Platform for immobilization and observation of subcellular processes

A method of immobilizing matter for imaging that includes providing an array of nanofibers and directing matter to the array of the nanofibers. The matter is immobilized when contacting at least three nanofibers of the array of nanofibers simultaneously. Adjacent nanofibers in the array of nanofibers may be separated by a pitch as great as 100 microns. The immobilized matter on the array of nanofibers may then be imaged. In some examples, the matter may be cell matter, such as protoplasts.

BACKGROUND

The present disclosure is related to observation of cellular materials, and more particularly to platforms for observing cellular materials.

One requirement for investigating cellular-level processes, such as cell growth, cell wall or cellulose biosynthesis and other dynamic responses, is the ability to monitor large numbers of individual cells, tissue fragments, and/or protoplasts over extended time periods. To achieve maximum resolution, high powered objectives are necessary which can typically only capture small numbers of cells in any single field of view. Therefore, it can be necessary to iteratively image different regions of a culture over the course of the experiment. Translation of the sample stage is typically required to observe several different specimens in different fields of view over the course of the experiment. If the specimens, such as protoplasts, that are being observed are not immobilized, they will typically move around within the culture chamber during these manipulations. Even without macroscale movement of the experiment, protoplasts may shift position just due to natural convection and/or vibrations of the systems.

SUMMARY

In one embodiment, nanostructured, high-aspect-ratio spike arrays are provided for immobilizing matter, such as cell matter, e.g., protoplasts, for temporal evaluation. Spike arrays may be fabricated at defined interspike dimensions to provide three or four point pinning of individual protoplasts. In one embodiment, the method of immobilizing cell matter for imaging includes providing an array of nanofibers, in which adjacent nanofibers in the array of nanofibers are separated by a pitch of less than 100 microns. Cell matter may then be directed to the array of the nanofibers, wherein the cell matter is immobilized when simultaneously in contact with at least three nanofibers of the array of nanofibers. The cell matter that is immobilized in the array of nanofibers may then be imaged.

In another aspect, a structure is provided for immobilizing matter, such as cell matter, multicellular tissues, extracellular matrices, and organic and inorganic solids. In one embodiment, the structure for immobilizing matter includes a channel for delivery of a fluid containing matter, and an array of nanofibers positioned within the channel having a pitch between adjacent nanofibers in the array of nanofibers ranging from 3 microns to 20 microns. Each of the nanofibers in the array of nanofibers has a tip diameter of 150 nm or less, and each of the nanofibers has an increasing diameter from the tip diameter to a base of each of the nanofibers. The combination of the pitch between adjacent nanofibers and the increasing diameter of the nanofibers provides dimensions that physically engage matter from the fluid containing matter that is delivered to the array of nanofibers. The matter is physically engaged when in simultaneous contact with at least three nanofibers of the array of nanofibers.

In another aspect, a method of analyzing matter response to external stimuli is provided that includes providing a platform including a channel and an array of nanofibers within the channel. In one embodiment, the method for analyzing matter includes providing a channel having an array of nanofibers within the channel. A first portion of the array of nanofibers has a pitch between adjacent nanofibers that immobilizes matter. A second portion of the array of nanofibers is separated from walls of the channel and provides at least one electrode. A fluid containing matter is passed through the channel, wherein the fluid containing matter is traversed across the first portion of the array of nanofibers before the second portion of the array of nanofibers. As the fluid containing matter is traversed past the first portion of the array of nanofibers, at least a portion of the matter contained within the fluid containing matter is immobilized by the first portion of the array of nanofibers. A stimuli is then applied to the matter that is immobilized by the first portion of the array of nanofibers. Emissions and secretions by the matter in response to the stimuli may be measured with the at least one electrode that is provided by the second portion of the array of nanofibers. The matter may be cell matter, such as protoplasts.

In another aspect, a structure is provided for immobilizing matter, and measuring emissions by the immobilized matter in response to stimuli that is applied to the immobilized matter. In one embodiment, the structure for analyzing matter response to external stimuli includes a channel for delivery of a fluid containing matter and an array of nanofibers that is present within the channel. The array of nanofibers includes a first portion that is present at an opening of the channel and a second portion that is present at the exit of the channel. The first portion of the array of nanofibers has a pitch between adjacent nanofibers to physically engage matter from the fluid containing matter being traversed through the channel from the opening to the exit. The second portion of the nanofibers provides at least one electrode that is physically separated from the sidewalls of the channel. The matter being immobilized by the structure may be cell matter, e.g., protoplasts.

DETAILED DESCRIPTION

Detailed embodiments of the present disclosure are described herein; however, it is to be understood that the disclosed embodiments are merely illustrative of the compositions, structures and methods of the disclosure that may be embodied in various forms. In addition, each of the examples given in connection with the various embodiments are intended to be illustrative, and not restrictive. Further, the figures are not necessarily to scale, some features may be exaggerated to show details of particular components. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ the compositions, structures and methods disclosed herein. References in the specification to “one embodiment”, “an embodiment”, “an example embodiment”, etc., indicate that the embodiment described may include a particular feature, structure, or characteristic, but every embodiment may not necessarily include the particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment.

The present disclosure relates to immobilizing matter for observation. Examples of matter that may be immobilized include cell matter and nutrients. As used herein, the term “cell matter” includes cells, a cell, and the components of a cell, such as organelles. For example, the term “cell matter” may include the nucleus, vacuoles, and mitochondria, all of which may be enclosed within the cell membrane and immersed in cytoplasm. Both animal and plant cells can provide cell matter that is suitable for use with the methods and structures disclosed herein. In some embodiments, the cell matter is provided by protoplasts. A “protoplast” is a plant cell in which the cell wall has been removed. For example, a protoplast may be a plant cell in which the cell wall has degraded away. Following degradation of the cell wall, a plasma remains which provides the protoplast. Protoplasts are typically fragile, and are not easily immobilized for observation. In some embodiments, the structures and methods disclosed herein are suitable for immobilizing matter for observation and treatment with reagents. Although the following disclosure describes the immobilization of cell matter, the present disclosure is not limited to only this type of matter. As the present disclosure is also applicable to other types of matter, such as extracellular materials, and organic/inorganic solids.

In one embodiment, a structure is provided for immobilizing cell matter that includes nano-structured, high-aspect-ratio nanofiber arrays, which may provide for temporal evaluation of cellular-level processes, such as cellulose biosynthesis (used as a specific example here). The term “array” as used to describe an array of nanofibers, i.e., nanofibers array, denotes a plurality of nanofibers. Nanofiber arrays10may be fabricated at defined pitch dimensions to provide three or four point pinning (also referred to as immobilization) of individual cell matter, such as protoplasts, as depicted inFIGS. 1A-3. As used throughout the present disclosure, the terms “pinning”, “immobilizing” and/or “immobilization” means that cell matter20is being obstructed from movement so that the cell matter20is maintained in a fixed position. Immobilization may include elimination of both translation and rotation of the cell matter20.FIGS. 1A and 1Bdepict immobilization of cell matter20by three point pinning. By “three point pinning” it is meant that the frictional force of the three nanofibers15that are in direct contact with the cell matter20physically immobilizes the cell matter20.FIGS. 2A,2B and3depict immobilization of cell matter20by four point pinning. By “four point pinning” it is meant that the frictional force of the four nanofibers15that are in direct contact with the cell matter20physically immobilizes the cell matter20. In both three point and four point pinning the cell matter20may also be in direct contact with the substrate5that is present between the adjacent nanofibers15of the nanofiber array10. During three point pinning an immobilized cell matter20is in contact with three nanofibers simultaneously, and during four point pinning an immobilized cell matter20is in contact with four nanofibers simultaneously. The term “simultaneously” as used to describe the contact of at least three adjacent nanofibers15in the array of nanofibers10when immobilizing cell matter20means that the at least three nanofibers15are in contact with the immobilized cell matter20at the same time.

The number of nanofibers15that are present in the nanofiber array10may be selected to correspond to the number of specimens of cell matter20that is intended to be immobilized in the nanofibers array10, and whether immobilization is provided by three point pinning, as depicted inFIGS. 1A and 1B, and/or four point pinning, as depicted inFIGS. 2A,2B and3. In one embodiment, the number of nanofibers15that are present within the nanofibers array10may range from 10 nanofibers15to 100 nanofibers15. In one example, the number of nanofibers15that are present in the nanofiber array10is equal to 25. The above examples for the number of nanofibers15that are present in the nanofibers array10is provided for illustrative purposes only, and is not intended to limit the present disclosure, as any number of nanofibers15may be present in the nanofibers array10.

The term “nanofiber” denotes a structure having an aspect ratio that is greater than 25:1 (height: width) and has a tip with a width of 1 micron or less. The inter-nanofiber dimension (also referred to as interspike dimension) between each of the adjacent nanofibers15may be referred to as the “pitch” between adjacent nanofibers15. Referring toFIGS. 1A-3, the pitch P1, P2, P3, P4, P5is the center to center distance, i.e., nanofiber tip to adjacent nanofiber tip, between adjacent nanofibers15. The pitch P1, P2, P3, P4, P5in combination with the geometry and dimensions of each nanofiber15may be selected to correspond to the dimensions of the cell matter20that is intended to be immobilized. In some embodiments, the pitch P1, P2, P3, P4, P5that is separating adjacent nanofibers15may be as great as 25 microns. In another embodiment, the pitch P1, P2, P3, P4, P5may range from 10 microns to 20 microns. In one example, when the cell matter20being immobilized is protoplast, the pitch P1, P2, P3, P4, P5is selected to immobilize cell matter20have a diameter ranging from 5 microns to 20 microns. In another example, when the cell matter20being immobilized is a yeast cell, the pitch P1, P2, P3, P4, P5is selected to immobilize cell matter20have a diameter ranging from 2 microns to 5 microns.

The nanofiber15may be composed of carbon, and in some embodiments may be referred to as a “carbon nanofiber”. The carbon nanofiber that provides the nanofibers14in the array of nanofibers may be a vertically aligned carbon nanofiber (VACNF). A vertically aligned carbon nanofiber is a carbon nanofiber in the height H1of the carbon nanofiber is substantially perpendicular to the upper surface of the substrate5on which the nanofiber15is present. By substantially perpendicular it is meant that the angle defined at the intersection of the height H1of the nanofiber15and the upper surface of the substrate5is +/−5° from normal.

Referring toFIGS. 1B and 2B, the aspect ratio of the nanofiber15is a ratio of the height H1of the nanofiber15to the width W1of the base of the nanofiber15. In some embodiments, the nanofibers15that are employed to provide the array of nanofibers10in the methods and structures disclosed herein have an aspect ratio that is greater than 50:1, which in some examples may be greater than 100:1. The height H1of the nanofiber15is measured from the tip of the nanofiber15to the base of the nanofiber15that is present in direct contact with the substrate5on which nanofiber15is present. In some embodiments, the height H1of the nanofiber15may be as great as 100 microns. In one embodiment, the height H1of the nanofiber15may range from 25 microns to 75 microns. In yet another embodiment, the height H1of the nanofiber15may range from 35 microns to 65 microns. The width W1of the base of the nanofiber15may be as great as 350 nm. In one embodiment, the width W1of the base of the nanofiber15may range from 100 nm to 300 nm. In another embodiment, the width W1of the base of the nanofiber15may range from 150 nm to 250 nm. The tip T1of each nanofiber15may have a diameter that is as great as 150 nm. In another embodiment, the tip T1of each nanofibers may range from 10 nm to 100 nm. It is noted that the aforementioned dimensions for the nanofibers15are provided for illustrative purposes only, and are not intended to limit the present disclosure to only the dimensions described above.

Referring toFIGS. 1A-3, in one embodiment, the array of nanofibers15is present on a substrate5. The substrate5is typically transparent. The substrate5may be composed of a semiconductor material, such as a silicon-containing material, or a non-conductor, such as fused silica. Silicon-containing materials that are suitable for the substrate5include, but are not limited to, silicon (Si), single crystal silicon, polycrystalline silicon, amorphous silicon, and silicon-containing materials with some or all of the Si replaced by Ge. In one example, the silicon-containing material that provides the substrate5is silicon having a [100] crystal orientation. In another example, the substrate5is composed of fused silica. In one example, the substrate5is provided by a 4″ wafer of [100] silicon. The thickness of the substrate5may range from 10 microns to 200 microns. In one example, the substrate5has a thickness of 170 microns.

In one embodiment, formation of the nanofiber array10may include the forming metal catalyst dots on a substrate5by with electron beam lithography (EBL) in conjunction with electron-gun metal evaporation, and catalytic growth of the nanofibers10on the substrate5using plasma enhanced chemical vapor deposition (PE-CVD). Growth of nanofibers10, e.g., carbon nanofibers, may begin with the formation of a catalytic precursor. Nickel (Ni) can be used as a catalyst. However, other metals such as iron (Fe) and cobalt (Co) can also be utilized as the catalyst with these procedures.

The catalyst for nanofiber10growth, e.g., carbon nanofiber growth, may be formed on the substrate5by forming a metal catalyst dot. A catalyst dot is fabricated on the substrate5using photo or electron beam (e-beam) lithography and electron gun (e-gun) metal evaporation. More specifically, in one embodiment, the substrate5is first coated with a photoresist material, e.g., poly(methyl methacrylate) PMMA, and is then either photolithographically or e-beam exposed and developed to produce a small openings in the photoresist. Typically, each opening in the photoresist corresponds to the positioning of a later formed nanofiber15in the nanofiber array10. Therefore, the pitch, i.e., center to center distance between adjacent openings in the photoresist, of the openings will correspond to the pitch of the nanofibers15in the nanofibers array10.

Following the formation of the openings through the photoresist, a buffer layer is deposited within the openings in direct contact with the substrate5to prevent the formation of catalyst silicide and to impede catalyst diffusion at elevated temperatures. In one example, the buffer layer may be composed of titanium (Ti) or chrome (Cr). The buffer layer may have a thickness of 50 nm. The catalyst layer may then be deposited atop the buffer layer, wherein at least a portion of the catalyst layer is present in the openings. In one embodiment, the catalyst layer is composed of nickel (Ni), but the catalyst may also be composed of other metals suitable for growing nanofibers, e.g., carbon nanofibers, such as iron (Fe) and cobalt (Co). The catalyst layer may have a thickness of 50 nm.

In a following process step, isolated catalysts dots may be formed from the catalyst layer and the buffer layer on the substrate5by lifting the photoresist layer from the substrate5. The photoresist layer may be lifted off the substrate in acetone, wherein the portion of the buffer layer and the catalyst layer that is present in the openings remains on the substrate to provide the metal catalyst dots for nanofibers growth. The portions of the catalyst layer and the buffer layer that are present on the portions of the photoresist layer between the openings is removed as the photoresist layers is lifted from the substrate5. In one embodiment, the isolated catalyst dots may each have a diameter ranging from 350 nm to 650 nm. In another embodiment, the isolated catalyst dots have a diameter ranging from 400 nm to 600 nm. In one example, the isolated catalyst dots each have a diameter of 500 nm. By “isolated” it is meant that each catalyst dot is an island of material that is separate from an adjacent catalyst dot. The pitch separating adjacent catalyst dots may be as great as 100 microns. In one embodiment, the pitch separating adjacent catalyst dots may range from 3 microns to 20 microns. In another embodiment, the pitch separating adjacent catalyst dots may range from 5 microns to 10 microns.

In one embodiment, forming nanofibers15, e.g., vertically aligned carbon nanofibers (VACNF), from the isolated catalyst dots includes the use of a direct current plasma enhanced chemical vapor deposition (DC-PECVD) that includes a vacuum chamber having an anode and a cathode present therein, in which the cathode may function as a heater. The details of one embodiment of DC plasma enhanced chemical vapor deposition a vacuum chamber including an anode and cathode for use in a plasma enhanced chemical vapor deposition have been described in U.S. Pat. No. 6,649,431, which is incorporated herein by reference. Although, the vertically aligned carbon nanofibers (VACNF) are described as being formed using DC-PECVD, embodiments have been contemplated in which radio-frequency (RF) or microwave plasmas also can be employed.

In some embodiments, for VACNF growth, a mixture of a carbonaceous gas and an etchant (e.g., acetylene and ammonia) can be used as the gas source. The etchant is needed to etch away graphitic carbon film that continuously forms during the growth from the plasma discharge. If not removed, the role of the film will be passivating the catalyst and thereby preventing the formation of VACNFs. Just prior to the VACNF growth process, ammonia can be introduced into the chamber and a plasma created. After the plasma is started, acetylene can be introduced and the VACNF growth can begin. Each catalyst dot, i.e., Ni (nickel) catalyst dot, initiates the formation of an individual VACNF. The catalyst dot can reside on top of the VACNF and provides for its continued catalytic growth upwards. In some embodiments, the VACNFs are oriented along plasma field lines and normally grow perpendicular to the substrate.

In some embodiments, the growth parameters may be adjusted to provide a nanofiber15, e.g., VACNF, having an increasing diameter from the tip of the nanofiber to the base of the nanofiber15. A nanofiber15having a base with a greater diameter than the tip of the nanofiber may be referred to as a carbon nanocone (CNC). One example of a growth parameter that may be adjusted to increase the diameter of the nanofiber15is the ratio of acetylene to ammonia. In this way, a CNC rather than a CNF can be formed. If the acetylene content is increased relative to that of ammonia (in addition to just diffusing through the Ni particle and precipitating at its bottom, thus providing for the growth in the vertical direction) carbon also begins to precipitate at the walls of the growing, initially cylindrical VACNF. Precipitation occurs due to the insufficient amount of the etchant (ammonia), which leads to the deposition rate of carbon being higher than the etching rate. Thus growth in two dimensions (vertical due to the catalytic growth through the catalyst dot, e.g., Ni particle, and lateral due to the carbon precipitation at the walls) occurs. The tip diameter of the CNC remains constant during the growth process and is determined only by the size of the catalyst dot. In contrast, at a given acetylene content the base diameter of the CNC increases with growth time. Furthermore, by changing growth parameters, such as the relative acetylene content, the angle of the sidewall (also referred to a cone angle) of the CNC can be changed. Higher acetylene content and higher pressure yield higher cone angles and vice versa. The CNC height is proportional to the growth time. The pitch of the carbon nanofibers, i.e., VACNF nanofibers or CNC nanofibers, is controlled by the patterning of the catalyst dots, as described above.

Referring toFIGS. 1A-3, in one specific example, formation of the nanofibers15in the array of nanofibers10may begin with forming a 50 nm thick nickel catalyst layer, on a 50 nm thick chrome buffer layer that is present on a 4″ silicon [100] or transparent fused silica substrate5. The catalyst layer and the buffer layer may then be photolithographically patterned or e-beam patterned to provide catalyst dots with a 500 nm diameter at a desired pitch (i.e. 5, 10, or 20 microns) over the entire surface, or select regions, of the substrate5. Nanofibers15, i.e., carbon nanofibers, may then be synthesized in a DC-PECVD reactor at a temperature of 650° C., 10 torr, 2 A, using a mixture of a carbonaceous source gas (acetylene) and an etch gas (ammonia). Growth time is selected to provide nanofibers15of desired length, typically ranging from approximately 10 microns to 17 microns tall, with tip diameters of approximately 100 nm.

Referring toFIGS. 1A,2A and3, the pitch P1, P2, P3, P4, P5separating the adjacent nanofibers15in the array of nanofibers10is typically equal to the pitch separating the adjacent catalyst dots. The pitch P1, P2separating adjacent nanofibers15in the array of nanofibers10may be uniform (also referred to as homogenous) as depicted inFIGS. 1A and 2A, or the pitch P3, P4, P5may be graded (also referred to as varied) to correspond to cell matter20of different diameters, as depicted inFIG. 3. The pitch P1, P2, P3, P4, P5on nanofiber arrays can be homogenous across the entire substrate5, or may be spatially varied in discrete increments or continuously varied across the substrate5.

These defined nanofiber pitches may be used to immobilize cell matter20, such as protoplasts, in various geometric configurations. Referring toFIGS. 1A and 2B, homogenous pitches P1, P2will tend to immobilize cell matter20, such as protoplasts, of a specific diameter, based upon the pitch and wedging of the protoplasts between nanofibers. Referring toFIG. 3, variable nanofiber pitch P3, P4, P5across a nanofiber array10can be used to immobilize cell matter20, such as protoplasts, of varying diameters. Referring toFIGS. 1A-3, the placement of nanofibers15may also be used to hold cell matter20in close proximity to one another, or with discrete intervals between individual or groups of cell matter20. In one example, such spatial variation of protoplast immobilization can be important when communication or diffusion of growth factors and other species between protoplasts may influence the digestion or synthesis of cellulose of individual protoplasts. In some embodiments, the pitch P1, P2, P3, P4, P5may be as great as 100 microns. In one embodiment, the pitch P1, P2, P3, P4, P5separating adjacent nanofibers15in the array of nanofibers10may range from 3 microns to 20 microns. In another embodiment, the pitch P1, P2, P3, P4, P5separating adjacent nanofibers15in the array of nanofibers10may range from 5 microns to 10 microns.

Referring toFIGS. 4-8B, the array of nanofibers10a,10b,10c,10d,10e,10f,10gmay be positioned within a fluidic channel25or is in communication with a series of fluidic channels25. In some embodiments, the fluidic channel25may be employed for delivering the cell matter20to the array of nanofibers10a,10b,10c,10d,10e,10f,10g, or for delivering reagents R1, R2, R3to the cell matter20that is immobilized on the array of nanofibers10a,10b,10c,10d,10e,10f. In one embodiment, the fluidic channel25is provided by walls26that formed on the substrate5using photolithography. For example, the walls26that define the fluidic channel25may be formed by depositing a photoresist layer on the substrate5, patterning the photoresist layer, and then developing the photoresist layer. Photoresists are classified into two groups: positive resists and negative resists. A positive resist is a type of photoresist in which the portion of the photoresist that is exposed to light becomes soluble to the photoresist developer. The portion of the photoresist that is unexposed remains insoluble to the photoresist developer. A negative resist is a type of photoresist in which the portion of the photoresist that is exposed to light becomes insoluble to the photoresist developer. The unexposed portion of the photoresist is dissolved by the photoresist developer.

In some embodiments, the photoresist may be composed of the Poly(vinyl cinnamate), Poly(methyl methacrylate) (PMMA), Poly(methyl glutarimide) (PMGI), Phenol formaldehyde resin (DNQ/Novolac) or multi-layers and combinations thereof. In one example, the photoresist that provides the walls26of the micro-channel is provided by an epoxy-based negative photoresist that is commonly referred to as SU-8. SU-8 is explained in more detail in U.S. Pat. No. 4,882,245, which is incorporated herein by reference. One derivative of SU-8 that is suitable for use with the methods and structures of the present disclosure is SU8: 2020.

In one embodiment, to form the walls26of the fluidic channel25, a layer of photoresist layer may be deposited on the upper surface of the substrate5using spin on deposition, spray coating or chemical solution deposition. The layer of photoresist may have a thickness ranging from 20 microns to 200 microns. In one example, the layer of photoresist that provides the walls26of the micro-fluidic channel25may have a thickness of 60 microns. The layer of photoresist may be patterned and developed to form walls26that define the fluidic channel25. By “patterned” it is meant that the layer of photoresist is selectively irradiated to light through a stencil, e.g., reticle, that is designed to allow light to fall only on preselected areas of the layer of the photoresist, e.g., areas that define a plurality of walls26that provide the fluidic channel25. The light causes a chemical change, e.g., cross-linking, in the layer of photoresist. The stencil (hereafter referred to as a reticle) may include a transparent substrate and a pattern layer. The radiant energy blocking portion may be comprised of chrome, chrome oxide, chromium nitride, iron oxide, silicon or a number of other opaque materials.

The radiation, i.e., light, that may be used to expose the layer of photoresist through the reticle may include UV, DUV, and the H and I lines of a mercury-vapor lamp. In another embodiment, the layer of photoresist may be exposed with an ion beam. Depending upon whether the photoresist is a positive resist or a negative resist, either the exposed portion or the unexposed portion of the layer of photoresist may be washed away, i.e., removed, using a developer. Examples of developers for use with positive resists include sodium hydroxide (NaOH) and tetramethyl ammonium hydroxide (TMAH) to be used in combination with a water rinse (H2O). Examples of developers for use with negative resists include xylene with a rinse composed of n-Butylacetate. In one embodiment, the developer for SU-8 is 1-Methoxy-2-propanol acetate.

In one embodiment, the walls26of the fluidic channel25may be provided by a dielectric material, such as an oxide, nitride or oxynitride, that is patterned and etched using photolithography. In this embodiment, the material layer that provides the dielectric material may first be deposited on the substrate, a mask of photoresist may be formed on the dielectric material, and the exposed portions of the dielectric material may be etched while the portions of the dielectric material are under and protected by the photoresist mask. The exposed portions of the dielectric material may be removed by an etch process that is selective to the photoresist mask and the substrate. The remaining portions of the dielectric material following selective etching provides the walls26of the fluidic channel25.

Referring toFIGS. 4-8B, in some embodiments, the walls26of the fluidic channel25may provide a chamber for housing the array of nanofibers10a,10b,10c,10d,10e,10f,10g, and a plurality of inlets for introducing a suspension of cell matter20to be caged on the array of nanofibers10a,10b,10c,10d,10e,10ffor optical viewing. The inlets27of the micro-fluidic channel25may also be employed to introduce reagents and buffer solutions to the array of nanofibers10a,10b,10c,10d,10e,10fthat are housed by the chamber of the fluidic channel25.

The walls26of the fluidic channel25may be formed after forming the array of nanofibers10. In one example, following the formation of the array of nanofibers10a,10b,10c,10d,10e,10f,10g, the micro-fluidic channel25is fabricated upon a 170 micron thick fused silica substrate, in which the walls26of the micro-fluidic channel25are composed of SU-8 crosslinkable epoxy photoresist that has been photolithographically patterned with UV light. In one embodiment, prior to depositing the photoresist material that provides the walls26, the array of nanofibers10a,10b,10c,10d,10e,10f,10gare first protected beneath a thin, uniform protective layer of photoresist, such as SU8 epoxy. In one example, to deposit the protective layer of photoresist, the substrate5including the array of nanofibers10a,10b,10c,10d,10e,10f,10gmay be spun with a photoresist of a 1:3 dilution of SU8 2002:SU8 thinner. The deposited protective photoresist may then be cured. In one embodiment, the deposited protective photoresist may be cured with a first prebaked at 60° C. for 1 minute followed by a second prebake at 90° C. for 2 minutes. The protective photoresist may then be exposed to 10 seconds of UV light. The wafer is then treated with a post baked that may include a first heat treatment for 1 minute at 60° C. followed by a second heat treatment for 2 minutes at 90° C. In some embodiments, the protective layer of photoresist anchors the base of the nanofibers15in the array of nanofibers10a,10b,10c,10d,10e,10f,10gto the substrate5. In some examples, this anchorage step protects the nanofibers15from shearing off from the substrate5during handling.

Following the formation of the protective layer of photoresist, the fluidic channels25may then be defined on the substrate5including the array of nanofibers10a,10b,10c,10d,10e,10f,10g. In one example, the walls26of the micro-fluidic channel25may be formed by depositing layers of photoresist, such as SU8 epoxy. The layers of photoresist that provide the walls26of the micro-fluidic channel25may then be cured using a pre-exposure bake including a first treatment at 60° C. for 1 minute followed by a second treatment at 90° C. for 3 minutes. The layers of photoresist that provide the walls26of the fluidic channel25may then be lithography patterned with a desired channel layout. The substrate5is then post exposure baked at 60° C. for 1 minute and 90° C. for 4 minutes. The substrate5including the array of nanofibers10a,10b,10c,10d,10e,10f,10gand the patterned photoresist layers for the walls26of the fluidic chamber25are then developed in SU8 developer for 2 minutes and rinsed in isopropyl alcohol. The above process sequence, in which the array of nanofibers10a,10b,10c,10d,10e,10f,10gis formed before the walls26of the fluidic channel25is provided for illustrative purposes only, and is not intended to limit the present disclosure. For example, embodiments have been contemplated, in which the walls26of the fluidic channel25are formed before the array of nanofibers10a,10b,10c,10d,10e,10f,10g.

Immobilization of cell matter20within the array of nanofibers10,10a,10b,10c,10d,10e,10f,10gdepicted inFIGS. 1-8Bcan be achieved using various techniques. In one embodiment, cell matter20may be immobilized onto the array of nanofibers10,10a,10b,10c,10d,10e,10f,10gusing a centrifuge. A centrifuge is a piece of equipment, generally driven by a motor, that puts an object in rotation around a fixed axis, applying a force perpendicular to the axis. The centrifuge works using sedimentation principles, where the centripetal acceleration causes more dense substances to separate out along the radial direction, i.e., toward the bottom of the centrifuge tube. By the same principle, lighter objects will tend to move to the top, i.e., to the top of the centrifuge tube (in the rotating picture, move to the center). In one embodiment, the nanofiber array may be positioned flat at the bottom of a centrifuge tube facing upwards, and a fluid containing cell matter20is positioned within the centrifuge tube, wherein as the centrifuge tube is run through the centrifuge, the cell matter20, e.g., protoplasts, are forced from the fluid containing the cell matter onto the array of nanofibers10,10a,10b,10c,10d,10e,10f,10g. The cell matter20is immobilized when in contact with three nanofibers in the embodiments in which the array of nanofibers10provides three point pinning, as depicted inFIGS. 1A-1B, and is immobilized when in contact with four nanofibers in the embodiments in which the array of nanofibers10,10a,10b,10c,10d,10e,10f,10gprovide four point pinning, as depicted inFIGS. 2A-8B.

The fluid containing cell matter may be a suspension of cell matter20. A suspension is a heterogeneous fluid containing solid particles that are sufficiently large for sedimentation. For example, solids particles suitable for sedimentation may be larger than 1 micrometer. The suspension of cell matter20may include any dispersion medium. For example, when the cell matter20is protoplasts the dispersion medium may consist of an aqueous solution containing an osmoticum (example mannitol or magnesium sulfate) and a protectant (calcium chloride). The solution may also include other additives to avoid flocculation of the cell matter, such as dispersants.

Various commercially available tubes are available for swinging bucket centrifuges that facilitate orienting a substrate upon a platform so that may then be placed into a centrifuge, which orients the centripetal force normal to the flat substrate surface during the centrifugation procedure. For fixed angle rotors (not swinging bucket), substrates5including nanofiber arrays10, such as those described above with reference toFIGS. 1A-3may be fixed within centrifuge tubes first filling the centrifuge tube with a small amount of uncrosslinked polydimethylsiloxane (PDMS) (silicone rubber) and curing the PDMS while spinning the centrifuge, thereby creating a slanted substrate5that the nanofiber arrays10may be positioned upon, normal to the centripetal force. It is noted that the present disclosure is not limited to only uncrosslinked PDMS, as other materials may be converted from a liquid to a solid material within the centrifuge tube during spinning, to provide a surface normal to the centripetal force.

Referring toFIGS. 1A-8B, in another embodiment, immobilization of the cell matter20onto the array of nanofibers10,10a,10b,10c,10d,10e,10f,10gmay be achieved by pipetting a suspension of the cell matter20directly down onto the array of nanofibers10,10a,10b,10c,10d,10e,10f,10g. In this embodiment, the suspension of the cell matter20may be allowed to settle upon the array of nanofibers10,10a,10b,10c,10d,10e,10f,10gwithout additional centripetal force. More secure immobilization of the settled cell matter20from the suspension can be achieved by pipetting a liquid against the settled cell matter20to further press the cell matter20into the array of nanofibers10,10a,10b,10c,10d,10e,10f,10g. The density of the solution used should not exceed the density of the cell matter being delivered in order to avoid buoyant forces which would interfere with immobilization of the material within the Nanofiber matrices.

An advantage of using immobilized cell matter20upon the arrays of nanofibers10,10a,10b,10c,10d,10e,10f,10gdepicted inFIGS. 1-8Bis that they may be integrated with fluidic channels25to facilitate reagent delivery, exchange, active transport studies, and diffusional transport studies of the immobilized cell matter20, e.g., protoplasts. Micro-fluidic reagent delivery can be used to perfuse the sample, i.e., immobilized cell matter20, with various nutrients, growth factors, digestive enzymes, or other relevant species. In some embodiments, the use of transparent substrates5, such as fused silica substrates, enables the use of laser scanning microscopy on the immobilized cell matter20. In the embodiments, in which the immobilized cell matter20is a protoplast, laser scanning microscopy may provide for high resolution imaging of cellulose degradation or biosynthesis. The fluidic channels25may be configured to provide a closed system or an open system.

Referring toFIG. 4, fluidic channels25having arrays of nanotubes10a,10b,10cpresent therein may be loaded with cell matter20, such as protoplasts, using a variety of methods including centrifugation in a swinging bucket centrifuge, as described above with reference toFIGS. 1A-3, or by allowing settling of a protoplasts suspension, or by pipetting a protoplast suspension down onto the array of nanofibers10a,10b,10cpresent within the chamber of the fluidic channel25. Once the cell matter20has been immobilized on the array of nanofibers10a,10b,10cthe fluidic channel25may be sealed. For example, the fluidic channel25may be sealed by aspirating excess buffer solution from the portions of the fluidic channel25that dose not include the chamber in which the array of nanofibers10a,10b,10cis present, and placing a cover onto the device. In one example, the cover that seals the fluidic channel25may be composed of silicone, e.g., polydimethylsiloxane (PDMS). In some embodiments, the cover that seals the fluidic channel25may feature at least one opening therethrough to provide for fluidic access ports to the chamber of the fluidic channel25in which immobilized cell matter20is present on the array of nanofibers10a,10b,10c.

In one embodiment, the fluidic channels25may be used to control the delivery of nutrients and/or growth factors to the immobilized cell matter20, e.g., immobilized protoplasts, that are present on the array of nanofiber10a,10b,10cthat are present in the chamber of the micro-fluidic channel25, and to control the flow of metabolic by-products between immobilized cell matter20. In one embodiment, in which the cell matter20is provided by protoplasts, based upon the micro-fluidic layout and cell matter immobilization locations, protoplasts may be located downstream of one another at downstream arrays of nanofibers10b,10csuch that downstream protoplasts can experience the metabolic byproducts of upstream cell matter20that is immobilized on upstream arrays of nanofibers10a.

In another embodiment, arrays of nanofibers may also be located so that the cell matter20, such as protoplasts, do not experience one another's metabolic byproducts, either by locating them in isolated fluidic channels (not depicted). In yet another embodiment, the laminar flow characteristics of the fluidic channels25may be employed to minimize movement of reagents and metabolic byproducts within the chamber of the fluidic chamber25in which the arrays of nanofibers10a,10b,10care present, as depicted inFIG. 5. For example, referring toFIG. 5, the first and second inlets27a,27bthat are present at opposing sides of the chamber containing the arrays of nanofibers10a,10b,10cmay be employed to apply reagents R1, R2to the cell matter20that is immobilized on the arrays of nanofibers10a,10b,10c, and a buffer solution B1may be introduced to a chamber by the inlet27cthat is between the first and second inlets27a,27b, wherein the buffer solution B1separates the first and second reagents R1as they flow down the fluidic chamber25.

In the embodiment depicted inFIG. 5, the cell matter20, e.g., protoplasts, that are immobilized on the side of the chamber of the fluidic channel25that is closest to the first inlet27ais only subjected to the first reagent R1that is introduced to the chamber through the first inlet27a, and the cell matter20, e.g., protoplasts, that are immobilized on the side of the chamber of the fluidic channel25that is closest to the second inlet27bis only subjected to the second reagent R2that is introduced to the chamber through the second inlet27b. The buffer solution B1that is introduced to the chamber by the third inlet27bseparates the first reagent R1from the second reagent R2, wherein the cell matter20that is immobilized on the arrays of nanofibers10a,10b,10cin the center of the chamber are only subjected to the buffer solution B1, and not the first and second reagents R1, R2. Examples of the first and second reagents R1, R2that may be applied to the cell matter20include at least one reagent selected from the group consisting of nutrients, growth factors, digestive enzymes and combinations thereof. For example, the first reagent may be a cell wall digesting/degrading enzyme, cellulase; the second reagent may be pectinase, separated by a buffer solution without a digestive component. The first reagent may be cellulase and the second reagent may be a combination of cellulase and hemicellulase; thereby enabling the observation of how the addition of hemicellulase impacts cell wall digestion on one side of the plant cell vs. not having hemicellulase in the microfluidic flow on the other side of the same cell. The buffer solution B1may include at least one fluid that is selected from the group consisting of water, and water with osmoticums and stabilizers to preserve the integrity of a fully protoplasted plant cell, including mannitol, sorbital, magnesium sulfate, calcium chloride, and combinations thereof.

In another embodiment of the present disclosure, techniques may be used to control reagent delivery with spatial resolution at or below the diameter of an individually selected cell matter20, such as protoplasts, that are immobilized on an array of nanofibers10d, as depicted inFIG. 6. For example, by using pinched injection techniques, one may expose immobilized protoplasts to highly spatially resolved reagent delivery. In one example in which the cell matter20is provided by protoplasts, a reagent such as 3-IAA (indole acetic acid) or a molecular agent can be delivered to just one side of an appropriately positioned protoplast or set of protoplasts to observe the impact of this reagent on those protoplasts in comparison to the protoplasts immediately adjacent thereto that are not exposed to the reagent due to the fluid delivery arrangement.

In one example, in which the immobilized cell matter20is protoplasts, the micro-fluidic mixing techniques described above with reference toFIGS. 1-8Bmay be used to evaluate the impact of serial dilutions of growth factors, or reagents on cellulose regeneration, or the dilution of digestive enzymes on cellulose degradation of the protoplasts. The fluid paths to the immobilized cell matter20, e.g., protoplasts, may be configured to provide serial dilution either using electrokinetic or hydrodynamic pumping techniques. The cell matter20that is immobilized in the fluid paths may be treated with specific dilutions of species and can be evaluated either in real-time, or at discrete time intervals using optical microscopy. For example, immobilized protoplasts may be exposed to a steady stream of indole-3-acetic acid as various dilutions in protoplast buffer solution for a 24 hr period, followed by brief exposure to fluorescein diacetate and calcofluor to observe cell viability and cellulose synthesis. Following dye delivery, the dye may be washed from the system using neat protoplast buffer. Following imaging, the protoplasts can once again be perfused with 3-IAA in protoplast buffer for an extended period.

Referring toFIGS. 7-8B, in addition to immobilization scaffolding, the arrays of nanofibers10e,10gmay provide active electrochemical probing elements to locally and temporally monitor specific analytes with high spatial resolution. In one embodiment, individual or groups of nanofibers15may be configured with electrical contacts (hereafter referred to as interconnect) such that the nanofiber15may be used as an electrode. In one embodiment, nanofibers within the arrays of nanofibers10e,10gmay provide an electrode by forming an electrically conductive interconnect30on at least a part of the substrate5, and growing at least one nanofiber15, e.g., carbon nanofiber, that is coupled to the electrically conductive interconnect30. As used herein, the term “electrically conductive” means a material having a room temperature conductivity of greater than 10−8(Ω−m)−1. The electrically conductive interconnect30can be made from any metal or combination of metals that survive the deposition process for forming the nanofibers15, such as the temperature of the DC-PECVD process used to provide VACNF. For example, the electrically conductive interconnect30may include one, or more, refractory metal(s), such as, for example, tungsten (W), molybdenum (Mo), tantalum (Ta), niobium (Nb), platinum (Pt), aluminum (Al) and combinations thereof. The electrically conductive interconnect30may be formed using deposition processes, such as physical vapor deposition (PVD), e.g., sputtering. Following deposition, the metal layer may be patterned and etched to provide the geometry that is desired for the electrically conductive interconnect30. One example of an etch process for etching the metal that provides the electrically conductive interconnect30is reactive ion etch.

In some embodiments, an insulating layer (not shown) may be formed on the substrate5prior to the formation of the interconnect structure30, in which the insulating layer may be patterned and provides for electrical isolation between adjacent interconnects30. Following the formation of the interconnects30on the substrate5, the nanofibers, e.g., vertically aligned carbon nanofibers (VACNF), may be formed on the interconnects30using the methods described above. To summarize, to form the nanofibers15, a buffer layer is formed in direct contact with the with the electrically conductive interconnects30, followed by the formation of a catalyst layer, wherein the stack of the buffer layer and the catalyst layer is patterned to provide catalyst dots. In the embodiments in which the nanofibers function as electrodes, the catalyst dots are in direct contact with the electrically conductive interconnects30. Following formation of the catalyst dots, the nanofibers15, e.g., carbon nanofibers, may be formed using a chemical vapor deposition process, such as direct current plasma enhanced chemical vapor deposition (DC-PECVD), using a mixture of a carbonaceous gas and an etchant, e.g., acetylene and ammonia. The details for forming the nanofibers15have been described above with reference toFIGS. 1-3. Carbon nanofibers are suitable for use as electrodes, because of their electrical conductivity, but other materials have been contemplated for the nanofibers15that provide the electrodes, and are within the scope of the present disclosure.

In some embodiments, the active probe site of the electrode is provided by only the tip T1of the nanofiber15. To provide that only the tip T1of the nanofiber15is the active probe site for the electrodes, the sidewalls of the nanofibers15may be passivated with a dielectric material. For example, a dielectric sheath35may be present on the sidewalls of the nanofibers15in the array of nanofibers10e,10gthat provide the electrodes. The tip T1of the nanofiber15that is not covered by the dielectric sheath35and provides the active probe side of the electrode may have a diameter ranging from 3 nm to 150 nm. In another embodiment, the tip of the nanofiber15may have a diameter ranging from 10 nm to 100 nm. The length of the tip of the nanofiber15that provides the active probe site may be as great as 200 microns. In one embodiment, the tip of the nanofiber15that provides the active probe site may range from 50 microns to 100 microns.

In some embodiments, the dielectric sheath35may be provided by silicon oxide (SiO2), silicon nitride, and/or an insulating polymer. The dielectric sheath35may be deposited on the nanofibers15of the arrays of nanofibers10e,10gusing a conformal deposition process, such as spin on deposition or chemical vapor deposition (CVD). The thickness of the dielectric sheath35may range from 25 nm to 100 nm. In another embodiment, the thickness of the dielectric sheath35may range from 50 nm to 75 nm. In some embodiments, the dielectric sheath35may be removed from the tip of the nanofibers15using an etch process. Typically, the etch process for removing the material of the dielectric sheath35from the tip T1of the nanofibers15is an anisotropic etch. For example, the tip of the nanofibers may be exposed by removing the dielectric sheath35with reactive ion etch (RIE). The etch process for removing the material of the dielectric sheath35is typically selective to the nanofiber15, e.g., carbon nanofiber. In one embodiment, the etch chemistry for removing the portion of the dielectric sheath35from the tip of the nanofibers15comprises CF4:O2and/or CF4:SF6. Further details for depositing the material layer that provides the dielectric sheath35, and the etch process for removing the material layer that provides the dielectric sheath from the tip T1of the nanofibers15are described in greater detail in U.S. Pat. No. 6,9892,519, which is incorporated herein by reference.

Typically, the tip T1of the nanofiber15that provides the active probe site of the electrodes provided by the array of nanofibers10e,10gis separated from the base and sidewalls26of the channel25. By positioning the active probe site of the electrodes centrally within the channel25, the electrodes provided by the arrays of nanofibers10e,10gdisclosed herein reduce measurement errors that results from interactions between the subject matter being measured and the sidewalls26and the base of the channel25. More specifically, it has been determined that interactions between the cell matter20and emissions by the immobilized cell matter20when interacting with the sidewalls26and the base of the channel25result in band broadening of the signals being electrochemically measured. By moving the active probe site of the electrodes to only the tip T1of the nanofibers15in the arrays of nanofibers10e,10g, the active probe site of the electrodes is moved away from the structures of the channel25that interfere with the cell matter20and the emissions by the immobilized cell matter20that is being electrochemically measured. More specifically, in some embodiments, by moving the active probe site of the electrodes to only the tip T1of the nanofibers15, the electrodes provided by the nanofiber arrays10e,10bsubstantially eliminates resolution degradation that occurs from smearing of the cell matter20and smearing of emissions by the cell matter against the sidewalls26and base of the channel25, substantially eliminating band-broadening effects.

FIGS. 8A and 8Bdepict one embodiment of a structure for analyzing cell matter response to external stimuli. In some embodiment, the structure for analyzing cell matter includes a fluidic channel25for delivery of a fluid containing cell matter, and an array of nanofibers10f,10gthat is present within the fluidic channel25. The fluidic channel25that is depicted inFIGS. 8A and 8Bis similar to the fluidic channel25that has been described above with reference toFIGS. 1-7. Therefore, the description of the fluidic channel25depicted inFIGS. 1-7is suitable for the fluidic channel25that is depicted inFIGS. 8A and 8B.

The array of nanofibers10f,10gincludes a first portion of nanofibers10fthat is present at an opening of the fluidic channel25and a second portion of nanofibers10gthat is present at the exit of the fluidic channel25. The first portion of nanofibers10fhas a pitch to physically engage cell matter20from the fluid containing cell matter being traversed through the fluidic channel25from the opening to the exit. The first portion of nanofibers10ffor immobilizing the cell matter20is similar to the array of nanofibers10,10a,10b,10c,10dthat have been described above with reference toFIGS. 1-6. Therefore, the description of the array of nanofibers10,10a,10b,10c,10ddepicted inFIGS. 1-6is suitable for the first portion nanofibers10fthat are depicted inFIGS. 8A and 8B. In one example, the adjacent nanofibers15in the first portion nanofibers10fhave a pitch that ranges from 5 microns to 20 microns. In one example, the tip diameter of each nanofiber15in the first portion nanofibers10franges from 10 nm to 100 nm, and the base of each nanofiber15in the first portion of nanofibers10fis as great as 350 nm.

The second portion nanofibers10gwithin the fluidic channel25is downstream of the first portion of nanofibers10fand provides electrodes having active probe sites that are physically separated from sidewalls26and base of the fluidic channel25. The electrodes provided by the second portion nanofibers10gmay be employed for analyzing emissions by the cell matter20that is immobilized on the first portion of nanofibers10f. In some embodiments, block masks composed of photoresist may be employed to allow for separate processing of the regions of the substrate5in which the first portion of the nanofibers10fand the second portion of the nanofibers10gare present.

In some embodiments, the structures depicted inFIGS. 8A and 8Bmay be employed to optically and electrochemically analyze cell matter20simultaneously. For example, fluid containing cell matter may be traversed through the fluidic channel25across the first portion of nanofibers10fbefore being traversed across the second portion of nanofibers10g. As the fluid containing cell matter is traversed past the first portion of nanofibers10f, at least a portion of the cell matter10contained within the fluid containing cell mater is immobilized by the first portion of nanofibers10f. The immobilized cell matter10that is present on the first portion of nanofibers10fmay be imaged using laser scanning microscopy, scanning electron microscope (SEM), atomic force microscope or a combination thereof.

Once the cell matter20is immobilized on the first portion of nanofibers10f, a stimuli may be applied to the cell matter20. Stimuli may be applied to the cell matter20by dispersing the stimuli in a liquid medium and flowing the stimuli through the channel25across the immobilized cell matter20. The stimuli may include nutrients, growth factors, digestive enzymes, or other relevant species. In one example, when the cell matter is a plant cell, the stimuli may be an enzyme that degrades the cell wall of the plant cell, including but not limited to cellulase, hemicellulase, pectinase. The stimuli may be hormones that modulate cellulosic synthesis of the cell wall, including auxins and synthetic auxins such at naphthalene-1 acetic acid. The stimuli can include antibodies, which bind to the surface of the cell, and interact with the cell. The stimuli can include fluorescently labeled species, which interact or bind with specific receptors on the cell surface.

In some embodiments, the cell matter20that is engaged to the first portion nanofibers10fmay be imaged while applying of the stimuli to the immobilized cell matter20.

Emissions by the immobilized cell matter20in response to the stimuli may be measured electrochemically by the second portion of nanofibers10gthat is present in the fluidic channel25downstream from the first portion of nanofibers10f. Some examples of emissions by the immobilized cell matter20that can be measured by the electrodes provided by the second portion of nanofibers10ginclude electroactive species such as some auxins, easily oxidized or reduced degradation products of the cell wall, electroactive peptides, and electroactive signaling species, and alditols and carbohydrates derives from cell wall digestion. Some examples of electrical measurements that can be taken by the electrodes provided by the second portion of nanofibers10gincludes fast scan cyclic voltammetry, amperometry, cyclic voltammetry, differential pulse voltammetry and combinations thereof. In one example, when the cell matter20is a plant cell and the stimuli applied to the immobilized plant cell degrades the cell wall of the plant cell, the emissions being measured by the electrodes of the second portion of nanofibers10gcan be the fragments of the plant cell's degraded cell wall, which can be measured by amperometry by clamping the electrode nanofiber at sufficiently high potential to oxidize the components digesting from the cell well (i.e. carbohydrate and glycoproteins).