Nanoneedle surfaces fabricated by inverted-glancing angle deposition

Abstract

Present embodiments are directed to a novel technique for inverted Glancing Angle Deposition (I-GLAD) that employs a bottom-up physical vapor deposition process for creating three-dimensional nanofeature arrays. These fabricated nanostructures are referred to as nanoneedles, in which sheets comprising the nanoneedles possess antimicrobial properties in a cost-effective and scalable fashion compared to other systems which have attempted to recreate bio-inspired antipathogenic surfaces.

Description

NANONEEDLE SURFACES FABRICATED BYINVERTED-GLANCING ANGLE DEPOSITIONUNITED STATES PATENT AND TRADEMARK OFFICE AS RECEIVING OFFICEPCT INTERNATIONAL PATENT APPLICATIONSTATEMENT OF GOVERNMENT INTEREST

[0001] This work is supported by the National Science Foundation (ECCS-GRANT No. 2025075) and National Science Foundation Cooperative Agreement (No. 1849213).CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This international patent application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 467,434 filed May 18, 2023, the teachings and entire disclosure of which are fully incorporated herein by reference.FIELD OF INVENTION

[0003] Novel nanofabrication processes are disclosed herein for creating nanoneedle surfaces, along with surfaces formed by such processes, which may be used as antimicrobial (i.e., antibacterial and antifungal) surfaces, through inverted-glancing angle deposition CIGLAD).BACKGROUND

[0004] Antimicrobial resistance (AMR) has become a global health and development threat, with total death directly and indirectly related to bacterial infection worldwide estimated over 6 million in 2019. AMR comes from the overuse of antibiotics, which is an economic way to treat bacterial infection. The treatment of bacterial infection becomes more challenging once the bacteria developed resistance and become superbugs, such as Methicillin-resistantStaphylococcus aureus. Bacterial infections occur in hospitals, such as by catheters, ventilators and bio-implants, as non-limiting examples. Therefore, there is a need for developing new approaches to prevent or reduce bacterial infections in the clinical setting, while reducing the reliance on antibiotics.

[0005] Antimicrobial surfaces are divided into two broad categories according to their mechanisms of killing microbes. One involves a physical mechanism, and the other involves a chemical mechanism. In addition, antimicrobial surfaces can be either naturally occurring or artificially created.

[0006] Naturally occurring antimicrobial surfaces are found in nature, and include cicada wings and copper sheets, as non-limiting examples. Cicada wings and dragonfly wings provide examples of a physical antimicrobial mechanism, while copper sheets provide an example of a chemical antimicrobial mechanism; they represent the two basic mechanisms of antimicrobial surfaces for killing microbes. The chemical mechanism may involve surfaces with metallic coatings (such as silver, titanium, and copper) or organic compounds (such as poly(acrylic acid) and various peptides; for example arginine-tryptophan-rich antimicrobial peptides, or CWR1 1 are reported effective against microbes.

[0007] On the other hand, physical antimicrobial mechanisms using nanofeatures is a potential approach to kill bacteria. Besides cicada wings and dragonfly wings, other surfaces with nanofeature arrays occur in nature that kill bacteria. These include some insect eyes (e.g., moth) and plant leaves (lotus). Even so, their limited surface area makes such sources less ideal for practical antimicrobial, antibacterial, and antifungal applications.

[0008] Accordingly, one of the main challenges is whether one can design and fabricate larger area surfaces comprising nanofeature arrays for practical applications. For example, attempts to use three-dimensional nanofeature features on the naturally occurring surfaces. such as nanocones and nanopillars, would present challenges in that they require fine controland scalability of advanced nanomanufacturing processing. Further, three-dimensional features are challenging to achieve through traditional nanofabrication methods, especially top- down nanofabrication techniques. Some conventional top-down nanofabrication techniques, such as electron beam lithography, have been tried for fabricating synthetic antimicrobial surfaces, but such techniques require a trade-off between the feature size of the needle-like structures that provide physical antimicrobial properties versus scalability. On the other hand, bottom-up fabrication processes, such as nano-additive manufacturing using two-photon polymerization (2PP), are challenging for achieving sub- 10 nm features and, therefore, impractical.

[0009] One such technique is GLancing Angle Deposition (GLAD), which w orks from the principle that separated nanofeatures can be formed by ballistic shadowing effect with directional vapor depositions when the substrate is tilted and rotated during the deposition. As known to persons having ordinary skill in the art, various features can be obtained by GLAD, including nanopillars, nanosprings, nanochevrons, and porous structures inside membranes. As the needle-like structures form, the seed layer - whether natural or synthetic - establishes a growth pattern of these nanofeatures and ultimately lead to a distribution of surface needle-like structures. During GLAD, a substrate is maneuvered (tilted and rotated); natural or synthetic nucleation sites cast shadows due to the tilt of the substrate, prompting separate nanofeatures to form on the substrates instead of as a continuous film.

[0010] Notwithstanding any awareness of artificial antimicrobial surfaces that could be fabncated. problems have persisted. Because the features needed for antimicrobial and superhydrophobic surfaces require the base, or bottom, to be broader than the tips, and the tips to be much finer than the base, a nanostructure more resembling a needle is required that will mechanically puncture and disintegrate microorganisms such as bacteria. However, to the inventors' knowledge, no practical methodology has emerged yet to achieve this morphologyin any effective way to create these nanostructures at scale including conventional GLAD processes. Indeed, due to broadening effects during the GLAD growth phase, the nanostructures can become up to 10 times broader as they grow up, hardly resembling a needlelike structure.

[0011] Moreover, certain artificial surfaces fabricated as thin films (“TF”) are not sufficiently robust to exert the mechanical forces needed to stay intact and kill the bacteria, and challenges are presented with adhesion of such films to various substrates. Based on such challenges associated with conventional GLAD processes, there is a need to engineer larger area surfaces composed of the nanofeature arrays for practical uses.SUMMARY OF EMBODIMENTS

[0012] Present embodiments include nanoneedle sheets and methods of forming such nanoneedle sheets, which may include and / or be used as antipathogenic sheets (e.g., providing antimicrobial and antifungal properties). Such methods comprise growing needle-like nano structures, or nanoneedles as referred to herein. The nanoneedles have a base and a tip. The nanoneedles originate as a seed layer on a substrate, and as they are grown in accordance with embodiments described herein, the nanoneedles are loosely adhered at their tip upon a substrate. At least one capping layer is formed at the base of the nanoneedles. Then at least one capping layer imparts mechanical strength to the collection of nanoneedles formed, essentially making the nanoneedles perform as a unitary piece that can be separated from the substrate and transported as a single unit more easily. In some embodiments, a support film formed from polyimide, such as Kapton®, is applied over the at least one capping layer. In accordance with multiple embodiments and alternatives, except where expressly stated otherwise, a support film can be either a flexible support film or a rigid support film.

[0013] Accordingly, embodiments of the present application include sheets comprising nanoneedles, a support film, and at least one capping layer. Each nanoneedle comprises a baseand a tip, wherein the tip of each nanoneedle is located more distal to the at least one capping layer compared to the base (i.e., the base is more proximal to the at least one capping layer than the tip), and the at least one capping layer is positioned between the nanoneedles and support film. In some embodiments, the nanoneedles are arrayed three-dimensionally with at least one capping layer that contacts the support film. The nanostructure achieved with these novel sheets enables the nanoneedles to impart antimicrobial properties to surfaces over which the sheets are applied or placed. In some embodiments, the support film-comprising apparatus is formed in different dimensions and shapes, e.g., flat or curved, to match a variety of surfaces over which it is applied.

[0014] In addition, the present embodiments and alternatives provide methods of forming cost effective and scalable antimicrobial surfaces that are easily transported to be placed over large surfaces in need of antimicrobial protection. Accordingly, methods described herein create antimicrobial surfaces through glancing angle deposition (GLAD) plus an inversion step (or, I-GLAD) to create nanoneedle-containing sheets for delivering antimicrobial properties upon large surfaces.

[0015] Glancing angle deposition (GLAD) can be initially employed as a vapor deposition process for creating three-dimensional nanofeature arrays. One such process is electron beam (E-beam) evaporation performed in a vacuum chamber creating ultrahigh negative pressures. In some embodiments, an incident angle changes with time as tilt is modified. Tilt is a deviation in the direction of vapor incidence relative to the substrate. Various known techniques can be used to achieve a range of incident angles during the vapor deposition process, one of which involves positioning the substrate on a standard mount rotational / tilt that moves through a range of angles in all three dimensions. The incident angle is defined as the angle between the vapor and the substrate normal. For example, a 0° incident angle means the incident vapor is perpendicular to the surface of a substrate. The substrate is first tilted to anangle higher than or equal to 85° for a nominal thickness, e.g., approximately 15 kA, whereby a natural seed layer is formed on the substrate, as will be discussed in a later section in connection with Figure 1, panel (a). In some embodiments, the needle-like structures continue to grow in circumference and when the needle length exceeds 200 nm, the capping process begins by decreasing the substrate tilt to a lower incident angle, normally 60-70°, for example 65°, for 0.5 pm nominal thickness, and finally 0° for 0.5 pm nominal thickness.

[0016] In some embodiments, after GLAD, the sample is taken out of the vacuum chamber and placed inside a parylene coating system. In an exemplary embodiment, parylene C monomers of approximately 10 mg in weight was used as a coating layer over the base of the nanoneedles to obtain a thickness of approximately 24 pm as non-limiting ranges; the layer of parylene provides an advantage of a thicker capping layer, which imparts mechanical strength that helps the sheet containing the nanoneedles remain crack-free while increasing the hydrophilicity of the surface.

[0017] In some embodiments, the film is inverted with a suitably thick Kapton* tape as a support film; the use of this or similar material over the inverted nanoneedles provides additional mechanical strengthening to the layers. As discussed further herein, the antimicrobial property of sheets formed using I-GLAD are characterized by applying Gram negative bacteria (E. coll) and also Gram positive bacteria (5. aureus) tests, whereby the results show the I-GLAD surfaces are antimicrobial against both bacteria.BRIEF DESCRIPTION OF THE FIGURES

[0018] The drawings, schematics, figures, and descriptions herein are to be understood as illustrative of structures, features and aspects of the present embodiments and do not limit the scope of the embodiments. Where the figures provide or suggest dimensional information, the scope of the application is not limited to the precise arrangements, scales, or dimensions as shown in the drawings, nor as discussed in the textual descriptions.

[0019] Figure 1 comprises panels (a)-(f) and exhibits the fabrication flow of the surfaces with nanoneedles formed by present embodiments.

[0020] Figure 2 comprises panels (a)-(g) and shows the bacteria test process where two common bacteria of E. coli (Gram negative) and S. aureus (Gram positive) are tested using four samples, including an I-GLAD sample.

[0021] Figure 3 shows an illustrative SEM cross section image of exemplary7I-GLAD nanoneedles, with inset showing a magnified image of a portion of the nanoneedles.

[0022] Figure 4 shows an illustrative SEM image of I-GLAD nanoneedles and parylene capping layers, with inset showing a magnified portion of these layers denoted by the rectangle.

[0023] Figure 5A and Figure 5B plot the grow th curves for E. coli with optical density7(y- axis) versus test time (x-axis). The figure illustrates that E. coli did not grow on I-GLAD samples and copper sheets (positive control), but did grow on Kapton (negative control); it proves the I-GLAD sheets are antimicrobial. There is also E. coli growth on Ge and Ti thin films, indicating that both Ge and Ti do not naturally possess antimicrobial properties, and the antimicrobial properties of the I-GLAD samples are only dependent on the nanostructures on the surfaces (nanoneedles).

[0024] Figure 6 shows close up SEM side by side cross section images of I-GLAD nanoneedles before application (panel a) and after application (panel b) of E. coli over the antimicrobial sheets.

[0025] Figure 7A and Figure 7B plots the optical density (y-axis) versus test time (x-axis) for S. aureus, which shows similar results against S. aureus as E. coli. The figure illustrates that 5. aureus did not grow on I-GLAD samples as well as copper sheets (positive control), but did grow on Kapton (negative control); it proves the I-GLAD sheets are antimicrobial against S. aureus. There is also 5. aureus growth on Ge and Ti thin films, indicating that both Ge andTi do not naturally possess antimicrobial properties, and the antimicrobial properties of the I- GLAD samples are only dependent on the nanostructures on the surfaces (nanoneedles).

[0026] Figure 8 comprises panels (a)-(d) and shows four side by side images of S', aureus on I-GLAD surface with different magnifications to indicate the killing effect on bacteria of the I-GLAD nanoneedles via puncturing mechanism.

[0027] Figure 9A shows a percent coverage, p, of GLAD natural seeds versus an incident angle, a, with insets represented by Figures 9B-9D indicating specific points on the curve at various scales.

[0028] Figure 10 is a graph showing the mass (g) at which an inversion of a film was observed in relation to various incident angles of deposition.

[0029] Figure 11 comprises panels (a)-(d) and provides SEM images showing an amount of yeast cells found on various surfaces, as part of experimental testing described herein.

[0030] Figure 12 is a graph showing effects on Colony Forming Units of yeasts associated with various nanoneedle surfaces.

[0031] Figures 13A, 13B, 14A, and 14B depict cell counts of dead and alive cells that were extracted from nanoneedle surfaces following yeast contact with the surfaces for various times at various optical densities as described therein.MULTIPLE EMBODIMENTS AND ALTERNATIVES

[0032] Disclosed herein are nanoneedle sheets, which include, but are not limited to, antimicrobial sheets, imparting antimicrobial properties to surfaces over which they are applied. Also disclosed herein are methods of forming such nanoneedle sheets, including antimicrobial sheets. Although some of the embodiments discussed herein are described as “antimicrobial sheets’7and data reported in the Examples focuses on effects, it will be understood that the scope of multiple embodiments and alternatives herein includes nanoneedle sheets for use in other applications, some but not all of which are identified herein.

[0033] In some embodiments, the antimicrobial sheets comprise nanoneedles, a support film, and at least one capping layer. The capping layer may comprise the same material or a different material as the nanoneedles. The nanoneedles may be grown from a natural seed layer arrayed over a substrate, although the substrate forms no part of the finished antimicrobial sheets. Rather, each nanoneedle comprises a base 17 at a first end and a tip 19 at a second end, which are labeled in Figure 1, panel (f).

[0034] At a beginning stage of production of the antimicrobial sheets, the natural seed layer that grows into the nanoneedles is formed upon a substrate. At an intermediate stage of production, each nanoneedle will have grown so that the tip of each nanoneedle contacts the substrate while the base of each nanoneedle by comparison is located more distal to the substrate (at this intermediate stage).

[0035] At completion of the antimicrobial sheets, the substrate will have been separated from the nanoneedles, w hich advantageously leaves the fine tips of the nanoneedles exposed to the environment. Before separating from the substrate, however, the nanoneedles are capped, and the entire array has a support film applied over the cap. In this way, the overall structure is a sheet comprising nanoneedles, a support film, and at least one capping layer positioned between the nanoneedles and support film. In some embodiments, the support film can also be considered a capping layer.

[0036] In some embodiments, a natural seed layer is formed at the early stage of the GLAD process, and nanoneedles 13 are grown from this natural seed layer. In some embodiments, the nanoneedles are formed from one or more vacuum-deposited materials grown from a seed layer into nanoneedles, with such materials including but not limited to germanium (Ge), titanium (Ti), or silicon dioxide (SiCh). In some embodiments, the flexible support film is a polyimide, for example poly (4.4'-oxydiphenylene-pyromellitimide) known commercially asKapton®.

[0037] Likewise, in some embodiments, there is at least one capping layer comprised of two layers, one being a nanoneedle capping layer formed from the same material as the nanoneedles, and the other being a coating layer which increases mechanical strength of the nanoneedle array. In some embodiments, the first layer is the nanoneedle capping layer made from the same material as the nanoneedles after they are grown, e.g., germanium (Ge) or titanium (Ti), as well as copper (Cu), aluminum (Al), and silicon dioxide (SiCh). Also, in some embodiments, the second layer is a coating layer 16 comprising a pary lene, which optionally may be a parylene chosen from the parylene C, pary lene AF-4, parylene F, parylene N, and polyimide (e.g., PI 2600), to provide mechanical strength for the film.

[0038] Additional embodiments will now be described in relation to the nanofabrication of the antimicrobial sheets. Figure 1 illustrates an exemplary fabrication flow of an inventive antimicrobial sheet 8, in accordance with multiple embodiments and alternatives. In some respects, such an exemplary method can be thought of as tw o phases. Phase 1 of an exemplary method uses a GLAD (Glancing Angel Deposition) technique.

[0039] A natural seed layer 12 is formed over a substrate 10 at panel (a). Silicon wafers are an exemplary material for the substrate, but many others can be used provided such material allows suitable adherence of the seed layer to the substrate so the seed layer. The natural seeds are a nucleation site of the deposited material that randomly formed at the early stage of the e- beam evaporation process (Figure 1, Panel (a), 12). Once the early nucleation sites are formed, they cast shadows to create deposition-free areas around them when a high incident angle (usually over 75°) is used during the deposition, and the vapor are only grown on top of the nucleation sites (seeds). Instead of forming a continuous film, the film with separate nanoneedles are formed.

[0040] In this regard, it will be appreciated that a seed layer is formed over the substrate as an array. For ease of reference, only one seed in the seed layer in panel 1(a) is labeled, but itwill be understood that the reference numeral 12 applies to the entire seed layer depicted in the figure panel. Likewise, as grow th of the seed layer occurs, they become nanoneedles 13 as depicted in panels (b) - (f of Figure 1. Again, for ease of reference, in each of these panels, only one of the nanoneedles is labeled, but it will be understood that the reference numeral 13 applies to all other nanoneedles depicted in panels (b) - (1) of Figure 1.

[0041] Turning back to the seed layer, any material that can be deposited for e-beam evaporation and other physical vapor deposition techniques such as metal or metalloid, e.g., Ti or Ge, may be deposited on the substrate as represented by panel (a) of Figure 1, with the metal or metalloid serving as the seed layer that grows into nanoneedles under the growth conditions described herein.

[0042] Following deposition, a suitable form of Physical Vapor Deposition, for example an electron beam (E-beam) evaporator with ultrahigh vacuum (~1.0e‘6Torr) is applied to the seed layer. The application of GLAD proceeds at an incident angle, alpha (a), of 85° to being growing the seed layer into nanoneedles. The growth of the nanoneedles continues as the incident angle is kept substantially the same for another 1.5 pm nominal thickness of deposition. As shown in panel (b) of Figure 1 , the circumferences of the nanoneedles increases as the vapor deposition process continues.

[0043] At panel (c) of Figure 1, the length of the nanoneedles has achieved approximately 500 nm. This is a point at which it is suitable to begin the capping process for the purpose of achieving a nanoneedle surface for a particular purposes and applications, a non-limiting example being antimicrobial, antibacterial, and antifungal surfaces.

[0044] It at this point that a departure from conventional GLAD techniques begins to occur. In some embodiments provided for herein, a decrease in the incident angle from 85° to 65° is used to commence the capping process. The incident angle may be reduced to approximately65° which begins to induce the nanoneedles merge as they grow wider at the base end.Reducing the incident angle further, e.g. to approximately 0°, helps sen e to fill in voids, enhancing the capping process and providing further mechanical support to the nanoneedle array. In an exemplary fabrication, the incident angle is set at 65° for 0.5 pm nominal thickness and finally 0° for another 0.5 pm nominal thickness. All references to thickness or other dimensions involved in describing a fabrication are meant only as exemplary' and are not intended to limit the scope of the embodiments described and claimed. At this point, the additional thickness is seen at the base end of the nanoneedles, which forms a nanoneedle capping layer 14, best seen in panels (c) - (1) of Figure 1, which imparts a unitary feature to the aggregate number of nanoneedles. Accordingly, nanoneedle capping layer 14 is referenced in that way because it can be formed from the same material as the nanoneedles, e.g., Ge, Ti, or S1O2.

[0045] In some embodiments, following formation of the nanoneedle capping layer 14, a coating layer 16 is formed over the nanoneedle capping layer 14 as best seen in panel (d) of Figure 1. This coating layer 16 also is depicted in Figure 1, panels (e) - (f). Together with nanoneedle capping layer, coating layer 16 increases mechanical strength of the now-capped nanoneedles to augment the at least one capping layer referred to herein. Further, in some embodiments, coating layer 16 prepares the nanoneedles to be separated from substrate 10, a stage during an exemplary process in which the nanoneedles 13 are separated at their tip end from substrate 10. In Figure 1, this separation is what occurs between panel (e) and panel (f). Advantageously, heating helps with the separation of the nanoneedle structure tips and the Si wafer substrate, as one example. At panel (1), the nanostructure is in the form of a sheet, which is more easily transported for use in a variety of environments, for example hospital or food preparation facility.

[0046] In some embodiments, for example as represented by panels (d) - (1) of Figure 1. a parylene C monomers is used for the coating layer. In some embodiments, parylene Cmonomer adds a thickness of about 24 pm to the coating layer. As desired, application of the coating layer may be achieved through known techniques, for example SCS vapor deposition. Other coating layers besides pary lene C monomer may be employed to increase the thickness and mechanical strength of the nanostructure in preparation for the support fdm. These may be polymeric or monomeric. Besides pary lene and polyimide, other materials can be used for the coating depending on the intended application of the nanoneedle sheets. For example, other materials might include aluminum, copper, or gold if one desires to impart conductive properties to the nanoneedle sheets, to name some alternative materials.

[0047] At Figure 1, panel (e), a support film 18 is applied over the coating layer 16. Optionally, the support film 18 is a polyimide film such as a 2-mil-thick Kapton® tape layer. Because the continuous top layer of the nanostructure, including the coating layer 16, adheres more strongly to the Kapton® tape layer than the tips of the nanoneedles adhere to the Si substrate 10, the nanoneedles are removed relatively easily from the substrate as show in panel (f) of Figure 1.

[0048] Accordingly, and as depicted at Figure 1 , panel (f), there is a step to invert the entire nanostructure. This achieves separation of the tip 17 of the nanoneedles and results in the nanostructure being in the nature of a sheet comprising a support film 18, with at least one capping layer between the support film and the nanoneedles. In some embodiments, the at least one capping layer comprises nanoneedle capping layer 14. Moreover, the tips of the needles now are exposed to the environment, where the fine tips of the nanoneedles can make contact with or be contacted by microorganisms in the environment, which are punctured and disintegrated when landing on the fine, nanoneedle tips. As shown in Figure 1, panel (1). the nanoneedle structure is separated from the substrate, and the process is referred to as “inverted” glancing angle deposition or, “I-GLAD.” In this configuration, the antimicrobial sheets that are formed are portable and able be applied to a variety of surfaces, including flat and curvedsurfaces. Figure 1, therefore, is illustrative of an effective, scalable way to create a variety' of nanoneedle surfaces with a broad range of applications, including but not limited to providing antimicrobial, antibacterial, and antifungal surfaces. By allowing the seeds in the seed layer to grow with a smaller diameter at the substrate than the broader diameter moving away from the substrate, once inversion occurs it achieves a needle structure with a fine tip end, where the tip end is exposed to the environment and otherwise unencumbered by the substrate.

[0049] All examples provided herein are meant as illustrative of various aspects of multiple embodiments and alternatives of any or all of inventive sheets comprising nanoneedles, at least one capping layer, and a support film, which may be obtained using an inverted glancing angle deposition process. These examples are non-limiting and merely characteristic of multiple alternative embodiments herein.Example 1 - Testing of antimicrobial sheets against E. coli and S. aureus

[0050] To compare the performance of antimicrobial sheets formed by the methods provided for herein, a control having no antimicrobial properties, namely LB deposited on a glass slide, also was analyzed. Escherichia coli (E. coli), a common Gram-negative bacterium, and Staphylococcus aureus (S. aureus) a common Gram-positive bacterium, were used on all antimicrobial sheets tested. E. coli strain BL21 (Thermo Fisher) was grown from glycerol stock (stored in -80 °C) by inoculating Lysogeny Broth (LB) at 37 °C for 24 h until growth is saturated in LB. S. aureus was also grown in similar conditions.

[0051] After 24 hours, the E. coli and S. aureus in LB were diluted to an optical density of 0.1. The bacteria were then aliquoted onto the deposited samples including the antibacterial sheets, and positive and negative controls. These samples with bacterium were then incubated at 37 °C for 4 h. After the incubation period, the bacteria were harvested using 10 microliters of LB from the surfaces they were grown on.

[0052] Growth curves were determined using aliquots of the harvested bacteria placed in a 96-well optical density plate (Greiner Bio-One) for spectroscopy. In parallel, 300 pL of LB were placed into each well in triplicate to each harvested E. colHS. aureus sample. The 96-well plate also had LB blanks present for calibration. The spectrophotometer (SpectraMax M2, Molecular Devices) was programmed to maintain a temperature of 37 °C for 20 h, to shake the plate for 5 s before a reading took place, to read each well at 600 nm wavelength, and to read every' 30 min. After 20 h, the raw data was analyzed by averaging the triplicate data, and the growth curves of the E. coli / S. aureus were obtained.

[0053] Figure 2 provides a schematic of the antimicrobial test process specific to E. coli (Gram negative) and also applicable for testing of S. aureus (Gram positive) pathogens which were tested. These pathogens were tested against four samples including a sample formed with the LGLAD technique. The pathogens in LB (Figure 2, panel a) were separated into aliquots (panel b) and placed on surfaces to form samples (panel c), each of which was washed using pure LB for sterilization before application of the bacteria. The bacteria-containing samples were then incubated (panel d), harvested (panel e), and placed in wells (panel f). Microbial content was measured by changes in incoming light compared to outgoing light passing through the samples (panel g) using known techniques, and the bacterial grow th curves were plotted as seen in this panel. In connection with the present Example, Figure 3 then provides an illustrative SEM cross section image of I-GLAD nanoneedles comprising the surfaces that w ere contacted by the samples. Similarly, Figure 4 provides an illustrative SEM image of I-GLAD nanoneedles and further illustrates the nanoneedle and parylene capping layers. Returning to Figure 2. panel (h). the growth patterns of E. coli are differentiated for the respective samples. The lower curve represents the surfaces in a bacteria-free state following washing with LB, while the upper and middle curves reflect the levels of growth for control and nanoneedlecontaining surfaces, respectively.

[0054] To further understand the growth parameters of E. coli used in these studies. Figure 5A and Figure 5B plot the optical density versus test time for E. coli. The LB media used for cultivating the bacteria is proved to be sterile before use (black line with star symbols). All sample surfaces are also tested using pure LB for sterilization before the application of the bacteria, and the green curves in the figures are reflecting the surfaces are bacteria-free. The growth patterns of E. coli are differentiated on the samples, as the red curves indicated in the figures. Figures 5 A and 5B show the level of optical density, which is associated with increased antibacterial properties in accordance with the examples, increases with each step of the I- GLAD process. With the grow th of the nanoneedle structure (I-GLAD curves), the capping layer added to the base of the needles (Ge TF curves), and the addition of a polyimide film, such as Kapton® (Kapton curves), increased antibacterial properties are achieved.

[0055] Additionally, Figure 6 shows close up SEM before (panel a) and after (panel b) results in cross section images of antimicrobial killing, with the use of nanoneedles formed by the inventive methods described herein. These nanoneedles comprise the structures shown in panel (f) of Figure 1, and the nanoneedles themselves were formed from Ge.

[0056] Similar to Figures 5 A and 5B, Figure 7 A and Figure 7B plot the optical density (y- axis) versus test time (x-axis) for A aureus, which again shows similar results against S. aureus as E. coli. The results of these studies indicate that I-GLAD samples show ed no growth of S. aureus for Ge or Ti, while growth of S. aureus is observed on the corresponding continuous films (Ge, TF and Ti TF). Accordingly, the data indicate that I-GLAD samples show antibacterial behavior for both Gram positive and Gram negative bacteria.

[0057] Figure 8 is separated into four panels representing A. aureus impacted by an antimicrobial surface fabricated as nanoneedles, using methods provided herein, at different magnifications. Panel (b) of Figure 8 shows the bacteria that were punctured (with tips of the nanoneedles through the bodies) and disintegrated (decomposed). Last, Figure 8, panel (d)shows biofilm layer (cytoplasm) at the base of the bacteria, the cytoplasm shows the bacteria is punctured through.

[0058] In view of the above, antibacterial surfaces created with I-GLAD are effective against Gram negative and Gram positive bacteria. These work by mechanically puncturing and disintegrating the pathogens. In this way, then, the approaches disclosed herein are not prone to common downsides associated with AMR wherein the pathogen may mutate and become resistant to chemical mechanisms of killing the bacteria using chemical antibiotics. Moreover, the methods and apparatuses described herein provide an ability to effectively, economically and practically fabricate larger area surfaces composed of the nanoneedle arrays for these beneficial uses. It also will be noted that, while Cicada wings w ere the first naturally occurring structures to confer biocidal activity based solely on physical surface morphology, they have been found ineffective against Gram positive bacteria. Accordingly, in addition to the present embodiments being more scalable than animal and insect structures to cover large areas where antimicrobial surfaces will provide great benefit, the present embodiments are effective against a wider range of bacterium than are Cicada w ings, as just one example. These and other advantages and benefits are to be gained by practicing the present embodiments alternatives, as provided for herein.Example 2 - Nanoneedle porosity

[0059] The inverting process combined with glancing angle deposition was tested to evaluate the porosity of the nanoneedles. As Figure 9 shows, the percent coverage, p, of GLAD natural seeds depends on the incident angle, a, whereby higher a results in lower percent coverage p on the substrate. Accordingly. Figure 9 shows the dependence of percent coverage p of GLAD natural seeds on the incident angle a from both theory (line) and experiment(scatters), at a scale bar of 500 nm.

[0060] An experimental setup was used to further evaluate the properties of nanoneedles formed according to present embodiments. The tests used samples of nanoneedle-containing substrates cut into rectangular shapes (2 cm x 6 cm). 2-mil-thick Kapton tapes were added over the samples, and the samples were placed into a rectangular support with the tape layer facing down, and with a longer portion of the tape not covering the samples partially suspended with a support clamp similar to a pulley, where a length of the tape was allowed to dangle over the far side of the support clamp, terminating in a free end. A clip w as added to the free end of the tape, allowing weights to be applied to the free end of the dangling tape. Accordingly, weights were applied, and the effects were observed in terms of achieving inversion of the film.

[0061] Figure 10 show-s the results of this test by graphing the mass (g) at which inversion of the film (which facilitates the creation of isolated nanoneedles) was observed with 4 samples for each incident angle (amax=75°, 77°, 79°, 81°, 83°, 85°). Accordingly, utilizing specific incident angles, the I-GLAD films will invert as shown in Figure 1. Experimentally, it was observed that for incident angles (amax) that were much lower than 79°, the I-GLAD films did not invert, so in some embodiments a first incident angle will be in a range from about 79° - 89°.

[0062] In some embodiments, following the first incident angle and inversion, the nanoneedles are formed into a final configuration through additional depositions at progressively smaller incident angles. For example, in some embodiments a second incident angle may be employed at an angle between about 60° - 65° and optionally a third incident angle between 0° - 30°.Example 3 - Antifungal properties - Colony Forming Units

[0063] An antifungal effect of the I-GLAD surfaces was experimentally verified. In one aspect. Figure 11 provides SEM images showing an amount of yeast cells on I-GLAD surfaces. at different scales after inoculation of the surfaces. A scale bar in panel (a) of this figure isused to indicate 10 pm. In turn, a scale bar is used in panels (b) and (d), respectively, to indicate greater magnification at 4 pm, and the scale bar I panel (c) indicates 5 pm. A region, 110, indicated in panel (a) is depicted in panel (b). In turn, a region, 111, also indicated in panel (a) is depicted in panel (c), with panel (d) showing the surface at a greater magnification than panel (a). A hemocytometer was used following normal procedure for stained cells. The number of cells counted were normalized to a volume of 0.01 mL, which was the amount administered to each surface. The surfaces were inoculated for 24 hours to an optical density7of 0.5 (optical density7units), compared to yeast not diluted typically at about 1.5.

[0064] Figure 12 shows the effects on Colony Forming Units associated with various surfaces following this 24-hour interaction. From left to right along the x-axis, the controls are referred to as Tape, GeO, and TiO, and tested against samples of Ge85 and Ti85.Example 4 - Additional testing of Antifungal properties

[0065] Figures 13A- 14B depict cell counts of dead and alive cells that were extracted from the different control and experimental nanoneedle surfaces following yeast contact with the surfaces for either 24 hours or 4 hours. The yeast Saccharomyces cerevisiae was grown in a peptone dextrose broth solution at 30 C for 24 hours. The yeast was aliquoted during the growth phase onto the sample surface. Methylene blue staining was used for this assay, and the yeast dilution aliquoted onto the surfaces were at two different dilutions, one with an optical density of about 2.0 (undiluted yeast, UDY) and the other with an optical density' of 0.5 (diluted yeast, DY). The controls were Tape, TiO, and GeO, and these were tested against Ti85 and Ge85 with live cell and dead cell counts depicted in the figures. On each of Figures 13A-14B. the y-axis indicates the cell counts for the 5 samples (3 control samples and 2 inventive surfaces) shown across the x-axis. For the controls, a higher content of the yeast cells on the control surfaces survived than on inventive surfaces, Ti85 and Ge85. The latter showed a dramatic increase in cell death with lower numbers, and in some cases very few, survivingcontact with the surface. Table 1, below, summarizes the parameters indicated by the graphs shown in Figures 13A-14B.Table 1

[0066] More specifically, Figures 13A-14B depict cell counts of dead and alive cells that were extracted from the different control (i.e., Tape, TiO, and GeO) and inventive (Ti85 and Ge85) surfaces after contact with the surface for the contact periods noted (4 hours or 24 hours) with the yeast dilution aliquoted onto the surfaces having an optical density of either 2.0 or 0.5.

[0067] The examples discussed above are in a context of antibacterial and antifungal properties of nanoneedle surfaces, i.e., nanoneedles arrays, formed in accordance with present embodiments. While specific examples have been provided, these have many applications. As mentioned herein, they may be used to physically kill bacteria, fungi, viruses, or other pathogens by mechanical puncturing. These surfaces and arrays may find uses in hospitals and other settings that seek or require antipathogenic surfaces. Alternatively, they may be used for drug delivery' applications where the needles penetrate a biological layer, such as skin or a cell wall; and alternatively in optical applications, where the cone shape of the needles results in an effective refractive index that varies as a function of depth; and alternatively in electrical applications, where the sharp tip concentrates the electric field, such as for field-emission tips; and alternatively in applications where the increase of surface area is beneficial, such as the anodes and cathodes of batteries and capacitors; and alternatively in changing a contact angleof surfaces, making the surface more hydrophobic or more hydrophilic: and alternatively as electrodes that penetrate cell walls to measure intracellular properties, to name some relevant applications.

[0068] It will be understood that the embodiments described herein are not limited in their application to the details of the teachings and descriptions set forth, or as illustrated in the accompanying figures. Rather, it will be understood that the present embodiments and alternatives, as described and claimed herein, are capable of being practiced or carried out in various ways. Also, it is to be understood that words and phrases used herein are for the purpose of description and should not be regarded as limiting. The use herein of such words and phrases as “such as,” “comprising,” “e.g.,” “containing,” or “having” and variations of those words is meant to encompass the items listed thereafter, and equivalents of those, as well as additional items. The use of “including” (or, “include,” etc.) should be interpreted as “including but not limited to.”

[0069] Accordingly, the foregoing descriptions of several embodiments and alternatives are meant to illustrate, rather than to sen e as limits on the scope of what has been disclosed herein. It will be understood by those having ordinary skill in the art that modifications and variations of these embodiments are reasonably possible in light of the above teachings and descriptions.

Claims

CLAIMSWhat is claimed is:

1. An antipathogenic sheet, comprising: nanoneedles and at least one capping layer; wherein each nanoneedle comprises a base and a tip; and wherein the base of each nanoneedle is located more proximal to the at least one capping layer than is the tip.

2. The antipathogenic sheet of claim 1, wherein the nanoneedles are formed from at least one of Ge, Ti, or SiCh, and are grown from a natural seed layer into nanoneedles.

3. The antipathogenic sheet of claim 1, wherein the nanoneedles are formed from a vacuum-deposited material grown from a seed layer into nanoneedles.

4. The antipathogenic sheet of claim 1, further comprising one or both of a coating layer and a support film, wherein the at least one capping layer is positioned more proximal to the tip of the nanoneedles than is the coating layer or the support film.

5. The antipathogenic sheet of claim 4, wherein the support film is a flexible support film and comprises polyimide or parylene.

6. The antipathogenic sheet of claim 1, wherein the at least one capping layer comprises a nanoneedle capping layer made from the same material as the nanoneedles, and further comprising a coating layer.

7. The antipathogenic sheet of claim 1, wherein the at least one capping layer comprises a nanoneedle capping layer made from a different material as the nanoneedles, and further comprising a coating layer.

8. The antipathogenic sheet of claim 6, wherein the coating layer is a flexible material comprising one or more of parylene C, parylene AF-4, parylene F, parylene N, and polyimide.

9. The antipathogenic sheet of claim 7, wherein the coating layer is a flexible material comprising one or more of parylene C, parylene AF-4, parylene F, parylene N, and polyimide.

10. The antipathogenic sheet of claim 1, wherein the sheet is capable of killing bacteria, fungi, and other pathogens.

11. A method for producing a nanoneedle sheet, comprising: forming a GLAD layer by: forming a seed layer on a substrate, growing the seed layer into nanoneedles using an energy source directed at the substrate according to a first incident angle in a range of about 79° to about 89°, initiating a nanoneedle capping process resulting in a nanoneedle capping layer, wherein the nanoneedle capping process is initiated by reducing the incident angle to a second incident angle in a range of about 60° to about 70°, and completing the nanoneedle capping process by reducing the incident angle to a third incident angle below the range of the second incident angle; applying a coating layer over the nanoneedles and applying a support film over the coating layer; and forming an I-GLAD layer by removing and inverting the nanoneedles from the substrate.

12. The method of claim 11, wherein the second incident angle is 60°-65°.

13. The method of claim 11 , wherein the third incident angle is 0° - 30°.

14. The method of claim 1 1, wherein the nanoneedle sheet is for killing bacteria, fungi and other pathogens.

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