ,  40  or  38 ′,  40 ′ may be grafted after the second functionalized layer  26  is applied (e.g., at  FIG.  14 H  or  FIG.  14 I ) as long as i) the functionalized layer  26  has different functional groups (than functionalized layer  24 ) for attaching the primers  38 ,  40  or  38 ′,  40 ′ or ii) any unreacted functional groups of the functionalized layer  24  have been quenched, e.g., using the Staudinger reduction to amines or additional click reaction with a passive molecule such as hexynoic acid. 
     When grafting is performed during the method, grafting may be accomplished using any of the grafting techniques described herein. 
     While a single set of the functionalized layers  24 ,  26  is shown in  FIG.  14 I , it is to be understood that the method described in reference to  FIG.  10 A  through  FIG.  10 C  or  FIG.  10 A ,  FIG.  10 D ,  FIG.  10 B , and  FIG.  10 C  in combination with  FIG.  14 A  through  FIG.  14 I  may be performed to generate an array of depressions  20  (each having functionalized layers  24 ,  26  therein) across the resin layer  14 ′,  18 ′. 
     The method described in either  FIG.  10 A  through  FIG.  10 C  or  FIG.  10 A ,  FIG.  10 D ,  FIG.  10 B , and  FIG.  10 C , in conjunction with  FIG.  15 A  through  FIG.  15 F  generally includes: forming a metal film  62  over a resin layer  14 ′,  18 ′ including a plurality of multi-depth depressions  20 ′ separated by interstitial regions  22 , each multi-depth depression  20 ′ including a deep portion  48  and a shallow portion  50  adjacent to the deep portion  48  defined by the resin layer  14 ′,  18 ′ (shown in  FIG.  10 B ); forming a sacrificial layer  52  over the metal film  62  ( FIG.  10 B ); sequentially dry etching the sacrificial layer  52  and the metal film  62  to expose the shallow portion  50  and the interstitial regions  22  (which forms metal film  62 ′,  FIG.  10 C ); lifting off the sacrificial layer  52  to expose the metal film  62 ′ ( FIG.  15 A ); depositing a first functionalized layer  24  over the metal film  62 ′ and the interstitial regions  22  and in the shallow portion  50  ( FIG.  15 A ); depositing a negative photoresist  60  over the first functionalized layer  24  ( FIG.  15 A ); directing, through the resin layer  14 ′,  18 ′, an ultraviolet light dosage, thereby forming an insoluble negative photoresist  60 ′ over interstitial regions  22  and in the shallow portion  50  and a soluble negative photoresist  60 ″ over the first functionalized layer  24  over the metal film  62 ′ ( FIG.  15 B ); ashing the first functionalized layer  24  from over the metal film  62 ′ ( FIG.  15 C ); etching the metal film  62 ′ from the deep portion  48  ( FIG.  15 C ); depositing a second functionalized layer  26  over the insoluble negative photoresist  60 ′ and in the deep portion  48  ( FIG.  15 D ); lifting off the insoluble negative photoresist  60 ′ ( FIG.  15 E ); and polishing the first functionalized layer  24  from the interstitial regions  22  ( FIG.  15 F ). 
     The metal film  62 ′ (shown in  FIG.  10 C ) may be formed over the resin layer  14 ′, or alternatively over the base support  17 ′ and resin layer  18 ′ as described herein in reference to, respectively,  FIG.  10 A  through  FIG.  10 C  or  FIG.  10 A ,  FIG.  10 D ,  FIG.  10 B , and  FIG.  10 C . Any of the materials and methods described in reference to these figures may be used. 
     The method then continues at  FIG.  15 A , which depicts several of the processes, including the removal of the sacrificial layer  52  to expose the metal film  62 ′ in the deep portion  48 , the application of the first functionalized layer  24 , and the application of the negative photoresist  60 . 
     The sacrificial layer  52  (shown in  FIG.  10 C ) may be removed via a lift-off process. The lift-off process may be performed with any suitable remover, which depends, in part, on the type of sacrificial layer  52  used. Upon removal of the sacrificial layer  52 , the underling metal film  62 ′ is exposed. 
       FIG.  15 A  depicts the first functionalized layer  24  deposited over the metal film  62 ′, over the interstitial regions  22 , and over the shallow portion  50 . When the resin layer  14 ′ is used, the applied functionalized layer  24  is positioned over the metal film  62 ′ and over exposed surfaces of the resin layer  14 ′ (including over surface  66  and the interstitial regions  22 ). When the multi-layer structure  16 ′ is used, the applied functionalized layer  24  is also positioned over the metal film  62 ′, and over exposed surfaces of the resin layer  18 ′ (including over surface  66  and the interstitial regions  22 ). The functionalized layer  24  may be any of the gel materials described herein and may be applied using any suitable deposition technique. A curing process may be performed after deposition. The functionalized layer  24  covalently attaches to the exposed surfaces of the resin layer  14 ′,  18 ′. 
       FIG.  15 A  also depicts the negative photoresist  60  deposited over the first functionalized layer  24 . The negative photoresist  60  may be any of the negative photoresists described herein. The deposited negative photoresist  60  is then exposed to an ultraviolet light dosage through the resin layer  14 ′, or alternatively, the base support  17 ′ and resin layer  18 ′, thereby forming an insoluble negative photoresist  60 ′ over the interstitial regions  22  and in the shallow portion  50 , and a soluble negative photoresist  60 ″ over the first functionalized layer  24  over the metal film  62 ′. The metal film  62 ′ blocks the light from reaching the negative photoresist  60  overlying the metal film  62 ′, and thus this portion becomes soluble. The soluble negative photoresist  60 ″ is then removed, using any suitable developer described herein for negative photoresists  60 . 
     After developer exposure, the insoluble negative photoresist  60 ′ remains over the first functionalized layer  24  at the shallow portion  50 , and the interstitial regions  22 . The insoluble negative photoresist  60 ′ is shown in  FIG.  15 B . 
     The method then continues at  FIG.  15 C , which depicts several of the processes, including the sequential removal of the first functionalized layer  24  and the metal film  62 ′ to expose the surface  64  of the resin layer  14 ′ or the surface  74  of the base support  17 ′. 
       FIG.  15 C  depicts when the first functionalized layer  24  is removed from the metal film  62 ′ positioned over the surface  64 ,  74  of the deep portion  48 . As depicted, the first functionalized layer  24  is also removed from a portion of the perimeter  29 , P that defines the deep portion  48 . The first functionalized layer  24  may be removed via an ashing process. The ashing process may be performed as described herein, and removes the first functionalized layer  24  to expose the metal film  62 ′. The insoluble negative photoresist  60 ′ is not susceptible to the ashing process, and thus the insoluble negative photoresist  60 ′ and the underlying first functionalized layer  24  remain in the shallow portion  50  and over the interstitial regions  22  after the ashing process. 
       FIG.  15 C  also depicts when the metal film  62 ′ is removed from the deep portion  48 . The metal film  62 ′ may be removed by a wet etching or lift-off process, which depends upon the material of the metal film  62 ′. As examples, an aluminum metal film  62 ′ can be removed in acidic or basic conditions, a copper metal film  62 ′ can be removed using FeCl 3 , a copper, gold or silver sacrificial layer can be removed in an iodine and iodide solution, and a silicon metal film  62 ′ can be removed in basic (pH) conditions. The removal of the metal film  62 ′ exposes the surface  64  of the resin layer  14 ′ at the deep portion  48  when the resin layer  14 ′ is used. The removal of the metal film  62 ′ exposes the surface  74  of the base support  17 ′ at the deep portion  48  when the resin layer  18 ′ is used. The removal of the metal film  62 ′ also exposes the remainder of the perimeter  29 , P that defines the deep portion  48  as well as the interior wall  29 , I. 
       FIG.  15 D  depicts the deposition of the second functionalized layer  26 , which is applied over the exposed surface  64 ,  74  and the insoluble negative photoresist  60 ′. The second functionalized layer  26  (e.g., the gel material that forms the second functionalized layer  26 ) may be applied using any suitable deposition technique. The second functionalized layer  26  does not contaminate the first functionalized layer  24 , which is covered by the insoluble negative photoresist  60 ′. 
     Referring now to  FIG.  15 E , the insoluble negative photoresist  60 ′ is removed through a lift-off process. The lift-off process may be any suitable lift-off process described herein. The lift-off process involves exposing the insoluble negative photoresist  60 ′ to a suitable remover for the type of negative photoresist  60  used. As shown in  FIG.  15 E , the removal process removes i) at least 99% of the insoluble negative photoresist  60 ′ and ii) the second functionalized layer  26  thereon. This removal process leaves the second functionalized layer  26  that is positioned on the surface  64  or  74 , and also leaves the first functionalized layer  24  intact. These portions of the functionalized layers  24 ,  26  remain intact, in part because they are covalently attached to the resin layer  14 ′ or  18 ′ and/or base support  17 ′. 
     In  FIG.  15 F , the functionalized layer  24  that is positioned over the interstitial regions  22  is removed, e.g., using a polishing process as described, for example, in reference to  FIG.  9 H . 
     Cleaning and drying processes may be performed after polishing. The cleaning process may utilize a water bath and sonication. The water bath may be maintained at a relatively low temperature ranging from about 22° C. to about 30° C. The drying process may involve spin drying, or drying via another suitable technique. 
     As depicted in  FIG.  15 F , the functionalized layer  24  is positioned on one half of the multi-depth depression  20 ′ (at the shallow portion  50  and the adjacent portion of the perimeter  29 , P) and the functionalized layer  26  is positioned on the other half of the multi-depth depression  20 ′ (at the deep portion  48  and the adjacent portion of the perimeter  29 , P). As such, the padlock like conformation  33  is eliminated. 
     While not shown, the methods of  FIG.  10 A  through  FIG.  10 C  or  FIG.  10 A ,  FIG.  10 D ,  FIG.  10 B , and  FIG.  10 C  in combination with  FIG.  15 A  through  FIG.  15 F  also include attaching respective primer sets  30 ,  32  to the functionalized layers  24 ,  26 . In some examples, the primers  34 ,  36  or  34 ′,  36 ′ (not shown in  FIG.  10 A  through  FIG.  10 D  or in  FIG.  15 A  through  FIG.  15 F ) may be pre-grafted to the functionalized layer  24 . Similarly, the primers  38 ,  40  or  38 ′,  40 ′ (not shown in  FIG.  10 A  through  FIG.  10 D  or in  FIG.  15 A  through  FIG.  15 F ) may be pre-grafted to the functionalized layer  26 . In these examples, additional primer grafting is not performed. 
     In other examples, the primers  34 ,  36  or  34 ′,  36 ′ are not pre-grafted to the functionalized layer  24 . In these examples, the primers  34 ,  36  or  34 ′,  36 ′ may be grafted after the functionalized layer  24  is applied (e.g., at  FIG.  15 A ). In these examples, the primers  38 ,  40  or  38 ′,  40 ′ may be pre-grafted to the second functionalized layer  26 . Alternatively, in these examples, the primers  38 ,  40  or  38 ′,  40 ′ may not be pre-grafted to the second functionalized layer  26 . Rather, the primers  38 ,  40  or  38 ′,  40 ′ may be grafted immediately after the second functionalized layer  26  is applied (e.g., at  FIG.  15 D ); or after insoluble negative photoresist  60 ′ removal (e.g., at  FIG.  15 E  or  FIG.  15 F ) as long as i) the functionalized layer  26  has different functional groups (than functionalized layer  24 ) for attaching the primers  38 ,  40  or  38 ′,  40 ′ or ii) any unreacted functional groups of the functionalized layer  24  have been quenched, e.g., using the Staudinger reduction to amines or additional click reaction with a passive molecule such as hexynoic acid. 
     When grafting is performed during the method, grafting may be accomplished using any of the grafting techniques described herein. 
     While a single set of the functionalized layers  24 ,  26  is shown in  FIG.  15 F , it is to be understood that the method described in reference to  FIG.  10 A  through  FIG.  10 C  or  FIG.  10 A ,  FIG.  10 D ,  FIG.  10 B , and  FIG.  10 C  in combination with  FIG.  15 A  through  FIG.  15 F  may be performed to generate an array of depressions  20 ′ (each having functionalized layers  24 ,  26  therein) across the resin layer  14 ′,  18 ′. 
     Methods with Varying Thickness 
     Other examples of the methods disclosed herein use a resin layer with varying thickness and UV transmission characteristics to create a mask that is used to pattern a photoresist  51 , which, in turn, is used to pattern the functionalized layer(s)  24 ,  26 . 
     Two examples of these methods are shown in  FIG.  16 A  through  FIG.  16 M , with one example including  FIG.  16 A  through  FIG.  16 H  and the other example including  FIG.  16 A  through  FIG.  16 C  and  FIG.  16 I  through  FIG.  16 M . 
     In the series of figures from  FIG.  16 A  through  FIG.  16 C , the method shown generally includes: depositing a first functionalized layer  24  over a resin layer  14 ′,  18 ′ including a plurality of multi-depth depressions  20 ′ separated by interstitial regions  22 , each multi-depth depression  20 ′ including a deep portion  48  and a shallow portion  50  adjacent to the deep portion  48  ( FIG.  16 B ); depositing a photoresist  51  over the first functionalized layer  24  ( FIG.  16 C ); and directing, through the resin layer  14 ′,  18 ′, an ultraviolet light dosage, whereby a first portion of the photoresist  51  generates an insoluble photoresist  51 ′ and a second portion becomes a soluble photoresist  51 ″ ( FIG.  16 C ). 
     One example of the method continues at  FIG.  16 D  through  FIG.  16 H , which includes: removing the soluble photoresist  51 ″, thereby exposing a portion of the first functionalized layer  24  ( FIG.  16 D ); removing the portion of the first functionalized layer  24 , thereby exposing a portion of resin layer  14 ′,  18 ′ ( FIG.  16 E ); depositing a second functionalized layer  26  over the insoluble photoresist  51 ′, and over the exposed portion of the resin layer  14 ′,  18 ′ ( FIG.  16 F ); removing the insoluble photoresist  51 ′, thereby exposing the first functionalized layer  24  ( FIG.  16 G ); and polishing the first functionalized layer  24  or the second functionalized layer  26  from the interstitial regions  22  ( FIG.  16 H ). 
     In this specific series, the method shown at  FIG.  16 A  through  FIG.  16 H  depicts when the photoresist  51  is a positive photoresist  56 ; and as a result of the ultraviolet light dosage, the positive photoresist  56  in the shallow portion  50  and the interstitial regions  22  becomes the insoluble photoresist  51 ′,  56 ′ and the positive photoresist  56  in the deep portion  48  becomes the soluble photoresist  51 ″; the deep portion  48  is exposed upon removal of the soluble photoresist  51 ″; the second functionalized layer  26  is deposited in the deep portion  48  and over the insoluble photoresist  51 ′,  56 ′; the removal of the insoluble photoresist  51 ′,  56 ′ exposes the first functionalized layer  24  in the shallow portion  50  and over the interstitial regions  22 ; and the polishing removes the first functionalized layer  24  from the interstitial regions  22 . 
       FIG.  16 A  depicts the multi-depth depression  20 ′, with a deep portion  48  and a shallow portion  50  adjacent to the deep portion  48 . The multi-depth depression  20 ′ is defined in either the single layer base support  14 ′ or the resin layer  18 ′ of the multi-layered structure  16 ′ as described herein. As such, the term “resin layer” may be referred to as “resin layer  14 ′,  18 ” throughout the description of this method. The underlying base support  17 ′ of the multi-layered structure  16 ′ is not shown in  FIG.  16 A  through  FIG.  16 M . 
     As mentioned, the resin layer  14 ′,  18 ′ has varying thicknesses and UV transmission characteristics to create a mask that is used to pattern the photoresist  51 . In this example, the deep portion  48  overlies a first resin portion  88  having a first thickness t 1  and the interstitial regions  22  overlie a second resin portion  90  having a second thickness t 2  that is greater than the first thickness t 1 . The first thickness t 1  is selected to allow UV light to transmit through the resin layer  14 ′,  18 ′ at the first resin portion  88  and the second thickness t 2  is selected to block UV light from transmitting through the resin layer  14 ′,  18 ′ at the second resin portion  90 . The shallow portion  50  overlies a third resin portion  92  having a third thickness t 3 . The third thickness t 3  is selected to block UV light from transmitting through the resin layer  14 ′,  18 ′ at the third resin portion  92 . The varying thicknesses t 1 , t 2 , t 3  are obtained when the multi-depth depression  20 ′ is etched, imprinted, etc. 
       FIG.  16 B  depicts the deposition of a first functionalized layer  24  over the multi-depth depression  20 ′ and the interstitial regions  22 . The first functionalized layer  24  is deposited over the resin layer  14 ′, or alternatively, the resin layer  18 ′. The functionalized layer  24  may be any of the gel materials described herein and may be applied using any suitable deposition technique. A curing process may be performed after deposition. The functionalized layer  24  covalently attaches to the exposed surfaces of the resin layer  14 ′,  18 ′, such as the surface  64  of the deep portion  48 , the surface  66  of the shallow portion  50 , and the interstitial regions  22 . 
       FIG.  16 C  depicts the deposition of a photoresist  51 . The photoresist  51  may be any of the photoresists described herein, i.e., a positive photoresist  56  or a negative photoresist  60 . Ultraviolet light is then directed through the backside of the resin layer  14 ′ or the base support  17 ′ (not shown) and the resin layer  18 ′ to pattern the photoresist  51  and generate an insoluble photoresist  51 ′ or a soluble photoresist  51 ″. As described herein, the base support  17 ′, when used, is able to transmit the UV light used for the backside exposure. 
     In this specific example of the method, the photoresist  51  is a positive photoresist  56 . As described, the first thickness t 1  is selected to allow UV light to transmit through the resin layer  14 ′,  18 ′ and the second and third thicknesses t 2 , t 3  are selected to block UV light from transmitting through the resin layer  14 ′,  18 ′. As such, the portion of the photoresist  51 ,  56  overlying the first resin portion  88  becomes soluble due to the exposure to the UV light, and the portions of the photoresist  51 ,  56  overlying the second and third resin portions  90 ,  92  become insoluble due to the lack of exposure to the UV light. In other words, when exposed to the ultraviolet light dosage, the insoluble photoresist  51 ′,  56 ′ forms over the shallow portion  50  and the interstitial regions  22  and the soluble photoresist  51 ″ forms over the deep portion  48  and is removed (see  FIG.  16 D ). 
     As noted,  FIG.  16 D  also depicts the removal of the soluble photoresist  51 ″. The soluble photoresist  51 ″ is removed using any suitable developer described herein for positive photoresists  56 . The removal of the soluble photoresist  51 ″ exposes the first functionalized layer  24  in the deep portion  48 . 
       FIG.  16 E  depicts removing the portion of the first functionalized layer  24 , thereby exposing a portion of resin layer  14 ′,  18 ′. The functionalized layer  24  may be removed by ashing, as described in  FIG.  14 D . The ashing process removes the first functionalized layer  24  to expose the surface  64  of the deep portion  48 . The insoluble photoresist  51 ′,  56 ′ is not susceptible to the ashing process, and thus the insoluble photoresist  51 ′,  56 ′ and the first functionalized layer  24  underneath remain in the shallow portion  50  and over the interstitial regions  22  after the ashing process. 
       FIG.  16 F  depicts the second functionalized layer  26  deposited over the insoluble photoresist  51 ′ and the exposed surface  64  of the deep portion  48 . The second functionalized layer  26  may be any of the gel materials described herein, and may be deposited using any suitable technique. The second functionalized layer  26  does not contaminate the first functionalized layer  24 , which is covered by the insoluble photoresist  51 ′,  56 ′. 
       FIG.  16 G  depicts the removal of the insoluble photoresist  51 ′,  56 ′. The insoluble photoresist  51 ′,  56 ′ may be removed via a lift-off process. The lift-off process may be any suitable lift-off process described herein, and may involve exposing the insoluble photoresist  51 ′,  56 ′ to a suitable remover for the type of positive photoresist  56  used. As shown in  FIG.  16 G , the removal process removes i) at least 99% of the insoluble photoresist  51 ′,  56 ′ and ii) the second functionalized layer  26  thereon. This removal process leaves the second functionalized layer  26  that is positioned on the surface  64 , and also leaves the first functionalized layer  24  intact. These portions of the functionalized layers  24 ,  26  remain intact, in part because they are covalently attached to the resin layer  14 ′ or  18 ′. 
       FIG.  16 H  depicts the removal of the functionalized layer  24  that is positioned over the interstitial regions  22 , e.g., using a polishing process as described, for example, in reference to  FIG.  9 H . 
     Cleaning and drying processes may be performed after polishing. The cleaning process may utilize a water bath and sonication. The water bath may be maintained at a relatively low temperature ranging from about 22° C. to about 30° C. The drying process may involve spin drying, or drying via another suitable technique. 
     As depicted in  FIG.  16 H , the functionalized layer  24  is positioned on one half of the multi-depth depression  20 ′ (at the shallow portion  50  and the adjacent portion of the perimeter sidewall  29 , P), and the functionalized layer  26  is positioned on the other half of the multi-depth depression  20 ′ (at the deep portion  48  and the adjacent portion of the perimeter sidewall  29 , P). As such, the padlock like conformation  33  is eliminated. 
     While not shown, the method of  FIG.  16 A  through  FIG.  16 H  also includes attaching respective primer sets  30 ,  32  to the functionalized layers  24 ,  26 . In some examples, the primers  34 ,  36  or  34 ′,  36 ′ (not shown in  FIG.  16 A  through  FIG.  16 H ) may be pre-grafted to the functionalized layer  24 . Similarly, the primers  38 ,  40  or  38 ′,  40 ′ (not shown in  FIG.  16 A  through  FIG.  16 H ) may be pre-grafted to the functionalized layer  26 . In these examples, additional primer grafting is not performed. 
     In other examples, the primers  34 ,  36  or  34 ′,  36 ′ are not pre-grafted to the functionalized layer  24 . In these examples, the primers  34 ,  36  or  34 ′,  36 ′ may be grafted after the functionalized layer  24  is applied (e.g., at  FIG.  16 B ). In these examples, the primers  38 ,  40  or  38 ′,  40 ′ may be pre-grafted to the second functionalized layer  26 . Alternatively, in these examples, the primers  38 ,  40  or  38 ′,  40 ′ may not be pre-grafted to the second functionalized layer  26 . Rather, the primers  38 ,  40  or  38 ′,  40 ′ may be grafted immediately after the second functionalized layer  26  is applied (e.g., at  FIG.  16 F ); or after insoluble photoresist  51 ′ removal (e.g., at  FIG.  16 G  or  FIG.  16 H ) as long as i) the functionalized layer  26  has different functional groups (than functionalized layer  24 ) for attaching the primers  38 ,  40  or  38 ′,  40 ′ or ii) any unreacted functional groups of the functionalized layer  24  have been quenched, e.g., using the Staudinger reduction to amines or additional click reaction with a passive molecule such as hexynoic acid. 
     When grafting is performed during the method, grafting may be accomplished using any of the grafting techniques described herein. 
     While a single set of the functionalized layers  24 ,  26  is shown in  FIG.  16 H , it is to be understood that the method described in reference to  FIG.  16 A  through  FIG.  16 H  may be performed to generate an array of depressions  20 ′ (each having functionalized layers  24 ,  26  therein) across the resin layer  14 ′,  18 ′. 
     In addition to the processes described in reference to  FIG.  16 A  through  FIG.  16 C , another example of the method continues at  FIG.  16 I  through  FIG.  16 M . In this method, the soluble photoresist  51 ″ is removed, thereby exposing a portion of the first functionalized layer  24  ( FIG.  16 I ); removing the portion of the first functionalized layer  24 , thereby exposing a portion of resin layer  14 ′,  18 ′ ( FIG.  16 J ); depositing a second functionalized layer  26  over the insoluble photoresist  51 ′, and over the exposed portion of the resin layer  14 ′,  18 ′ ( FIG.  16 K ); removing the insoluble photoresist  51 ′, thereby exposing the first functionalized layer  24  ( FIG.  16 L ); and polishing the first functionalized layer  24  or the second functionalized layer  26  from the interstitial regions  22  ( FIG.  16 M ). 
     In this specific series, the method shown at  FIG.  16 A  through  FIG.  16 C  and continuing at  FIG.  16 I through  16 M  depicts when the photoresist  51  is a negative photoresist  60 ; and as a result of the ultraviolet light dosage, the negative photoresist  60  in the deep portion  48  becomes the insoluble photoresist  51 ′,  60 ′, and the negative photoresist  60  in the shallow portion  50  and over the interstitial regions  22  becomes the soluble photoresist  51 ″,  60 ″; the shallow portion  50  and the interstitial regions  22  are exposed upon removal of the soluble photoresist  51 ″,  60 ″; the second functionalized layer  26  is deposited over the shallow portion  50 , the interstitial regions  22 , and the insoluble photoresist  51 ′,  60 ′; the removal of the insoluble photoresist  51 ′,  60 ′ exposes the first functionalized layer  24  in the deep portion  48 ; and the polishing removes the second functionalized layer  26  from the interstitial regions  22 . 
     The steps of the method depicted in the series of  FIG.  16 A  through  FIG.  16 B  may be performed as described herein. 
       FIG.  16 C  depicts the deposition of a photoresist  51 . The photoresist  51  may be any of the photoresists described herein, i.e., a positive photoresist  56  or a negative photoresist  60 . Ultraviolet light is then directed through the backside of the resin layer  14 ′ or the base support  17 ′ (not shown) and the resin layer  18 ′ to pattern the photoresist  51  and generate an insoluble photoresist  51 ′ or a soluble photoresist  51 ″. As described herein, the base support  17 ′, when used, is able to transmit the UV light used for the backside exposure. 
     In the specific example of the method from  FIG.  16 I  through  FIG.  16 M , the photoresist  51  is a negative photoresist  60 . As described, the first thickness t 1  is selected to allow UV light to transmit through the resin layer  14 ′,  18 ′ and the second and third thicknesses t 2 , t 3  are selected to block UV light from transmitting through the resin layer  14 ′,  18 ′. As such, the portion of the photoresist  51 ,  60  overlying the first resin portion  88  becomes insoluble due to the exposure to the UV light, and the portions of the photoresist  51 ,  60  overlying the second and third resin portions  90 ,  92  become soluble due to the lack of exposure to the UV light. In other words, when exposed to the ultraviolet light dosage, the insoluble photoresist  60 ′ forms over the deep portion  48  and the soluble photoresist  60 ″ forms over the over the shallow portion  50  and the interstitial regions  22  and is removed (see  FIG.  16 I ). 
     As mentioned,  FIG.  16 I  also depicts the removal of the soluble photoresist  51 ″,  60 ″. The soluble photoresist  51 ″,  60 ″ is removed using any suitable developer described herein for negative photoresists  60 . The removal of the soluble photoresist  51 ″,  60 ″ exposes the first functionalized layer  24  in the shallow portion  50  and on the interstitial regions  22 . 
       FIG.  16 J  depicts removing the portion of the first functionalized layer  24 , thereby exposing a portion of resin layer  14 ′,  18 ′. The functionalized layer  24  may be removed by ashing, as described in  FIG.  14 D . The ashing process removes the first functionalized layer  24  to expose the surface  66  of the shallow portion  50  and the interstitial regions  22 . The insoluble photoresist  51 ′,  60 ′ is not susceptible to the ashing process, and thus the insoluble photoresist  51 ′,  60 ′ and the first functionalized layer  24  underneath remain in the deep portion  48  after the ashing process. 
       FIG.  16 K  depicts the second functionalized layer  26  deposited over the insoluble photoresist  51 ′,  60 ′ and the exposed surface  66  of the shallow portion  50 . The second functionalized layer  26  may be any of the gel materials described herein, and may be deposited using any suitable technique. The second functionalized layer  26  does not contaminate the first functionalized layer  24 , which is covered by the insoluble photoresist  51 ′,  60 ′. 
       FIG.  16 L  depicts the removal of the insoluble photoresist  51 ′,  60 ′. The insoluble photoresist  51 ′,  60 ′ may be removed via a lift-off process. The lift-off process may be any suitable lift-off process described herein, and may involve a suitable remover for the type of negative photoresist  60  used. As shown in  FIG.  16 L , the removal process removes i) at least 99% of the insoluble photoresist  51 ′,  60 ′ and ii) the second functionalized layer  26  thereon. This removal process leaves the second functionalized layer  26  that is positioned on the surface  66  and the interstitial regions  22 , and also leaves the first functionalized layer  24  intact. These portions of the functionalized layers  24 ,  26  remain intact, in part because they are covalently attached to the resin layer  14 ′ or  18 ′. 
       FIG.  16 M  depicts the removal of the functionalized layer  26  that is positioned over the interstitial regions  22 , e.g., using a polishing process as described, for example, in reference to  FIG.  9 H . 
     Cleaning and drying processes may be performed after polishing. The cleaning process may utilize a water bath and sonication. The water bath may be maintained at a relatively low temperature ranging from about 22° C. to about 30° C. The drying process may involve spin drying, or drying via another suitable technique. 
     As depicted in  FIG.  16 M , the functionalized layer  24  is positioned on one half of the multi-depth depression  20 ′ (at the deep portion  48  and the adjacent portion of the perimeter  29 , P) and the functionalized layer  26  is positioned on the other half of the multi-depth depression  20 ′ (at the shallow portion  50  and the adjacent portion of the perimeter  29 , P). As such, the padlock like conformation  33  is eliminated. 
     While not shown, the method of  FIG.  16 A  through  FIG.  16 C  and continuing at  FIG.  16 I  through  FIG.  16 M  also includes attaching respective primer sets  30 ,  32  to the functionalized layers  24 ,  26 . In some examples, the primers  34 ,  36  or  34 ′,  36 ′ (not shown in  FIG.  16 A through  16 C  and continuing at  FIG.  16 I  through  FIG.  16 M ) may be pre-grafted to the functionalized layer  24 . Similarly, the primers  38 ,  40  or  38 ′,  40 ′ (not shown in  FIG.  16 A  through  FIG.  16 C  and continuing at  FIG.  16 I  through  FIG.  16 M ) may be pre-grafted to the functionalized layer  26 . In these examples, additional primer grafting is not performed. 
     In other examples, the primers  34 ,  36  or  34 ′,  36 ′ are not pre-grafted to the functionalized layer  24 . In these examples, the primers  34 ,  36  or  34 ′,  36 ′ may be grafted after the functionalized layer  24  is applied (e.g., at  FIG.  16 B ). In these examples, the primers  38 ,  40  or  38 ′,  40 ′ may be pre-grafted to the second functionalized layer  26 . Alternatively, in these examples, the primers  38 ,  40  or  38 ′,  40 ′ may not be pre-grafted to the second functionalized layer  26 . Rather, the primers  38 ,  40  or  38 ′,  40 ′ may be grafted immediately after the second functionalized layer  26  is applied (e.g., at  FIG.  16 K ); or after insoluble negative photoresist  51 ′,  60 ′ removal (e.g., at  FIG.  16 L  or  FIG.  16 M ) as long as i) the functionalized layer  26  has different functional groups (than functionalized layer  24 ) for attaching the primers  38 ,  40  or  38 ′,  40 ′ or ii) any unreacted functional groups of the functionalized layer  24  have been quenched, e.g., using the Staudinger reduction to amines or additional click reaction with a passive molecule such as hexynoic acid. 
     When grafting is performed during the method, grafting may be accomplished using any of the grafting techniques described herein. 
     While a single set of the functionalized layers  24 ,  26  is shown in  FIG.  16 M , it is to be understood that the method described in reference to  FIG.  16 A  through  FIG.  16 C  and continuing at  FIG.  16 I  through  FIG.  16 M  may be performed to generate an array of depressions  20 ′ (each having functionalized layers  24 ,  26  therein) across the resin layer  14 ′,  18 ′. 
     Methods with Varying Metal Layer Thickness 
     In the series of figures from  FIG.  17 A  through  FIG.  17 K , the method generally includes: depositing a first functionalized layer  24  over a resin layer  14 ′,  18 ′ including a plurality of multi-depth depressions  20 ′ separated by interstitial regions  22 , each multi-depth depression  20 ′ including a deep portion  48  and a shallow portion  50  adjacent to the deep portion  48  ( FIG.  17 F ); depositing a photoresist  51  over the first functionalized layer  24  ( FIG.  17 F ); directing, through the resin layer  14 ′,  18 ′, an ultraviolet light dosage, whereby a first portion of the photoresist  51  generates an insoluble photoresist  51 ′ and a second portion becomes a soluble photoresist  51 ″ ( FIG.  17 F ,  FIG.  17 G ); removing the soluble photoresist  51 ″, thereby exposing a portion of the first functionalized layer  24  ( FIG.  17 G ); removing the portion of the first functionalized layer  24 , thereby exposing a portion of resin layer  14 ′,  18 ′ ( FIG.  17 H ); depositing a second functionalized layer  26  over the insoluble photoresist  51 ′, and over the exposed portion of the resin layer  14 ′,  18 ′ ( FIG.  17 I ); removing the insoluble photoresist  51 ′, thereby exposing the first functionalized layer  24  ( FIG.  17 J ); and polishing the first functionalized layer  24  or the second functionalized layer  26  from the interstitial regions  22  ( FIG.  17 K ). 
     In this specific example of the method, prior to the depositing of the first functionalized layer  24 , the method further includes: forming a metal film  62  by sputtering or thermally evaporating a metal material over the resin layer  14 ′,  18 ′, the metal film  62  having a first thickness T 1  over the interstitial regions  22 , a second thickness T 2  over the deep portion  48 , and a third thickness T 3  over the shallow portion  50 , wherein the second thickness T 2  is about 30 nm or less and is at least 10 nm thinner than the first thickness T 1  and the third thickness T 3  is less than the first thickness T 1  and greater than the second thickness T 2  ( FIG.  17 A ); depositing a negative photoresist  60  over the metal film  62  ( FIG.  17 B ); directing, through the resin layer  14 ′,  18 ′, a second ultraviolet light dosage to form an insoluble negative photoresist  60 ′ overlying the deep portion  48 , and a soluble negative photoresist  60 ″ in the shallow portion  50  and over the interstitial regions  22  (also shown in  FIG.  17 B ); removing the soluble negative photoresist  60 ″, thereby exposing the metal film  62  in the shallow portion  50  and over the interstitial regions  22  ( FIG.  17 C ); removing the metal film  62  (leaving a portion of the metal film  62 ′) to expose the resin layer  14 ′,  18 ′ in the shallow portion  50  and at the interstitial regions  22  ( FIG.  17 D ); and removing the insoluble negative photoresist  60 ′, thereby exposing the metal film  62 ′ in the deep portion  48  ( FIG.  17 E ); and wherein the first functionalized layer  24  is deposited over the metal film  62 ′ and the resin layer  14 ′,  18 ′ exposed in the shallow portion  50  and at the interstitial regions  22  ( FIG.  17 F ). 
     Further still, the example of the method shown in  FIG.  17 A  through  FIG.  17 K  depicts: wherein the photoresist  51  is a positive photoresist  56 ; as a result of the ultraviolet light dosage, the positive photoresist  56  in the shallow portion  50  and the interstitial regions  22  become the insoluble photoresist  51 ′,  56 ′, and the positive photoresist  56  in the deep portion  48  becomes the soluble photoresist  51 ″,  56 ″; the deep portion  48  is exposed upon removal of the soluble photoresist  51 ″,  56 ″; the second functionalized layer  26  is deposited in the deep portion  48  and over the insoluble photoresist  51 ′,  56 ′; the removal of the insoluble photoresist  51 ′,  56 ′ exposes the first functionalized layer  24  in the shallow portion  50  and over the interstitial regions  22 ; and the polishing removes the first functionalized layer  24  from the interstitial regions  22 . 
     The method of the series of  FIG.  17 A  through  FIG.  17 K  includes a metal material that is sputter coated or thermally evaporated on the surface of the resin layer  14 ′,  18 ′ of the multi-depth depression  20 ′. During sputtering, the metal material is deposited at an angle (e.g., 45° or 60°) relative to the surface(s) of the multi-depth depression  20 ′. This creates a shadow effect in the multi-depth depression  20 ′ where less or no metal material is deposited in an area of the multi-depth depression  20 ′ that is transverse to the incoming metal material. Thus, the substrate is rotated throughout sputtering to introduce the metal material to these area(s) of the multi-depth depression  20 ′. As the metal material continues to be applied to the interstitial regions  22  as the substrate is rotated, this process deposits more of the metal material on the interstitial regions  22  and less of the metal material in the depressions  20 ′ due, at least in part, to the shadow effect. The pressure may also be adjusted during sputtering. Low pressure (about 5 mTorr or less) renders sputtering more directional, which maximizes the shadow effect. A similar effect may be achieved with thermal evaporation (e.g., using low pressure), and thus this technique may be used instead of sputtering to create the metal film  62 . Thus, as a result of sputtering or thermal evaporation, a metal film  62  (see  FIG.  17 A ) is generated having a first thickness T 1  over the interstitial regions  22 , a second thickness T 2  over the surface  64  of the deep portion  48  of the multi-depth depression  20 ′, and a third thickness T 3  over the surface  66  of the shallow portion  50  of the multi-depth depression  20 ′. Sputtering or thermal evaporation is controlled so that the second thickness T 2  (which is at least ⅓ times smaller than the first thickness T 1 ) and the third thickness T 3  is less than the first thickness T 1  and greater than the second thickness T 2  (e.g., T 1 &gt;T 3 &gt;T 2 ). The second thickness T 2  may be coupled with a UV light dosage that is able to transmit through the metal film  62  at its thinner portion, i.e. at the deep portion  48 , while the first thickness T 1  and the third thickness T 3  are sufficient to block the same UV light dosage from transmitting through the metal film  62  at its thicker portions, i.e., the shallow portion  50  and the interstitial regions  22  ( FIG.  17 A ). In other examples, the second thickness T 2  may be coupled with a UV light dosage that is blocked by the metal film  62 ′ at its thinner portion, i.e., at the deep portion  48  (see, e.g.,  FIG.  17 G ). 
     The second thickness T 2  is about 30 nm or less and is at least 10 nm thinner than the first thickness T 1 . In some examples, the second T 2  is 20 nm or less (which provides desirable UV transmittance). As such, in some instances, T 2  20-10 nm. In one example, the first thickness T 1  is about 30 nm and the second thickness T 2  is at least 10 nm thinner (e.g., 20 nm or less (e.g., 8.5 nm, 15 nm, etc.). As other examples, T 1 =40 nm and T 2 =30 nm; =15 nm and T 2 =5 nm; =20 nm and T 2 =10 nm; and T 1 =25 nm and T 2 =15 nm. 
     The metal material used to form the metal film  62  in this example of the method may be titanium, chromium, aluminum, gold, or copper. In some examples, the metal material may be at least substantially pure (&lt;99% pure). In other examples, molecules or compounds of the listed elements may be used as long as the metal film  62  is i) opaque (non-transparent or having transmittance less than 0.25) to the light energy used for light sensitive material alteration in the thick regions and ii) transparent (having transmittance greater than 0.25) to the light energy used for light sensitive material alteration in the thin regions. For example, oxides of any of the listed metals (e.g., aluminum oxide, zinc oxide, titanium dioxide, etc.) may be used, alone or in combination with the listed metal. As a result of sputtering or thermal evaporation, the metal film  62  having varying thicknesses T 1 , T 2 , and T 3  is positioned over the resin layer  14 ′,  18 ′, as shown in each of the series of  FIG.  17 A  through  FIG.  17 G . 
       FIG.  17 A  also depicts the multi-depth depression  20 ′, with a deep portion  48  and a shallow portion  50  adjacent to the deep portion  48 . Whether the resin layer  14 ′ or  18 ′ is used, the surface at the deep portion  48  is the surface  64 , and the surface at the shallow portion  50  is the surface  66 . In this example method, the resin layer  14 ′,  18 ′ is to be transmissive to the ultraviolet light dosage and thus the material and/or thickness of the resin layer  14 ,  18 ′ may be appropriately selected. 
       FIG.  17 B  depicts the deposition of a photoresist  51 . The photoresist  51  in this specific example of the method is a negative photoresist  60 , and may be any of the negative photoresists  60  disclosed herein. As described, the first and third thicknesses T 1 , T 3  are selected to block the UV light dosage from transmitting through the metal film  62 , and the second thickness T 2  is selected to transmit the UV light dosage through the metal film  62 . As such, the portion of the photoresist  51 ,  60  overlying the metal film  62  with the second thickness T 2  becomes insoluble due to the exposure to the UV light, and the portions of the photoresist  51 ,  60  overlying the metal film  62  with the first and third thicknesses T 1 , T 3  become soluble due to the lack of exposure to the UV light. In other words, when exposed to the ultraviolet light dosage, the insoluble photoresist  60 ′ forms over the deep portion  48  and the soluble photoresist  60 ″ forms over the shallow portion  50  and the interstitial regions  22  (see  FIG.  17 B  and  FIG.  17 C ). 
       FIG.  17 C  depicts the removal of the soluble negative photoresist  60 ″. The soluble photoresist  60 ″ is removed using any suitable developer described herein for negative photoresists  60 . The removal of the soluble negative photoresist  60 ″ exposes the metal film  62  in the shallow portion  50  and at the interstitial regions  22  (see  FIG.  17 C ). 
       FIG.  17 D  depicts the removal of a portion of the metal film  62  in the shallow portion  50  and at the interstitial regions  22 . A portion of the metal film  62  may be removed with a wet etching process, as described herein, e.g. at  FIG.  8 G  and  FIG.  9 G . As shown in  FIG.  17 D , the metal film  62  removal exposes the surface  66  at the shallow portion  50 . The insoluble negative photoresist  60 ′ is not susceptible to the wet etching process, and thus the insoluble negative photoresist  60 ′ and the portion of the metal film  62 ′ underneath it in the deep portion  48  remain intact after the wet etching process. 
       FIG.  17 E  depicts the removal of the insoluble negative photoresist  60 ′, which exposes the metal film  62 ′ in the deep portion  48 . The insoluble negative photoresist  60 ′ may be removed via a lift-off process. The lift-off process may be any suitable lift-off process described herein. The lift-off process involves exposing the insoluble negative photoresist  60 ′ to a suitable remover for the type of negative photoresist  60  used. As shown in  FIG.  17 E , the removal process removes at least 99% of the insoluble negative photoresist  60 ′. This removal process leaves the portion of the metal film  62 ′ in the deep portion  48 . 
       FIG.  17 F  depicts the deposition of a first functionalized layer  24  over the metal film  62 ′ and the exposed resin layer  14 ′,  18 ′ at the shallow portion  50  and the interstitial regions  22 . The first functionalized layer  24  is deposited over the metal film  62 ′ and the resin layer  14 ′, or alternatively, the metal film  62 ′ and the resin layer  18 ′. The functionalized layer  24  may be any of the gel materials described herein and may be applied using any suitable deposition technique. A curing process may be performed after deposition. The functionalized layer  24  covalently attaches to the exposed surfaces of the resin layer  14 ′ or to the exposed surfaces of the resin layer  18 ′. Whether the resin layer  14 ′ or  18 ′ is used, the applied functionalized layer  24  is positioned over exposed surfaces of the resin layer  14 ′ or  18 ′, including the surface  66  of the shallow portion  50 , and the interstitial regions  22 . 
       FIG.  17 F  also depicts the application of another photoresist, which is a positive photoresist  56 . Any of the positive photoresists  56  described herein may be used. The positive photoresist  56  is then exposed to an ultraviolet light dosage to form an insoluble positive photoresist  56 ′ and a soluble positive photoresist  56 ″. When the ultraviolet light dosage is applied, the portion of the metal film  62 ′ in the deep portion  48  is thin enough to enable the ultraviolet light dosage to transmit therethrough, forming a soluble positive photoresist  56 ″ over the deep portion  48 . It is to be understood that the ultraviolet light dosage used in this step of the method is lower than the ultraviolet light dosage used in reference to  FIG.  17 B , and thus the thickness of the resin layer  14 ′,  18 ′ (underlying the interstitial regions  22  and the surface  66 ) is sufficient to block the ultraviolet light dosage. As such, the positive photoresist  56  overlying the interstitial regions  22  and the surface  66  are not exposed to the ultraviolet light and become insoluble. The insoluble positive photoresist  56 ′ forms over shallow portion  50  and the interstitial regions  22  (see  FIG.  17 G ). 
       FIG.  17 G  also depicts the removal of the soluble positive photoresist  56 ″. The soluble positive photoresist  56 ″ is removed using any suitable developer described herein for positive photoresists  56 . 
       FIG.  17 H  depicts several of the processes, including the sequential removal of the first functionalized layer  24  and the metal film  62 ′ to expose the resin layer  14 ′,  18 ′ in the deep portion  48 .  FIG.  17 H  depicts when the first functionalized layer  24  is removed from the metal film  62 ′ positioned over the surface  64  of the deep portion  48 . As depicted, the first functionalized layer  24  is also removed from a portion of the perimeter sidewall  29 , P that defines the deep portion  48 . The first functionalized layer  24  may be removed via an ashing process. The ashing process may be performed as described herein, and removes the first functionalized layer  24  to expose the metal film  62 ′. The insoluble positive photoresist  56 ′ is not susceptible to the ashing process, and thus the insoluble positive photoresist  56 ′ and the underlying first functionalized layer  24  remain in the shallow portion  50  and over the interstitial regions  22  after the ashing process. 
       FIG.  17 H  also depicts when the metal film  62 ′ is removed from the deep portion  48 . The metal film  62 ′ may be removed by a wet etching or lift-off process, which depends upon the material of the metal film  62 ′. As examples, an aluminum metal film  62 ′ can be removed in acidic or basic conditions, a copper metal film  62 ′ can be removed using FeCl 3 , a copper, gold or silver sacrificial layer can be removed in an iodine and iodide solution, and a silicon metal film  62 ′ can be removed in basic (pH) conditions. The removal of the metal film  62 ′ exposes the surface  64  of the resin layer  14 ′,  18 ′ at the deep portion  48 . The removal of the metal film  62 ′ also exposes the remainder of the perimeter sidewall  29 , P that defines the deep portion  48  as well as the interior wall  29 , I. 
       FIG.  17 I  depicts the deposition of the second functionalized layer  26 , which is applied over the exposed surface  64  in the deep portion  48  and the insoluble positive photoresist  56 ′. The second functionalized layer  26  (e.g., the gel material that forms the second functionalized layer  26 ) may be applied using any suitable deposition technique. The second functionalized layer  26  does not contaminate the first functionalized layer  24 , which is covered by the insoluble positive photoresist  56 ′. 
     Referring now to  FIG.  17 J , the insoluble positive photoresist  56 ′ is removed through a lift-off process. The lift-off process may be any suitable lift-off process described herein. The lift-off process involves exposing the insoluble positive photoresist  56 ′ to a suitable remover for the type of positive photoresist  56  used. As shown in  FIG.  17 J , the removal process removes i) at least 99% of the insoluble positive photoresist  56 ′ and ii) the second functionalized layer  26  thereon. This removal process leaves the second functionalized layer  26  that is positioned on the surface  64  and on the sidewalls  29 , P and  29 , I in the deep portion  48 . The removal process also leaves the first functionalized layer  24  intact over surface  66  of the shallow region  50  and at the interstitial regions  22 . These portions of the functionalized layers  24 ,  26  remain intact, in part because they are covalently attached to the resin layer  14 ′ or  18 ′. 
     In  FIG.  17 K , the functionalized layer  24  that is positioned over the interstitial regions  22  is removed, e.g., using a polishing process as described, for example, in reference to  FIG.  9 H . 
     Cleaning and drying processes may be performed after polishing. The cleaning process may utilize a water bath and sonication. The water bath may be maintained at a relatively low temperature ranging from about 22° C. to about 30° C. The drying process may involve spin drying, or drying via another suitable technique. 
     As depicted in  FIG.  17 K , the functionalized layer  24  is positioned on one half of the multi-depth depression  20 ′ (e.g., at the shallow portion  50  and the adjacent portion of the perimeter sidewall  29 , P), and the functionalized layer  26  is positioned on the other half of the multi-depth depression  20 ′ (e.g., at the deep portion  48  and the adjacent portion of the perimeter sidewall  29 , P). As such, the padlock like conformation  33  is eliminated 
     While not shown, the method of  FIG.  17 A  through  FIG.  17 K  also includes attaching respective primer sets  30 ,  32  to the functionalized layers  24 ,  26 . In some examples, the primers  34 ,  36  or  34 ′,  36 ′ (not shown in  FIG.  17 A  through  FIG.  17 K ) may be pre-grafted to the functionalized layer  24 . Similarly, the primers  38 ,  40  or  38 ′,  40 ′ (not shown in  FIG.  17 A  through  FIG.  17 K ) may be pre-grafted to the functionalized layer  26 . In these examples, additional primer grafting is not performed. 
     In other examples, the primers  34 ,  36  or  34 ′,  36 ′ are not pre-grafted to the functionalized layer  24 . In these examples, the primers  34 ,  36  or  34 ′,  36 ′ may be grafted after the functionalized layer  24  is applied (e.g., at  FIG.  17 F ). In these examples, the primers  38 ,  40  or  38 ′,  40 ′ may be pre-grafted to the second functionalized layer  26 . Alternatively, in these examples, the primers  38 ,  40  or  38 ′,  40 ′ may not be pre-grafted to the second functionalized layer  26 . Rather, the primers  38 ,  40  or  38 ′,  40 ′ may be grafted immediately after the second functionalized layer  26  is applied (e.g., at  FIG.  17 I ); or after insoluble positive photoresist  56 ′ removal (e.g., at  FIG.  17 J  or  FIG.  17 K ) as long as i) the functionalized layer  26  has different functional groups (than functionalized layer  24 ) for attaching the primers  38 ,  40  or  38 ′,  40 ′ or ii) any unreacted functional groups of the functionalized layer  24  have been quenched, e.g., using the Staudinger reduction to amines or additional click reaction with a passive molecule such as hexynoic acid. 
     When grafting is performed during the method, grafting may be accomplished using any of the grafting techniques described herein. 
     While a single set of the functionalized layers  24 ,  26  is shown in  FIG.  17 K , it is to be understood that the method described in reference to  FIG.  17 A  through  FIG.  17 K  may be performed to generate an array of depressions  20 ′ (each having functionalized layers  24 ,  26  therein) across the resin layer  14 ′,  18 ′. 
     Method to Form Trenches 
     As mentioned herein, some of the architecture within the flow channels  12  includes multi-depth trenches  21 .  FIG.  19 A  through  FIG.  19 K  depict top views of a portion of a channel  12  having a multi-depth trench  21  defined therein between two interstitial regions  22  as the trench  21  is processed to generated isolated areas  86 ,  86 ′ of the functionalized layers  24 ,  26 . Cross-sectional views of the portion of the channel  12  are shown in  FIG.  18 A  through  FIG.  18 I  to illustrate some of the processes.  FIG.  18 A  through  FIG.  18 I  and  FIG.  19 A  through  FIG.  19 K  together depict an example method for patterning isolated areas of the trenches  21  with the functionalized layers  24 ,  26  in a manner that reduces the padlock like configuration  33 . 
     The method generally includes: depositing a first functionalized layer  24  over a resin layer  14 ,  14 ′,  18 ,  18 ′ including a plurality of multi-depth trenches  21  separated by interstitial regions  22 , each multi-depth trench  21  including a deep portion  48 ′ and a shallow portion  50 ′ adjacent to the deep portion  48 ′ ( FIG.  18 A  and  FIG.  19 A ); patterning the first functionalized layer  24 , whereby a portion  25 ′ of the first functionalized layer  24  in the deep portion  48 ′ is covered by a region  53 ′ of a sacrificial layer  52 ′ and portions of the first functionalized layer  24  in the shallow portion  50 ′ and over the interstitial regions  22  are removed ( FIG.  18 D  and  FIG.  19 D ); depositing a second functionalized layer  26  over the region  53 ′ of the sacrificial layer  52 ′ and the interstitial regions  22  and in the shallow portion  50 ′ ( FIG.  18 E  and  FIG.  19 E ; lifting off the region  53 ′ of the sacrificial layer  52 ′, thereby exposing the portion  25 ′ of the first functionalized layer  24  in the deep portion  48 ′ ( FIG.  18 F  and  FIG.  19 F ); polishing the second functionalized layer  26  from the interstitial regions  22  ( FIG.  18 G  and  FIG.  19 G ); applying a photoresist  51  in a pattern of spatially separated stripes  82 ,  82 ′ that are at least substantially perpendicular to the multi-depth trenches  21  ( FIG.  19 I ); removing areas  84  of the first functionalized layer  24  and the second functionalized layer  26  that are exposed between the spatially separated stripes  82 ,  82 ′ ( FIG.  19 I  and  FIG.  19 J ); and removing the photoresist  51  ( FIG.  18 I  and  FIG.  19 K ). 
     As shown in  FIG.  18 A , the multi-depth trench  21  is defined in either the single layer base support  14 ,  14 ′ or the resin layer  18 ,  18 ′ of the multi-layered structure  16 ,  16 ′ as described herein. As such, the term “resin layer” may be referred to as “resin layer  14 ,  14 ′,  18 , or  18 ” throughout the description of this method. The underlying base support  17 ,  17 ′ of the multi-layered structure  16 ,  16 ′ is not shown in  FIG.  18 A  through  FIG.  18 I . 
     The multi-depth trench  21  may be etched, imprinted, or defined in the resin layer  14 ,  14 ′,  18 ,  18 ′ using any suitable technique. In one example, nanoimprint lithography is used. In this example, a working stamp is pressed into the resin layer  14 ,  14 ′,  18 ,  18 ′ while the material is soft, which creates an imprint (negative replica) of the working stamp features in the resin layer  14 ,  14 ′,  18 ,  18 ′. The resin layer  14 ,  14 ′,  18 ,  18 ′ may then be cured with the working stamp in place. Curing may be accomplished as described herein in reference to  FIG.  4 A . After curing, the working stamp is released. This creates topographic features in the resin layer  14 ,  14 ′,  18 ,  18 ′. In this example, as shown in  FIG.  18 A , the topographic features of the multi-depth trench  21  include the shallow portion  50 ′ (and its bottom surface  66 ′), the deep portion  48 ′ (and its bottom surface  68 ), the internal wall  29 , I separating the deep portion  48 ′ and the shallow portion  50 ′, and the opposed sidewalls  29 , E 1 , E 2 . The top view of the generated multi-depth trench  21  is shown in  FIG.  19 A . 
     While one multi-depth trench  21  is shown in  FIG.  18 A  and  FIG.  19 A , it is to be understood that the method may be performed to generate an array of multi-depth trenches  21  including respective deep portions  48 ′ and shallow portions  50 ′, separated by interstitial regions  22 , across the surface of the resin layer  14 ,  14 ′,  18 ,  18 ′. 
     If the resin layer  14 ,  14 ′,  18 ,  18 ′ does not include surface groups to covalently attach to the functionalized layers  24 ,  26 , the resin layer  14 ,  14 ′,  18 ,  18 ′ may first be activated, e.g., through silanization or plasma ashing. If the resin layer  14 ,  14 ′,  18 ,  18 ′ does include surface groups to covalently attach to the functionalized layers  24 ,  26 , the activation process is not performed 
       FIG.  18 B  and  FIG.  19 B  depict the first functionalized layer  24  deposited over the resin layer  14 ,  14 ′,  18 ,  18 ′. The functionalized layer  24  is deposited over the surface  64 ′,  66 ′ in the deep and shallow portions  48 ′,  50 ′, and over the interstitial regions  22 . As depicted in  FIG.  18 B , the functionalized layer  24  also deposits on the opposed sidewalls  29 , E 1 , E 2  and the interior side wall  29 , I. The functionalized layer  24  may be any of the gel materials described herein and may be applied using any suitable deposition technique. A curing process may be performed after deposition. The functionalized layer  24  covalently attaches to the exposed surfaces of the resin layer  14 ,  14 ′,  18 ,  18 ′. 
     The functionalized layer  24  is then patterned. Patterning the first functionalized layer  24  involves applying a sacrificial layer  52 ′ over the first functionalized layer  24  ( FIG.  18 C  and  FIG.  19 C ); and dry etching the sacrificial layer  52 ′ and the portions of the first functionalized layer  24  in the shallow portion  50 ′ and over the interstitial regions  22  ( FIG.  18 D  and  FIG.  19 D ). 
     Referring specifically to  FIG.  18 C  and  FIG.  19 C , the sacrificial layer  52 ′ is deposited over the first functionalized layer  24 . In this example, the sacrificial layer  52 ′ may be any example of the negative or positive photoresists disclosed herein or poly(methyl methacrylate), and may be applied and cured as described herein. 
     Referring now to  FIG.  18 D  and  FIG.  19 D , the sacrificial layer  52 ′ and the first functionalized layer  24  are dry etched to expose the surface  66 ′ in the shallow portion  50  and the interstitial regions  22 . This dry etching process is performed for a measured amount of time to expose the desired surfaces/regions  66 ′,  22 . As shown in  FIG.  18 D  and  FIG.  19 D , the timed dry etching is stopped so that the region  53 ′ of the sacrificial layer  52 ′ and underlying portion  25 ′ of the functionalized layer  24  remain in the portion of the deep portion  48 ′ that is next to the interior wall  29 , I. As such, the remaining sacrificial layer  52 ′ is at least substantially co-planar with the surface  66 ′ at the shallow portion  50 ′. In one example, the timed dry etch may involve a reactive ion etch (e.g., with 100% O 2  or 10% CF 4  and 90% O 2 ) where the sacrificial layer  52 ′ and functionalized layer  24  are etched at a rate of about 17 nm/min. In another example, the timed dry etch may involve a 100% O 2  plasma etch where the sacrificial layer  52 ′ and functionalized layer  24  are etched at a rate of about 98 nm/min. 
     As shown in  FIG.  18 E  and  FIG.  19 E , the second functionalized layer  26  is deposited over the region  53 ′ of the sacrificial layer  52 ′, exposed portions of the first functionalized layer  24 , and the interstitial regions  22 , and in the shallow portion  50 ′. In this example, “in the shallow portion,” means that the second functionalized layer  26  is deposited over portions of the resin layer  14 ,  14 ′,  18 ,  18 ′ that are exposed in the shallow portion  50 ′, e.g., the surface  66 ′ and the opposed sidewall  29 , E 2 . It is to be understood that the second functionalized layer  26  may also be deposited over other exposed portions of the resin layer  14 ,  14 ′,  18 ,  18 ′, such as some of the opposed sidewall  29 , E 1  and/or some of the internal wall  29 , I. 
     The second functionalized layer  26  may be any of the gel materials described herein and may be applied using any suitable deposition technique. A curing process, as described herein, may be performed after deposition. The second functionalized layer  26  covalently attaches to the resin layer  14 ,  14 ′,  18 ,  18 ′. 
     Referring specifically to  FIG.  18 F  and  FIG.  19 F , the sacrificial layer  52 ′ is removed in a lift-off process. The lift-off process may involve an organic solvent suitable for the sacrificial material that is used. Any of the removers set forth herein for the insoluble positive photoresist or the insoluble negative photoresist may be used when these materials are used as the sacrificial layer  52 . The lift-off process removes i) at least 99% of the region  53 ′ of the sacrificial layer  52 ′ and ii) the functionalized layer  26  positioned thereon. The lift-off process does not remove the portion  25 ′ of the functionalized layer  24  that had been in contact with the region  53 ′ of the sacrificial layer  52 ′. Thus, the lift-off process exposes the functionalized layer  24  at the surface  64 ′ of the resin layer  14 ,  14 ′,  18 ,  18 ′ at the deep portion  48 ′, as depicted in  FIG.  18 F  and  FIG.  19 F . 
     In  FIG.  18 G  and  FIG.  19 G , the functionalized layer  26  that is positioned over the interstitial regions  22  is removed, e.g., using a polishing process. The polishing process may be performed as described herein, e.g., in reference to  FIG.  9 H . 
     A photoresist  51  is then applied to generate a pattern of spatially separated stripes  82 ,  82 ′ that are at least substantially perpendicular to the multi-depth trenches  21 . The photoresist  51  may be a positive photoresist  56  or a negative photoresist  60 . 
     In one example, applying the photoresist  51  in the pattern of the spatially separated stripes  82 ,  82 ′ involves: depositing a positive photoresist  56  over the multi-depth trenches  21  and the interstitial regions  22  ( FIG.  19 H ); selectively exposing portions of the positive photoresist  56  to an ultraviolet light dosage, whereby the exposed portions become soluble (i.e., positive soluble photoresist  56 ″) and unexposed portions become the spatially separated stripes  82 ,  82 ′ (positive insoluble photoresist  56 ′); and removing the exposed, soluble portions  56 ″. In this example, a photomask is used to pattern the positive photoresist  56 . The photomask blocks UV light from reaching the portions of the positive photoresist  56  that are to become insoluble (i.e., that are to become the stripes  82 ,  82 ′), and allows UV light to reach the portions of the positive photoresist  56  that are to become soluble. A suitable positive photoresist developer is used to remove the positive soluble photoresist  56 ″. 
     In another example, applying the photoresist  51  in the pattern of the spatially separated stripes  82 ,  82 ′ involves: depositing a negative photoresist  60  over the multi-depth trenches  21  and the interstitial regions  22  ( FIG.  19 H ); selectively exposing portions of the negative photoresist  60  to an ultraviolet light dosage, whereby the exposed portions become the spatially separated stripes  82 ,  82 ′ (negative insoluble photoresist  60 ′) and unexposed portions become soluble (i.e., positive soluble photoresist  60 ″); and removing the unexposed, soluble portions  60 ″. In this example, a photomask is used to pattern the negative photoresist  60 . The photomask blocks UV light from reaching the portions of the negative photoresist  60  that are to become soluble, and allows UV light to reach the portions of the negative photoresist  60  that are to become insoluble (i.e., that are to become the stripes  82 ,  82 ′). A suitable negative photoresist developer is used to remove the soluble negative photoresist  60 ″. 
       FIG.  19 H  depicts an example of how the photoresist  51  can be patterned with UV light to form the positive/negative insoluble photoresist  56 ′,  60 ′ and the positive/negative soluble photoresists  56 ″,  60 ″. The width W s  of each portion of the positive/negative soluble photoresists  56 ″,  60 ″ is at least 100 nm. As the soluble portions are removed, this width W s  will provide a desirable distance between the isolated functionalized layers  86 ,  86 ′ that are ultimately formed. 
       FIG.  19 I  depicts the positive/negative insoluble photoresists  56 ′,  60 ′ after the positive/negative soluble photoresists  56 ″,  60 ″ are removed. Each of the remaining positive/negative insoluble photoresists  56 ′,  60 ′ corresponds with one of the spatially separated stripes  82 ,  82 ′. The spatially separated stripes  82 ,  82 ′ cover portions of the functionalized layers  24 ,  26  that form the isolated areas  86 ,  86 ′ (see  FIG.  18 H  and  FIG.  19 I  together). The width W l  of each spatially separated stripe  82 ,  82 ′ may be any of the widths set forth herein for the width W s  or the width of the protrusions  28 . This width W s  will provide desirable dimensions for the isolated areas  86 ,  86 ′ of the functionalized layers  24 ,  26  that are ultimately formed. As depicted in  FIG.  19 I , each spatially separated stripe  82 ,  82 ′ is at least substantially perpendicular to the length of the trench  21 . 
     Additionally, the pattern of the spatially separated stripes  82 ,  82 ′ leaves areas  84  of the first functionalized layer  24  and the second functionalized layer  26  that are exposed between the spatially separated stripes  82 ,  82 ′. The areas  84  of the first functionalized layer  24  and the second functionalized layer  26  that are exposed between the spatially separated stripes  82 ,  82 ′ are then removed, e.g., via ashing. Any of the plasma ashing processes set forth herein may be used. The removal of the areas  84  exposes the underlying surfaces  64 ′,  66 ′, as shown in  FIG.  19 J . The exposed surfaces  64 ′  66 ′ create interstitial-like regions between the isolated areas  86 ,  86 ′ of the functionalized layers  24 ,  26  that are ultimately formed (see  FIG.  19 K ). 
     The spatially separated stripes  82 ,  82 ′ are then removed, e.g., using a suitable remover for the insoluble photoresist  56 ′,  60 ′ that defines the stripes  82 ,  82 ′. 
     The functionalized layers  24 ,  26  underlying the spatially separated stripes  82 ,  82 ′ remain intact after removal of the spatially separated stripes  82 ,  82 ′, as shown in  FIG.  19 K . As such, the removal of the stripes  82 ,  82 ′ exposes the underlying portions of the functionalized layers  24 ,  26 , which are isolated areas  86 ,  86 ′ along the trench  21 .  FIG.  18 I  depicts a cross-section of one of the areas  86  of  FIG.  19 K . Even though the functionalized layer  24  is sandwiched by the portions of the functionalized layer  26 , the padlock like configuration  33  shown in  FIG.  1 A  is reduced by about 70%, in part because the functionalized layer  24  is not completely surrounded by the functionalized layer  26 . 
     While not shown, the method of  FIG.  18 A  through  FIG.  18 I  and  FIG.  19 A  through  FIG.  19 K  also includes attaching respective primer sets  30 ,  32  to the functionalized layers  24 ,  26 . In some examples, the primers  34 ,  36  or  34 ′,  36 ′ (not shown in  FIG.  18 A  through  FIG.  18 I  and  FIG.  19 A  through  FIG.  19 K ) may be pre-grafted to the functionalized layer  24 . Similarly, the primers  38 ,  40  or  38 ′,  40 ′ (not shown in  FIG.  18 A  through  FIG.  18 I  and  FIG.  19 A  through  FIG.  19 K ) may be pre-grafted to the functionalized layer  26 . In these examples, additional primer grafting is not performed. 
     In other examples, the primers  34 ,  36  or  34 ′,  36 ′ are not pre-grafted to the functionalized layer  24 . In these examples, the primers  34 ,  36  or  34 ′,  36 ′ may be grafted after the functionalized layer  24  is applied (e.g., at  FIG.  18 B  and  FIG.  19 B ). In these examples, the primers  38 ,  40  or  38 ′,  40 ′ may be pre-grafted to the second functionalized layer  26 . Alternatively, in these examples, the primers  38 ,  40  or  38 ′,  40 ′ may not be pre-grafted to the second functionalized layer  26 . Rather, the primers  38 ,  40  or  38 ′,  40 ′ may be grafted after the second functionalized layer  26  is applied and prior to subsequent processing (e.g., at  FIG.  18 E  and  FIG.  19 E ), or after the second functionalized layer  26  is applied and processed (e.g., at  FIG.  18 F  and  FIG.  19 F , or  FIG.  18 G  and  FIG.  19 G , or at  FIG.  18 I  and  FIG.  19 K ) as long as i) the functionalized layer  26  has different functional groups (than functionalized layer  24 ) for attaching the primers  38 ,  40  or  38 ′,  40 ′ or ii) any unreacted functional groups of the functionalized layer  24  have been quenched, e.g., using the Staudinger reduction to amines or additional click reaction with a passive molecule such as hexynoic acid. 
     When grafting is performed during the method, grafting may be accomplished using any of the grafting techniques described herein. While two areas  86 ,  86 ′ of the functionalized layers  24 ,  26  are shown in  FIG.  19 K , it is to be understood that the method described in reference to  FIG.  18 A  through  FIG.  18 I  and  FIG.  19 A  through  FIG.  19 K  may be performed to generate an array of depressions  21  (each having a desired number of areas  86 ,  86 ′ therein) across the resin layer  14 ,  14 ′,  18 ,  18 ′. 
     To further illustrate the present disclosure, examples are given herein. It is to be understood that these examples are provided for illustrative purposes and are not to be construed as limiting the scope of the present disclosure. 
     NON-LIMITING WORKING EXAMPLES 
     Example 1 
     An example method similar to that shown in  FIG.  4 A  through  FIG.  4 D  and  FIG.  6 A  through  FIG.  6 F  was performed, except that functionalized layers were not included. The method involved generating multi-depth depressions in a nanoimprint lithography resin using a working stamp and curing process. A negative photoresist (AZ1505 positive photoresist from MicroChemicals) was deposited in the multi-depth depressions and cured. A timed dry reactive ion etching process with 90% CF 4  and 10% O 2  was used to etch back the negative photoresist so that some remained in the deep portion of each multi-depth depression and a small portion remained over the surface in the shallow portion. A SEM image (magnification of about 127,000×) of one of the multi-depth depressions with the photoresist in the deep portion is shown in  FIG.  20   . 
     The nanoimprint lithography resin was then time dry reactive ion etched with 10% CF 4  and 90% O 2  to remove the interstitial regions. This dry etching process was performed until the photoresist in the multi-depth depression protruded above the etched portions of the nanoimprint lithography resin. Another SEM image (magnification of about 109,000×) of the multi-depth depression was then taken after the interstitial regions had been dry etched. This is shown in  FIG.  21   . As depicted, the negative photoresist remained in what had been, prior to dry etching, the multi-depth depression. 
     These results illustrate that a series of timed dry etching processes may be used reduce the perimeter sidewall of the multi-depth depressions. 
     Example 2 
     An example method similar to that shown in  FIG.  17 A  through  FIG.  17 K  was performed. The method involved generating multi-depth depressions in a nanoimprint lithography resin using a working stamp and curing process. Aluminum was then sputter coated (60° angle) on the multi-depth depressions at room temperature. The aluminum formed a metal film over the multi-depth depressions and the interstitial regions. The aluminum metal film had various thicknesses, i.e. a thicker film was formed over the interstitial regions and the shallow portion of the multi-depth depression, and a thinner film was formed over the deep portion of the multi-depth depression. 
     Then, a negative photoresist (NR9-1500PY from Futurrex) was deposited over the metal film. Ultraviolet light (365 nm) was directed through the backside of the nanoimprint lithography resin, and then soluble portions of the negative photoresist were removed in a developer (RD6 (a tetramethylammonium hydroxide (TMAH) based developer) from Futurrex). SEM images (about 70,000× magnification) of the top view of the patterned nanoimprint lithography resin were taken before ( FIG.  22 A ) and after ( FIG.  22 B ) photoresist development.  FIG.  22 A  illustrates the photoresist across the entire surface.  FIG.  22 B  clearly illustrates that the soluble portions of the photoresist were removed from the interstitial regions and from the shallow portion of each multi-depth depression after being developed, while the insoluble portions of the photoresist remained in the deep portion of each multi-depth depression after being developed. These results illustrate that the thicker portions of the metal mask blocked the UV light (rendering the negative photoresist soluble) and that the thinner portions of the metal mask enabled UV light transmission (render the negative photoresist insoluble). 
     ADDITIONAL NOTES 
     It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. It should also be appreciated that terminology explicitly employed herein that also may appear in any disclosure incorporated by reference should be accorded a meaning most consistent with the particular concepts disclosed herein. 
     Reference throughout the specification to “one example”, “another example”, “an example”, and so forth, means that a particular element (e.g., feature, structure, and/or characteristic) described in connection with the example is included in at least one example described herein, and may or may not be present in other examples. In addition, it is to be understood that the described elements for any example may be combined in any suitable manner in the various examples unless the context clearly dictates otherwise. 
     While several examples have been described in detail, it is to be understood that the disclosed examples may be modified. Therefore, the foregoing description is to be considered non-limiting.