Patent Publication Number: US-9416281-B1

Title: Making imprinted multi-layer biocidal particle structure

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     Reference is made to commonly-assigned co-pending U.S. patent application Ser. No. 14/607,100, filed Jan. 28, 2015, entitled Imprinted Multi-layer Biocidal Particle Structure, by Burberry et al, to commonly-assigned co-pending U.S. patent application Ser. No. 14/607,109, filed Jan. 28, 2015, entitled Using Imprinted Multi-layer Structure, by Cok et al, and to commonly-assigned co-pending U.S. patent application Ser. No. 14/526,619 filed Oct. 29, 2014, entitled Making Imprinted Multi-layer Biocidal Particle Structure, by Cok et al. 
     FIELD OF THE INVENTION 
     The present invention relates to biocidal layers having antimicrobial efficacy on a surface. 
     BACKGROUND OF THE INVENTION 
     Widespread attention has been focused in recent years on the consequences of bacterial and fungal contamination contracted by contact with common surfaces and objects. Some noteworthy examples include the sometimes fatal outcome from food poisoning due to the presence of particular strains of  Escherichia coli  in undercooked beef;  Salmonella  contamination in undercooked and unwashed poultry food products; as well as illnesses and skin irritations due to  Staphylococcus aureus  and other micro-organisms. Anthrax is an acute infectious disease caused by the spore-forming bacterium bacillus anthracis. Allergic reactions to molds and yeasts are a major concern to many consumers and insurance companies alike. In addition, significant fear has arisen in regard to the development of antibiotic-resistant strains of bacteria, such as methicillin-resistant  Staphylococcus aureus  (MRSA) and vancomycin-resistant  Enterococcus  (VRE). The U.S. Centers for Disease Control and Prevention estimates that 10% of patients contract additional diseases during their hospital stay and that the total deaths resulting from these nosocomially-contracted illnesses exceeds those suffered from vehicular traffic accidents and homicides. 
     In response to these concerns, manufacturers have begun incorporating antimicrobial agents into materials used to produce objects for commercial, institutional, residential, and personal use. Noble metal ions such as silver and gold ions are known for their antimicrobial properties and have been used in medical care for many years to prevent and treat infection. In recent years, this technology has been applied to consumer products to prevent the transmission of infectious disease and to kill harmful bacteria such as  Staphylococcus aureus  and  Salmonella.    
     In common practice, noble metals, metal ions, metal salts, or compounds containing metal ions having antimicrobial properties can be applied to surfaces to impart an antimicrobial property to the surface. If, or when, the surface is inoculated with harmful microbes, the antimicrobial metal ions or metal complexes, if present in effective concentrations, will slow or even prevent altogether the growth of those microbes. Recently, silver sulfate, Ag 2 SO 4 , described in U.S. Pat. No. 7,579,396, U.S. Patent Application Publication 2008/0242794, U.S. Patent Application Publication 2009/0291147, U.S. Patent Application Publication 2010/0093851, and U.S. Patent Application Publication 2010/0160486 has been shown to provide efficacious antimicrobial protection in polymer composites. The United States Environmental Protection Agency (EPA) evaluated silver sulfate as a biocide and registered its use as part of EPA Reg. No, 59441-8 EPA EST. NO. 59441-NY-001. In granting that registration, the EPA determined that silver sulfate was safe and effective in providing antibacterial and antifungal protection. Antimicrobial activity is not limited to noble metals but is also observed in other metals such as copper and organic materials such as triclosan, and some polymeric materials. 
     It is important that the antimicrobial active element, molecule, or compound be present on the surface of the article at a concentration sufficient to inhibit microbial growth. This concentration, for a particular antimicrobial agent and bacterium, is often referred to as the minimum inhibitory concentration (MIC). It is also important that the antimicrobial agent be present on the surface of the article at a concentration significantly below that which can be harmful to the user of the article. This prevents harmful side effects of the article and decreases the risk to the user, while providing the benefit of reducing microbial contamination. There is a problem in that the rate of release of antimicrobial ions from antimicrobial films can be too facile, such that the antimicrobial article can quickly be depleted of antimicrobial active materials and become inert or non-functional. Depletion results from rapid diffusion of the active materials into the biological environment with which they are in contact, for example, water soluble biocides exposed to aqueous or humid environments. It is desirable that the rate of release of the antimicrobial ions or molecules be controlled such that the concentration of antimicrobials remains above the MIC. The concentration should remain there over the duration of use of the antimicrobial article. The desired rate of exchange of the antimicrobial can depend upon a number of factors including the identity of the antimicrobial metal ion, the specific microbe to be targeted, and the intended use and duration of use of the antimicrobial article. 
     Antimicrobial coatings are known in the prior art, for example as described in U.S. Patent Application Publication 2010/0034900. This disclosure teaches a method of coating a substrate with biocide particles dispersed into a coating so that the particles are in contact with the environment. In other designs, for example as taught in U.S. Pat. No. 7,820,284, a polymeric overcoat is applied over a base coat including anti-microbial particles. The overcoat is permeable or semi-permeable to the agents released from the anti-microbial particles. Non-planar coatings are also known to provide surface topographies for non-toxic bio-adhesion control, for example as disclosed in U.S. Pat. No. 7,143,709. 
     Imprinting methods useful for forming surface topographies are taught in CN102063951. As discussed in CN102063951, a pattern of micro-channels are formed in a substrate using an embossing technique. Embossing methods are generally known in the prior art and typically include coating a curable liquid, such as a polymer, onto a rigid substrate. A pattern of micro-channels is embossed (impressed or imprinted) onto the polymer layer by a master having an inverted pattern of structures formed on its surface. The polymer is then cured. 
     Fabrics or materials incorporating biocidal elements are known in the art and commercially available. U.S. Pat. No. 5,662,991 describes a biocidal fabric with a pattern of biocidal beads. U.S. Pat. No. 5,980,620 discloses a means of inhibiting bacterial growth on a coated substrate comprising a substantially dry powder coating containing a biocide. U.S. Pat. No. 6,437,021 teaches a water-insoluble polymeric support containing a biocide. Methods for depositing thin silver-comprising films on non-conducting substrates are taught in U.S. Patent Application Publication No. 2014/0170298. 
     SUMMARY OF THE INVENTION 
     It is important that any anti-microbial surface coating or material be efficacious when it is provided in a desired environment, that it continues to be efficacious over a desired lifetime, and that the anti-microbial surface coating or material be robust in the presence of environmental contaminants, such as gases or liquids. In particular, it is useful to clean the anti-microbial surface coating or material, for example with water or other cleaners. 
     The efficacy of antimicrobial coatings and materials depend at least in part on their structure, surface area, and the rate at which and duration for which the antimicrobial material is exposed to microbes. There is a need, therefore, for antimicrobial coatings with improved efficacy, environmental robustness, and reduced costs. 
     In accordance with the present invention, a method of making a multi-layer biocidal structure includes: 
     providing a support; 
     locating a first curable layer on the support, the first curable layer including dispersed multiple biocidal particles; 
     locating a second curable layer on the first curable layer, wherein multiple biocidal particles are dispersed within only the first curable layer; 
     imprinting the first curable layer and the second curable layer in a single step with an imprinting stamp having a structure with a depth greater than the thickness of the second curable layer; 
     curing the first curable layer and the second curable layer in a single step to form a first cured layer and a second cured layer; and 
     removing the imprinting stamp. 
     The present invention provides a biocidal multi-layer structure that provides improved antimicrobial properties with thinner layers having increased surface area made in a cost-efficient process. The biocidal multi-layer structure is robust in the presence of environmental contaminants and cleaning agents. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The above and other features and advantages of the present invention will become more apparent when taken in conjunction with the following description and drawings wherein identical reference numerals have been used to designate identical features that are common to the figures, and wherein: 
         FIG. 1  is a cross section of a multi-layer structure illustrating an embodiment of the present invention; 
         FIGS. 2A-2G  are cross sections of sequential construction steps useful in a method of the present invention; 
         FIG. 3  is a cross section of an alternative embodiment of the present invention; and 
         FIGS. 4 and 5  are flow diagrams illustrating methods of the present invention. 
     
    
    
     The Figures are not drawn to scale since the variation in size of various elements in the Figures is too great to permit depiction to scale. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention provides a multi-layer structure useful in forming an antimicrobial or biocidal article on a support. Multi-layer structures of the present invention provide improved antimicrobial properties and usability made in a cost-efficient process. In useful methods of the present invention, multiple uncured coatings are formed on a support, imprinted together, and then cured together. A thin top layer can control the rate at which antimicrobial elements are exposed to the environment and also provide environmental protection to the antimicrobial materials, for example provide protection from cleaning agents. The imprinted layers provide a greater surface area for the antimicrobial materials and a topographical structure that inhibits the growth and reproduction of microbes. Coating and imprinting processes provide a cost-efficient manufacturing method. 
     Referring to  FIG. 1 , in an embodiment of the present invention, an imprinted multi-layer structure  5  includes a support  30  having a support thickness  36 . A bi-layer  7  having a topographical structure is located on or over the support  30 . The bi-layer  7  includes a first cured layer  10  on or over the support  30  and a second layer  20  on a side of the first cured layer  10  opposite the support  30 . In an embodiment, the second layer  20  is a second cured layer  20 . The first cured layer  10  has a first-layer thickness  16  and the second cured layer  20  has a second-layer thickness  26 . Multiple biocidal particles  60  are located in the first layer  10 . Indentations  80  with a depth  6  are located in the first and second cured layers  10 ,  20  to form a topographical structure in the bi-layer  7 . At least one depth  6  of the topographical structure is greater than the second-layer thickness  26 . By providing a topographical structure depth  6  that is greater than the second-layer thickness  26 , embodiments of the present invention provide both a topographical structure that inhibits the growth and reproduction of microbes and increased surface area having a reduced thickness through which biocidal agents (e.g. biocidal particles  60 ) are exposed to the environment. In some embodiments, the depth  6  is between 0.5 microns and 50 microns or between 0.5 microns and 10 microns, or between 0.5 microns and 5 microns, or 0.5 microns to 2 microns. 
     In a useful arrangement, the support  30  is adhered with an adhesive layer  50  to a surface  8  of a structure  40 . In embodiments, the adhesive is a binder or primer. Alternatively, or in addition, the adhesive layer  50 , binder, or primer can form the surface  8  on the support  30  on which the first cured layer  10  is readily coated, for example by controlling the surface energy of the support surface or the first cured layer  10 . In another embodiment, an adhesion-promoting layer is located between the first and second curable layers  10 ,  20  (not shown) to adhere the first cured layer  10  and the second cured layer  20  together and enable the second cured layer  20  to be coated over the first cured layer  10  before the first cured layer  10  and the second cured layer  20  are imprinted to form the indentations  80  of the bi-layer  7  and the imprinted multi-layer structure  5 . 
     In an embodiment of the present invention, the biocidal particles  60  are located only in the first cured layer  10 . Thus, the second cured layer  20  provides environmental protection to the biocidal particles  60  and protects the biocidal particles  60  from environmental contaminants, such as dirt, moisture, gases, and liquids including cleaning agents. In another embodiment, the first cured layer  10  includes a first material and the second cured layer  20  includes a second material that is different from the first material. Alternatively, the first cured layer  10  and the second cured layer  20  include one or more common materials. 
     Coating or other deposition methods for forming multiple layers on a substrate are known in the art, such as curtain or hopper coating or laminating, as are imprinting and curing methods useful for forming the indentations  80  in the first and second cured layers  10 ,  20 . Curable materials, for example heat or radiation sensitive resins are also known as are supports such as glass or plastic, adhesives, and surfaces such as walls, tables, cylinders, handles and the like. 
     In an embodiment, the second cured layer  20  is thinner than the first cured layer  10 . As shown in  FIG. 1 , the first cured layer  10  has portions having the first-layer thickness  16  that are thicker than the second-layer thickness  26 . A thin second cured layer  20  can provide protection from environmental contaminants while permitting biocidal agents to effectively enter the environment. In an alternative embodiment, the second-layer thickness  26  is greater than the first-layer thickness  16 . 
     As used herein, a structured layer is a layer that is not smooth or not planar on a microscopic scale corresponding to the magnitude of the indentations  80 . For example if the support  30  is planar, a structured layer formed on the support  30  according to the present invention is flat but non-planar and is not smooth. If the support  30  is not planar but is smooth, for example having a surface that is curved in one or more dimensions (such as a spherical section), a structured layer formed on the support  30  according to the present invention is not flat and is not smooth. Whether or not the support  30  is planar, the structured layer can include indentations  80 , channels, pits, holes, extended portions, mesas or other physical elements or structures. In one embodiment, the surface is rough. The depth  6  of the bi-layer  7  is the distance from an exposed surface of the portion of the bi-layer  7  furthest from the support  30  to an exposed surface of the portion of the bi-layer  7  that is closest to the support  30  in a direction that is orthogonal to a surface of the support  30 . 
     In an embodiment, the first cured layer  10  is located on or over the support  30 . The support  30  is any layer that is capable of supporting the first and second cured layers  10 ,  20  and in different embodiments is rigid, flexible, or transparent and, for example is a substrate made of glass, plastic, paper, or vinyl or combinations of such materials or other materials. In an embodiment, the first cured layer  10  is cross linked to the second cured layer  20  to provide rigidity and improved strength for the layers and to prevent delamination of the first cured layer  10  form the second cured layer  20 . 
     In a useful arrangement, the support  30  is adhered, for example with an adhesive layer  50  such as a pressure-sensitive adhesive or glue such as wall-paper glue, to the surface  8  of the structure  40 . The surface  8  is any surface  8 , planar or non-planar that is desired to resist the growth of biologically undesirable organisms, including microbes, bacteria, or fungi. In various applications, the structure  40  is a structure such as a wall, floor, table top, door, handle, cover, device, or any structure  40  having the surface  8  likely to come into contact with a human. The imprinted multi-layer structure  5  can form a wall paper or plastic wrap for structures  40 . 
     In a useful embodiment of the imprinted multi-layer structure  5  having the bi-layer  7 , the biocidal particles  60  include a silver component, have a sulfur or chlorine component, have a copper component, are a salt, are a silver sulfate salt, or are other biocidal particles  60 . In an embodiment, the first or second cured layers  10 ,  20  include phosphors. The biocidal particles  60  can have a distribution of sizes so that some of the biocidal particles  60  are large particles, for example from two microns to 20 microns, and other particles are small particles, for example from 100 nm to two microns. 
     By biocidal layer is meant herein any layer that resists the growth of undesirable biological organisms, including microbes, bacteria, or fungi or more generally, eukaryotes, prokaryotes, or viruses. In particular, the biocidal bi-layer  7  inhibits the growth, reproduction, or life of infectious micro-organisms that cause illness or death in humans or animals and especially antibiotic-resistant strains of bacteria. The bi-layer  7  is rendered biocidal by including biocidal particles  60  such as ionic metals or metal salts in the first cured layer  10 . Biocidal agents from the biocidal particles  60  can interact with any contaminants or biological organisms in the environment. The biocidal layer  7  or biocidal particle  60  is anti-microbial. 
     In other embodiments, the biocidal first cured layer  10  has a thickness that is less than at least one diameter of one or more of the biocidal particles  60 , has a thickness that is less than a mean diameter of the biocidal particles  60 , or has a thickness that is less than the median diameter of the biocidal particles  60 . Alternatively, the biocidal particles  60  have at least one diameter between 0.05 and 25 microns. In yet another arrangement, the second cured layer  20  is greater than or equal to 0.5 microns thick and less than or equal to 20 microns thick. 
     In yet another embodiment, the first or second cured layers  10 ,  20 , have a hydrophobic surface, for example by providing a roughened surface either by imprinting or by a treatment such as sandblasting or exposure to energetic gases or plasmas. 
     Referring to  FIGS. 2A to 2G  and  FIG. 4 , a method of the present invention includes making the imprinted bi-layer  7  on the support  30  ( FIG. 1 ) provided in step  100 . A dispersion of biocidal particles  60  is formed in step  120 , for example in a container  66  ( FIG. 2A ). The dispersion is coated over the support  30  to provide the first curable layer  13  in step  105  as shown in  FIG. 2B . In an alternative embodiment, an uncured layer including biocidal particles  60  is laminated on the support  30  to provide the first curable layer  13 . 
     In an embodiment, a dispersion of biocidal particles  60  is formed in a carrier such as a liquid, for example a curable resin, in the container  66  ( FIG. 2A ) in step  120  of  FIG. 4 . Making and coating liquids with dispersed particles is known in the art. A dispersion having biocidal particles  60  has been made. The dispersion included three-micron silver sulfate particles milled in an SU8 liquid to an average particle size of one micron, and successfully coated on glass and tested with  E. coli  and  S. aureus  bacteria. 
     In step  110  a second curable layer  23  is located over the first curable layer  13 , for example by coating, as illustrated in  FIG. 2C . In an alternative embodiment, the second curable layer  23  is made separately and laminated on or over the first curable layer  13  before the first curable layer  13  is cured. 
     The first curable layer  13  and the second curable layer  23  are formed in any of various ways, including extrusion or coating, for example spin coating, curtain coating, or hopper coating, or other methods known in the art. In other embodiments of the present invention, locating the first curable layer  13  includes laminating a first curable material on or over the support  30  or locating the second curable layer  23  includes laminating a second curable material on or over the first curable layer  13 . 
     Referring to  FIG. 2D , the first curable layer  13  and the second curable layer  23  are imprinted in a single step  125  with an imprinting stamp  90  having a structure with a structure depth  6  greater than the second layer thickness  26  of the second curable layer  23  ( FIG. 2E ) and then cured in a single step  130 , for example with heat or radiation  92  to form the first cured layer  10  and the second cured layer  20  ( FIG. 2F ). The imprinting stamp  90  is removed in step  135  to form an imprinted bi-layer  7  with a topographical structure having a depth  6  greater than the second-layer thickness  26  of the second cured layer  20  ( FIG. 2G ) to form the bi-layer  7  of the imprinted multi-layer structure  5  of the present invention. Imprinting methods using stamps  90  are known in the art. In an optional step  140 , a portion of the second cured layer  20  is removed, for example by etching or using energetic particles such as with plasma etching, reactive plasma etching, ion etching, or sandblasting the second cured layer  20 . Such a removal treatment can remove a portion of any coating over the biocidal particles  60  to more readily expose the biocidal particles  60  to the environment. The removal can render the second cured layer  20  thinner or expose portions of the first cured layer  10  or biocidal particles  60  to the environment. 
     The imprinted multi-layer structure  5  having the structured bi-layer  7  of the present invention has been constructed in a method of the present invention using cross-linkable materials such as curable resins (for example using SU8 at suitable viscosities and PEDOT) coated on a glass surface and imprinted using a PDMS stamp to form micro-structures in the bi-layer  7 . 
     Referring to  FIG. 3 , the imprinting step  125  forming the indentations  80  of the multi-layer structure  5  (shown in  FIGS. 2D and 2E ) can result in the movement of some biocidal particles  60  into the original second curable layer  27  so that the second-layer thickness  26  is reduced as indicated with the dashed lines illustrating the final second-layer thickness  28 . The second-layer thickness  26  is the portion of the second cured layer  20  between the biocidal particles  60  and the surface of the second cured layer  20  and can vary over the extent of the support  30 . 
     Referring further to  FIG. 4  in an embodiment of the present invention, the surface  8  of the structure  40  is identified in step  150 . The surface  8  is a surface which it is desired to keep free of microbes, for example a wall, floor, table top, door, handle, knob, cover, or device surface, especially any surface  8  found in any type of medical institution. In an embodiment, the surface  8  is planar; in another embodiment, the surface  8  is non-planar. In step  155 , an adhesive is located, for example on the surface  8  or on the side of the support  30  opposite the first cured layer  10 , to form the adhesive layer  50 . The support  30  is adhered to the surface  8  in step  160 . In a further embodiment, the support  30 , first cured layer  10 , and second cured layer  20  are heated to shrink the imprinted multi-layer structure  5  on the surface  8  if the surface  8  is non-planar. In an embodiment, the heating step (not shown separately) is also the adhesion step  160  and a separate adhesive layer  50  is not necessary or used. In an embodiment, the second cured layer  20  is thinner than the first cured layer  10 . 
     In an embodiment, the first cured layer  10  includes a first cross-linkable material, the second cured layer  20  includes a second cross-linkable material and the curing step  130  cross-links the first cross-linkable material to the second cross-linkable material. In another embodiment, the first material includes a first cross-linkable material and the second material includes a second cross-linkable material that is different from the first cross-linkable material and the curing step  130  cross-links the first cross-linkable material to the second cross-linkable material. Alternatively, the first material includes a first cross-linkable material, the second material includes a second cross-linkable material that is the same as the first cross-linkable material, and a third material is included in either the first material or the second material but not both the first and second materials and the curing step  130  cross-links the first cross-linkable material to the second cross-linkable material. 
     Referring to  FIG. 5 , in various embodiment of the present invention, the biocidal bi-layer  7  is located on the surface  8  in step  200  and observed or used over time in step  205 . Periodically or as needed, the imprinted multi-layer structure  5  is cleaned in step  210 , for example by washing with water or with a cleaning fluid, or wiping the multi-layer structure  5 . The imprinted multi-layer structure  5  is repeatedly observed or used (step  205 ) and cleaned (step  210 ) until it is no longer efficacious for its intended purpose. If the biocidal imprinted multi-layer structure  5  is determined (step  215 ) not to be efficacious, it is replaced, removed, or covered over in step  220 . 
     In an embodiment, the cleaning step removes dead micro-organisms or dirt from the surface of the bi-layer  7  so that the biocidal efficacy of the biocidal particles  60  is improved in the absence of the dead micro-organisms or dirt. Useful cleaners include hydrogen peroxide, for example 2% hydrogen peroxide, water, soap in water, or a citrus-based cleaner. In an embodiment, the 2% hydrogen peroxide solution is reactive to make oxygen radicals that improve the efficacy of biocidal particles  60 . In various embodiments, cleaning is accomplished by spraying the surface of the bi-layer  7  with a cleaner and then wiping or rubbing the surface. The cleaner can dissolve the second cured layer  20  material (e.g. cross linking material) and the wiping or rubbing can remove dissolved material or abrade the surface of the second cured layer  20  to expose other biocidal particles  60  or increase the exposed surface area of exposed biocidal particles  60 . 
     Alternatively, the cleaning or washing step  210  refreshes the biocidal particles  60 , for example by a chemical process, to improve their biocidal efficacy. This can be done, for example, by ionizing the biocidal particles  60 , by removing oxidation layers on the biocidal particles  60 , or by removing extraneous materials such as dust from the biocidal particles  60 . 
     Replacement of the bi-layer  7  can proceed in a variety of ways. In one embodiment, another biocidal imprinted multi-layer structure  5  is simply located over the biocidal imprinted multi-layer structure  5 . Thus, the biocidal multi-layer structure  5  becomes the structure  40  and another biocidal imprinted multi-layer structure  5  is applied to the structure  40 , for example with the adhesive layer  50  ( FIG. 1 ). In another embodiment, the biocidal imprinted multi-layer structure  5  is removed and another biocidal imprinted multi-layer structure  5  put in its place. As shown in  FIG. 1 , the support  30  is adhered to the structure  40  with the adhesive layer  50 . Chemical, mechanical, or heat treatments are applied to the biocidal multi-layer structure  5  to loosen, dissolve, or remove the adhesive layer  50  so the biocidal imprinted multi-layer structure  5  can be removed and another adhesive layer  50  applied to the structure  40  to adhere the biocidal imprinted multi-layer structure  5  to the structure  40 . In an embodiment, the biocidal imprinted multi-layer structure  5  is mechanically peeled from the structure  40  and another biocidal imprinted multi-layer structure  5  having the adhesive layer  50  is adhered to the structure  40 . 
     In another embodiment of the present invention, fluorescent or phosphorescent materials are included in the first cured layer  10  and are illuminated. The fluorescent or phosphorescent materials respond to ultra-violet, visible, or infrared illumination and emit light that can be seen or detected and compared to a threshold emission value. Thus, the continuing presence of the first cured layer  10  is observed. When light emission in response to illumination is no longer present at a desired level, the first cured layer  10  is replaced. 
     The present invention is useful in a wide variety of environments and on a wide variety of surfaces  8 , particularly surfaces  8  that are frequently handled by humans. The present invention can reduce the microbial load in an environment and is especially useful in medical facilities. 
     The invention has been described in detail with particular reference to certain embodiments thereof, but it will be understood that variations and modifications can be effected within the spirit and scope of the invention. 
     PARTS LIST 
     
         
           5  multi-layer structure 
           6  depth 
           7  bi-layer 
           8  surface 
           10  biocidal first cured layer 
           13  first curable layer 
           16  first-layer thickness 
           20  second layer/second cured layer 
           23  second curable layer 
           26  second-layer thickness 
           27  original second-layer thickness 
           28  final second-layer thickness 
           30  support 
           36  support thickness 
           40  structure 
           50  adhesive layer 
           60  biocidal particle 
           66  container 
           80  indentations 
           90  stamp 
           92  radiation 
           100  provide support step 
           105  locate first layer step 
           110  locate second layer step 
           120  form dispersion step 
           125  imprint first and second layers step 
           130  cure first and second layers step 
           135  remove stamp step 
           140  remove second layer portion step 
           150  identify surface step 
           155  locate adhesive step 
           160  adhere support to surface step 
           200  locate structure step 
           205  observe/use structure step 
           210  clean structure step 
           215  determination step 
           220  replace biocidal layer step