WO2010132610A2 - Thermoplastic substrates with wrinkled metallic surfaces for chemical and biological sensing - Google Patents

Abstract

Provided are sensors for detecting chemical and biological agents. The sensors include a heat-shrunk thermoplastic substrate and a film of metal disposed over the surface of the substrate, wherein the film of metal comprises a microstructure characterized by wrinkle-like features. Also provided are methods for making the sensors and methods for detecting chemical and biological agents with the sensors using backscattering spectrometry.

Description

THERMOPLASTIC SUBSTRATES WITH WRINKLED METALLIC SURFACES FOR CHEMICAL AND BIOLOGICAL SENSING

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U. S. C. § 119(e) of U.S. Provisional Serial No. 61/177,952, filed May 13, 2009, the content of which is incorporated by reference in its entirety.

BACKGROUND

[0002] Detection and discernment of nucleic acids, proteins, and other biologically relevant small molecules are of critical diagnostic importance [I]. The ability to identify such with high sensitivity enables accurate pathogen identification for environmental protection, food safety, and early disease diagnosis [2]. It allows also for better and faster response to bio-terrorism threats. Most molecular identification methods currently rely on fluorescence readouts in which fluorophores are coupled to specific biomolecules of interest for detection [3].

[0003] As the unique blueprint to every organism, nucleic acid sequences are important molecular signatures. The polymerase chain reaction (PCR) coupled with molecular fluorophore readouts enables rapid nucleic acid sequencing for high-sensitivity molecular identification [4]. However, the PCR is complex, costly, and sensitive to contamination. Moreover, it is limited in its ability to multiplex multiple targets [5].

[0004] Abnormal protein levels reflect infections and diseases. The gold standard for protein analysis is the fluorescent based enzyme-linked immunosorbent assay (ELISA), with detection limits typically in the picomolar range [5]. Higher sensitivity enables protein markers of infectious diseases and cancers to be detected earlier, at lower concentrations. Earlier diagnosis enables more effective treatment and therefore potentially higher patient survival rates [I]. Despite their exquisite sensitivity and prevalence in molecular detection, fluorophores have significant drawbacks, including photo-bleaching, broad absorption/emission bands, and dependence on expensive excitation and detection equipment. Moreover, fluorescence based labeling and detection typically requires multiple steps [6].

[0005] Label-free approaches are more adaptable to point-of care diagnostics, in which rapid, low-cost, low-powered, portable, and robust systems are required. This is particularly important for first responders of bio-terrorist threats as well as diagnostics for the developing world. Affinity bio-sensors allow for the real-time analysis of biospecific interactions without the need for labeling molecules. Various optical methods for label-free biomolecular detection have been explored. Plasmonics involves manipulating light in the subwavelength regime. Nano-structured free-electron metals can be resonantly excited using visible light to produce surface plasmon oscillations that lead to surface-bound electromagnetic fields; these fields can then be manipulated in various ways to detect bio- molecules [2,7,8]. For example, in surface plasmon resonance sensing, molecular adsorption can be detected through changes in the refractive index.

[0006] Surface plasmon excitations which leverage subwavelength field localization can also be used for enhanced fluorescence spectroscopy (metal-enhanced fluorescence) or label-free spectroscopy, such as surface-enhanced Raman spectroscopy (SERS) based on more efficient inelastic scattering of light by a molecule in proximity to nanostructured metals. The SERS provides chemical bond information and is considerably more sensitive (down to single molecule sensitivity) than either refractometric or colormetric assays [9]. While one of the best label-free approaches, the challenge with the SERS lies in the tradeoff between reliability (with structures made from surface roughening or colloids) and manufacturability (with structures requiring high cost ion beam or electron beam nano- fabrication approaches) [10,11].

[0007] Surface Plasmon Resonance (SPR) may be used to measure the adsorption of chemical and biological agents on dielectric substrates coated with a layer of metal. See

Maier, S.A., et al. (2005) J Appl Phys 98: 1-10. Surface plasmons, surface electromagnetic waves that propagate in a direction parallel to the metal/dielectric or metal/air interface, may be excited by illuminating metal coated dielectric substrate under the appropriate conditions. Chemical and biological agents on the substrate surfaces may be detected by SPR reflectivity measurements. SPR may also be used to enhance spectroscopy measurements such as fluorescence (when the agents are fluorescently labeled) and Raman scattering. Such techniques are known as metal-enhanced fluorescence (MEF) and surface- enhanced Raman scattering (SERS), respectively. See Xu, H., et al. (1999) Phys. Rev. Lett. 83: 4357-4360; Shuming N., et al. (1997) Science 275: 1102-1106; Song, J.H., et al. (2005) Nano Lett. 5: 1557-1561. However, these and other SPR experimental set-ups may be complex and costly, involving a considerable amount of instrumentation. Similarly, the production of substrates capable of supporting surface plasmons are labor-intensive and expensive. Moreover, for MEF, fluorescent labeling of agents can be expensive and can involve excessive preparation. In addition, chemical and biological agents in the natural environment are generally not fluorescently labeled and may not be inherently fluorescent. Therefore, there is a need for simple and low cost sensors that are capable of detecting unlabeled chemical and biological agents.

SUMMARY OF THE INVENTION

[0008] Provided herein are sensors, methods for making the sensors, and methods for using the sensors to detect and identify chemical and biological agents. The sensors comprise a heat-shrunk thermoplastic substrate coated with a film of metal having a rough surface. Specifically, the film of metal comprises a microstructure characterized by wrinkle-like features. The sensors may be used to detect chemical and biological agents, even those that are not fluorescently labeled, using backscattering spectrometry. By contrast to conventional SPR sensors, the disclosed sensors may be made using ultra-rapid processes and low cost, readily available materials. Moreover, the optical properties of the sensors may be tuned to correspond to the optical properties of the chemical and biological agents of interest, greatly increasing the accuracy and sensitivity of the sensors. Finally, compared to conventional SPR techniques, backscattering spectrometry is simple and requires only minimal instrumentation.

[0009] In one aspect, sensors are provided. The sensors may include a heat-shrunk thermoplastic substrate and a film of metal disposed over the surface of the substrate, wherein the film of metal includes a microstructure characterized by wrinkle-like features. The wrinkle-like features may be folded, wrinkle-like features or cracked, wrinkle-like features, both of which are further described below. The orientation of the wrinkle-like features across the surface of the substrates may vary, including both random and substantially parallel orientations. Similarly, the dimensions, including the height and spacing, of the wrinkle-like features may vary.

[0010] The composition of the thermoplastic substrates and the metallic film may vary. A variety of thermoplastic materials may be used, including, but not limited to, polystyrene. In the disclosed sensors, the thermoplastic substrate is heat shrunk, which means the thermoplastic substrate has been exposed to heat, resulting in a reduction of size of the substrate. The metallic film may comprise a variety of metals or combinations of metals, including, but not limited to silver, gold, and copper. The film may include single layers or multiple layers of metals. Similarly, the thicknesses of the metal layers may vary.

[0011] In another aspect, methods for making the sensors are provided. The methods involve depositing a film of metal over the surface of a thermoplastic substrate and shrinking the coated substrate. Wrinkles form in the metallic film due to the stiffness incompatibility between the metallic film and the thermoplastic substrate. As further described below, the characteristics of the wrinkles may be controlled by the various parameters of the heating process and thickness of the deposited metal film.

[0012] In yet another aspect, methods for using the sensors to detect chemical and biological agents using backscattering spectrometry are provided. The methods involve exposing any of the disclosed sensors to a chemical or biological agent; exposing the sensor to light, and measuring the backscattered light from the sensor to detect the chemical or biological agent. Backscattering spectrometry, and the information that the technique provides, is further described below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIGS. 1A-1C show images of exemplary sensors with metallic films having folded, wrinkle-like features.

[0014] FIGS. 2A-2B show images of exemplary sensors metallic films have cracked, wrinkle-like features.

[0015] FIG. 3 illustrates the process for fabricating the nanostructured metallic substrates of the present disclosure. A shape memory polymer is coated with a thin film of metal. Upon heating, the polymer retracts, but the metal does not lead to a buckling of the metal surface. The final image on the right shows a scanning electron micrograph of one such nano-structured metallic substrate fabricated using this method.

[0016] FIG. 4 is a sketch illustrating the physical problem. A wave is incident on several obstacles situated over a rough surface. The rough surface is given by the function z=βx, y). Light scatters from the obstacles and the rough surface.

[0017] FIG. 5 is a diagram showing the interactions between the point obstacle and the slightly rough surface given by Eq. (5.3). In the first diagram, the incident field and the incident field reflected by the rough surface excite the point obstacle. Next, that exciting field is scattered by the point obstacle and reflected by the rough surface to excite the point obstacle again. This series continues to include infinitely many interactions between the point obstacle and the slightly rough surface.

[0018] FIG. 6 is a plot of the rough surface and point obstacle shown on the y=0 plane. The point obstacle is located at position T₁ = (11.7, 0.0, 0.1) in units of wavelengths.

[0019] FIG. 7 are contour plots of the image /(x,y) defined in Eq. (5.5) corresponding to a single point obstacle shown in FIG. 6 for the Dirichlet (top) and Neumann (bottom) cases.

[0020] FIG. 8 is a plot of the rough surface and two point obstacles shown on the y=0 plane. The point obstacles are located at positions T₁=(11.7, 0.0, 0.1) and r₂ ⁼ (9.7, 0.0, 0.1) in units of wavelengths.

[0021] FIG. 9 are contour plots of the image /(x,y) defined in Eq. (5.5) corresponding to two point obstacles shown in FIG. 8 for the Dirichlet (top) and Neumann (bottom) cases.

[0022] FIG. 10 illustrates the process of fabrication of nanowrinkles. a) Scheme of fabrication of biaxial (left) and uniaxial (right) wrinkles. b,c) SEM images of biaxial (b) and uniaxial (c) wrinkles on shrunk polystyrene sheets covered with a 10 nm thick layer of gold.

[0023] FIG. 11 are demonstrates characterization of nanowrinkles. a) SEM image of biaxial wrinkles with IOnm thick gold. Inset: Its 2D FFT pattern, b) Wavelength distributions of biaxial wrinkles with various thickness of gold, c) Main panel: SEM image of uniaxial wrinkles with IOnm thick gold. Inset: Its 2D FFT pattern. Bottom panel: Cross- sectional SEM view, d) Wavelength distributions of uniaxial wrinkles with various thickness of gold, e) Plot of main wavelength of biaxial (black triangles) and uniaxial (red circles and blue squares) wrinkles as a function of gold layer thickness. The dashed lines show anticipated slopes from the theory. Note that " 1 " and "2" in (c), (d), and (e) indicate the first and the second populations of uniaxial wrinkle wavelengths.

[0024] FIG. 12 shows illustrated utilities of nano wrinkles, a) Scheme of fabrication of uniaxial wrinkles inside a Shrinky-Dinks-based microchannel. b) SEM image of uniaxial wrinkles, with 45nm thick silver layer, inside a channel. Right panel: Enlarged view of outlined area, c) Wide-field epifluorescence images of dyes on a glass plate (top) and on uniaxial wrinkles (bottom), d) Top: The corresponding intensity profiles along the arrows in (c). Bottom: Fluorescence lifetime measurements of dyes on a glass plate (blue) and on wrinkles (red). The black lines show that each time trace can be well fitted with exponential decays, e) SEM image of discrete "wrinkled flowers".

DETAILED DESCRIPTION

[0025] Provided herein are sensors, methods for making the sensors, and methods for using the sensors to detect and identify chemical and biological agents.

Definitions

[0026] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude others. "Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination when used for the intended purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants or inert carriers. "Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for preparing the micro fluidic device. Embodiments defined by each of these transition terms are within the scope of this invention.

[0027] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 0.1. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term "about". It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0028] As used in the specification and claims, the singular form "a," "an" and "the" include plural references unless the context clearly dictates otherwise.

[0029] A "thermoplastic material" is intended to mean a plastic material which shrinks upon heating. In one aspect, the thermoplastic materials are those which shrink uniformly without distortion. A "Shrinky-Dink" is a commercial thermoplastic which is used a childrens toy. The shrinking can be either bi-axially (isotropic) or uni-axial (anisotropic) and can be un-iaxially or bi-axially stressed prior to further shrinkage. Suitable thermoplastic materials for inclusion in the methods of this invention include, for example, high molecular weight polymers such as acrylonitrile butadiene styrene (ABS), acrylic, celluloid, cellulose acetate, ethylene-vinyl acetate (EVA), ethylene vinyl alcohol (EVAL), fluoroplastics (PTFEs, including FEP, PFA, CTFE, ECTFE, ETFE), ionomers kydex, a trademarked acrylic/PVC alloy, liquid crystal polymer (LCP), polyacetal (POM or Acetal), polyacrylates (Acrylic), polyacrylonitrile (PAN or Acrylonitrile), polyamide (PA or Nylon), polyamide-imide (PAI), polyaryletherketone (PAEK or Ketone), polybutadiene (PBD), polybutylene (PB), polybutylene terephthalate (PBT), polyethylene terephthalate (PET), Polycyclohexylene Dimethylene Terephthalate (PCT), polycarbonate (PC), polyhydroxyalkanoates (PHAs), polyketone (PK), polyester polyethylene (PE), polyetheretherketone (PEEK), polyetherimide (PEI), polyethersulfone (PES), polysulfone polyethylenechlorinates (PEC), polyimide (PI), polylactic acid (PLA), polymethylpentene (PMP), polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polyphthalamide (PPA), polypropylene (PP), polystyrene (PS), polysulfone (PSU), polyvinyl chloride (PVC), polyvinylidene chloride (PVDC) and spectralon.

[0030] A "metal" for use in this invention includes but is not limited to platinum, gold, titanium, silver, copper, a dielectric substance, a paste or any other suitable metal or combination thereof. Examples of suitable dielectric substances include metal oxides, such as aluminum oxide, titanium dioxide and silicon dioxide. Examples of suitable pastes include conductive pastes such as silver pastes. [0031] The metal can be applied to the thermoplastic material by a variety of methods known to one skilled in the art, such as printing, sputtering and evaporating. The term "evaporating" is intended to mean thermal evaporation, which is a physical vapor deposition method to deposit a thin film of metal on the surface of a substrate. By heating a metal in a vacuum chamber to a hot enough temperature, the vapor pressure of the metal becomes significant and the metal evaporated. It recondenses on the target substrate. As used herein, the term "sputtering" is intended to mean a physical vapor deposition method where atoms in the target material are ejected into the gas phase by high-energy ions and then land on the substrate to create the thin film of metal. Such methods are well known in the art (Bowden et al. (1998) Nature (London) 393: 146-149; Bowden et al. (1999) Appl. Phys. Lett. 75:

2557-2559; Yoo et al. (2002) Adv. Mater. 14: 1383-1387; Huck et al. (2000) Langmuir 16: 3497-3501; Watanabe et al. (2004) J. Polym. Sci. Part B: Polym. Phys. 42: 2460-2466; Volynskϋ et al. (2000) J. Mater. Sci. 35: 547-554; Stafford et al. (2004) Nature Mater. 3: 545-550; Watanabe et al. (2005) J. Polym. Sci. Part B: Polym. Phys. 43: 1532-1537; Lacour, et al. (2003) Appl. Phys. Lett. 82: 2404-2406.)

[0032] In addition, the metal can be applied to the thermoplastic material using "pattern transfer." The term "pattern transfer" refers to the process of contacting an image-forming device, such as a mold or stamp, containing the desired pattern with an image-forming material to the thermoplastic material. After releasing the mold, the pattern is transferred to the thermoplastic material. In general, high aspect ratio pattern and sub-nanometer patterns have been demonstrated. Such methods are well known in the art (Sakurai, et al., US Patent 7,412,926; Peterman, et al., US Patent 7,382,449; Nakamura, et al., US Patent 7,362,524; Tamada, US Patent 6,869,735).

[0033] Another method for applying the image forming material includes, for example "micro-contact printing". The term "micro -contact printing" refers to the use of the relief patterns on a PDMS stamp (also referred to as the thermoplastic material) to form patterns of self-assembled monolayers (SAMs) of an image-forming material on the surface of a thermoplastic material through conformal contact. Micro-contact printing differs from other printing methods, like inkjet printing or 3D printing, in the use of self-assembly (especially, the use of SAMs) to form micro patterns and microstructures of various image-forming materials. Such methods are well known in the art (Cracauer, et al., US Patent 6,981,445; Fujihira, et al, US Patent 6,868,786; Hall, et al, US Patent 6,792,856; Maracas, et al, US Patent 5,937,758).

[0034] A "patterning device" is intended to be broadly interpreted as referring to a device that can be used to convey a patterned cross-section, corresponding to a pattern that is to be created in a target portion of the substrate.

[0035] A "pattern" is intended to mean a mark or design.

[0036] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 0.1. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term "about". It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

Sensors

[0037] The sensors may include a heat-shrunk thermoplastic substrate and a film of metal disposed over the surface of the substrate. The film of metal includes a microstructure characterized by wrinkle-like features. By "microstructure" it is meant a structure comprising features on the micrometer scale. However, the microstructure may also include features on the nanometer scale. FIGS. 1 and 2 show images of a variety of substrates coated with wrinkled metallic surfaces. In FIG. 1, the wrinkle-like features are further characterized as folds in the metallic film, each fold having substantially rounded, smooth edges. As used herein, the types of wrinkled metallic films as shown in FIG. 1 will be referred to as a metallic films having folded, wrinkle-like features. In FIG. 2, the folds in the wrinkled metallic surface have cracked, resulting in petal-like (FIG. 2A) or ribbon-like (FIG. 2B) features. As compared to the edges of the folded, wrinkle-like features, the edges of the petal- like or ribbon- like features are relatively sharp. As used herein, the types of wrinkled metallic surfaces as shown in FIG. 2 will be referred to as metallic films having cracked, wrinkle-like features. Moreover, the wrinkled metallic films with petal-like features may also be referred to as nanopetal films.

[0038] As shown in FIGS. 1 and 2, the orientation of the wrinkles in the metallic film may vary. In some embodiments, the wrinkles are randomly oriented across the surface of the substrate. Such embodiments are shown in FIGS. IA, 1C, and 2A. In other embodiments, the wrinkles are oriented substantially parallel to one another across the surface of the substrate. Such embodiments are shown in FIGS. lB and 2B. By "substantially parallel," it is meant that the wrinkles of the wrinkled metallic film are parallel to one another, but not necessarily perfectly parallel. As shown in FIGS. IB and 2B, although each of the wrinkles are approximately aligned with one another, many of the wrinkles are not perfectly straight so that the wrinkles are not perfectly parallel to one another. In still other embodiments, the wrinkle-like features are localized to regions on the surface of the substrate and the regions are separated by areas in which the film of metal is substantially flat. Such an embodiment is shown in FIG. 1C.

[0039] The dimensions of the wrinkle-like features of the metallic film may vary. In some embodiments, the average height of the wrinkle-like features ranges from about 2 nm to about 100 nm. This includes average heights of about 10 nm, 25 nm, 50 nm, 75 nm, etc. By height, it is meant the distance between a low point on the top surface of the metallic film (i.e., a valley in the wrinkled metallic film or a flat region on the metallic film) to a high point on the top surface of the metallic film (i.e., the peak of a folded wrinkle or an edge of a cracked wrinkle). An average height may be obtained by averaging the heights of a plurality of wrinkles of the wrinkled metallic surface. In other embodiments, the average spacing of the wrinkle-like features ranges from about to 100 nm to about 3 μm. This includes average spacings of about 300 nm, 600 nm, 1 μm, 2 μm, etc. By spacing, it is meant the distance between the high point on one wrinkle (i.e., the peak of a folded wrinkle or an edge of a cracked wrinkle) and the high point on another wrinkle. An average spacing may be obtained similar to the average height described above.

[0040] The substrate of the sensor includes a thermoplastic material. As noted above, the term "thermoplastic material" encompasses those plastic materials that shrink upon heating. Suitable thermoplastic materials include, but are not limited to high molecular weight polymers such as acrylonitrile butadiene styrene (ABS), acrylic, celluloid, cellulose acetate, ethylene-vinyl acetate (EVA), ethylene vinyl alcohol (EVAL), fluoroplastics (PTFEs, including FEP, PFA, CTFE, ECTFE, ETFE), ionomers kydex, a trademarked acrylic/PVC alloy, liquid crystal polymer (LCP), polyacetal (POM or Acetal), polyacrylates (Acrylic), polyacrylonitrile (PAN or Acrylonitrile), polyamide (PA or Nylon), polyamide-imide (PAI), polyaryletherketone (PAEK or Ketone), polybutadiene (PBD), polybutylene (PB), polybutylene terephthalate (PBT), polyethylene terephthalate (PET), Polycyclohexylene Dimethylene Terephthalate (PCT), polycarbonate (PC), polyhydroxyalkanoates (PHAs), polyketone (PK), polyester polyethylene (PE), polyetheretherketone (PEEK), polyetherimide (PEI), polyethersulfone (PES), polysulfone polyethylenechlorinates (PEC), polyimide (PI), polylactic acid (PLA), polymethylpentene (PMP), polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polyphthalamide (PPA), polypropylene (PP), polystyrene (PS), polysulfone (PSU), polyvinyl chloride (PVC), polyvinylidene chloride (PVDC) and spectralon. In some embodiments, the substrate comprises polystyrene. Polystyrene, as well as a number of other thermoplastic materials, is flexible, durable, lightweight, and inexpensive, each of which is a desirable characteristic for a chemical and biological sensor.

[0041] In the disclosed sensors, the thermoplastic substrates are heat shrunk. By "heat shrunk" it is meant that the thermoplastic substrate has been exposed to heat, which reduces the size of the substrate as compared to the size of the substrate prior to exposure to heat. The size of the heat shrunk substrate may be reduced by a variety of amounts as compared to the size of the substrate prior to exposure to heat. In some embodiments, the size of the heat shrunk substrate is about 60%, 70%, 80%, or 90% the size of the substrate prior to exposure to heat. Heat shrinking is further described below.

[0042] The composition of the metallic film may vary. A variety of metals may be used, including, but not limited to platinum, gold, titanium, silver, copper, a dielectric substance, a paste or any other suitable metal or combination thereof. Examples of suitable dielectric substances include metal oxides, such as aluminum oxide, titanium dioxide and silicon dioxide. Examples of suitable pastes include conductive pastes such as silver pastes. In some embodiments, the metallic film includes a single layer of any of these metals or combinations of these metals. In other embodiments, the metallic film may include two or more adjacent layers of metal. By way of example only, a multi-layer metallic film may include a first layer of metal disposed over the substrate and a second layer of metal disposed over the first layer of metal. Other layers of metal may disposed over each previous layer of metal. The composition of the first layer of metal may be the same or different from the composition of the second layer of metal. [0043] Similarly, the thickness of the metallic film may vary. By "thickness of the metallic film," it is meant the thickness of the film prior to the heating of the substrate which leads to the formation of the wrinkle-like features, as further described below. The thickness of the metallic film may vary from about 1 nm to about 100 nm. This includes embodiments in which the thickness is about 10 nm, 25 nm, 50 nm, 75 nm, etc. For multilayer films, the thickness of each layer of metal may be the same or different. In some embodiments, the multi-layer film comprises two layers of different metals, wherein the first layer of the first metal has a thickness of about 30 nm to about 50 nm and the second layer of the second metal has a thickness of about 30 nm to about 50 nm. Such multi-layer films at these thicknesses are particularly suitable for forming metallic films have cracked, wrinkle-like features.

Methods for making sensors

[0044] The methods for making the disclosed sensors involve depositing a film of metal over the surface of a thermoplastic substrate and shrinking the coated substrate. Wrinkles form in the metallic film due to the stiffness incompatibility between the metallic film and the thermoplastic substrate. As further described below, the characteristics of the wrinkles may be controlled by the various parameters of the heating process and thickness of the deposited metal film.

[0045] Methods for depositing films of metal over substrates are known. By way of example only, physical vapor deposition (PVD) techniques or chemical vapor deposition (CVD) techniques may be used to deposit metal films of varying thicknesses on substrates. These techniques may also be used to form patterned metallic films, in which metal is deposited in specific regions on a substrate.

[0046] Shrinking of the coated thermoplastic substrates may be accomplished by exposing the coated thermoplastic substrates to heat. A variety of heat sources may be used, including, but not limited to an oven, such as a conventional oven or toaster oven. The temperature of the heating process may vary. In some embodiments, the temperature ranges from about 100⁰C to about 200⁰C. This includes a temperature of about 16O⁰C, although other temperatures are possible. The length of heating may also vary. In some embodiments, the length of heating may be from about 1 minute, 5 minutes, 10 minutes, or even more. Longer heating times increase the amount of shrinkage of the thermoplastic substrate. The ability to achieve wrinkled metallic surfaces using inexpensive substrates (e.g., polystyrene) and heating sources (e.g., toaster ovens) in a matter of minutes provides an ultra-rapid, ultra- low cost method of making chemical and biological sensors.

[0047] The coated thermoplastic substrates may be biaxially or uniaxially shrunk. By "biaxially" shrunk, it is meant that the shrinking of the thermoplastic material is isotropic. By "uniaxially" shrunk, it is meant that the shrinking of the thermoplastic material is anisotropic. Uniaxially shrinking may be accomplished by constraining a thermoplastic substrate at two edges during the heating process, resulting in shrinking along only one axis of the material.

[0048] Various parameters of the heating process may be adjusted to control the characteristics of the metallic wrinkles. By way of example only, the length of heating, which determines the degree of shrinkage, affects the wrinkle height and wrinkle spacing. As another example, the orientation of the wrinkles may be controlled through biaxial or uniaxial shrinking. As shown in FIGS. IA, 1C, and 2A, biaxially shrinking leads to the formation of wrinkles that are randomly oriented across the surface of the substrate. Uniaxially shrinking leads to the formation of wrinkles that are oriented substantially parallel across the surface of the substrate, as shown in FIGS. IB and 2B.

[0049] Similarly, the thickness of the metallic film affects the characteristics of the metallic wrinkles. The thickness of the metallic film affects the wrinkle spacing and the wrinkle height. The thickness of the metallic film also affects whether the metallic film will include folded wrinkle-like features or cracked wrinkle-like features. Thinner metallic films (i.e., those less than about 80 nm) tend to produce folded wrinkle-like features while thicker metallic films (i.e., those greater than about 80 nm) cracked wrinkle-like features.

[0050] Certain of the wrinkled metallic films and methods of making the films have been described in International Application No. PCT/US2008/083283, which is hereby incorporated by reference in its entirety.

[0051] The disclosed sensors may also be incorporated into a variety of devices, including, but not limited to micro fluidic devices. Micro fluidic devices may include micrometer-sized channels etched into the surface of a substrate. Exemplary microfluidic devices and methods for making the devices are described in International Application No. PCT/US2008/083283, which is hereby incorporated by reference in its entirety. The incorporation of sensors into such micro fluidic devices may be accomplished in a variety of ways. By way of example only, channels etched into a thermoplastic substrate may be coated with a film of metal and the coated thermoplastic substrate heated to provide channels having any of the wrinkled metallic surfaces described above. Integration with microfluidics will serve to reduce the target-probe reaction time based on reduced diffusion distance, enhanced mass transport and increases in the probability of collisions.

Methods for using sensors [0052] The disclosed sensors may be used to detect chemical and biological agents via backscattering spectrometry and enhanced backscattering spectrometry. These are known techniques. See, e.g., A. Ishimaru, Wave Propagation and Scattering in Random Media (IEEE Press, New York, 1996); L. Tsang, J. A. Kong and K. -H. Ding, Scattering of Electromagnetic Waves: Theories and Applications (John Wiley & Sons, Inc., New York, 2000); L. Tsang, J. A. Kong, K. -H. Ding and C. O. Ao, Scattering of Electromagnetic Waves: Numerical Simulations (John Wiley & Sons, Inc., New York, 2000); L. Tsang and J. A. Kong, Scattering of Electromagnetic Waves: Advanced Topics (John Wiley & Sons, Inc., New York, 2000); A. A. Maradudin, Light Scattering and Nanoscale Surface Roughness (Springer, New York, 2006);J. A. Sanchez-Gil, J. V. Garcia-Ramos and E. R. Mendez, "Electromagnetic mechanism in surface-enhanced Raman scattering from

Gaussian-correlated randomly rough metal substrates," Optics Express 10, 879-886 (2002); C. Kuo and M. Moghaddam, "A theoretical analysis of backscattering enhancement due to surface plasmons from multilayer structures with rough interfaces," IEEE Transactions on Antennas and Propagation 56, 1133-1143 (2008); A. A. Maradudin and E. R. Mendez, "Enhanced backscattering of light from weakly rough, random metal surfaces," Applied Optics 32, 3335-3343 (1993); C. S. West and K. A. O'Donnell, "Observations of backscattering enhancement from polaritons on a rough metal surface," Journal of the Optical Society ofAmerica A 12, 390-397 (1995); A. A. Maradudin, A. R. McGurn and E. R. Mendez, "Surface plasmon polariton mechanism for enhanced backscattering of light from one-dimensional randomly rough metal surfaces," Journal of the Optical Society of America A 12, 2500-2506 (1995); T.-K. Chan, Y. Kuga, and A. Ishimaru, "Subsurface detection of a buried object using angular correlation function measurement," Waves in Random Media 7, 457-465, (1997); A. Ishimaru, J. D. Rockway and Y. Kuga, "Rough surface Green's function based on first order modified perturbation and smoothed diagram methods," Waves in Random and Complex Media 10, 17-31 (2000); J. T. Johnson, "A numerical study of scattering from an object above a rough surface," IEEE Transactions on Antennas and Propagation 50, 1361-1367 (2002).

[0053] Briefly, backscattering enhancement, a phenomenon in which a well-defined peak in the backscattered direction off randomly rough surfaces is apparent, is the result of multiple scattering effects as well as surface plasmon interactions. When a chemical or biological agent is adsorbed to such a surface, interactions of light scattered by the agent and the surface as well as any self-interactions that might occur are encoded in the optical characteristics of the measured scattered light, such as wavelength, angle, spatial and polarization diversity. Both direct and inverse scattering measurements may be used. Thus, even unlabeled chemical and biological agents may be detected by comparing the optical characteristics of backscattered light from a surface having adsorbed agents to the optical characteristics of backscattered light from a bare surface. Moreover, plasmon resonance interactions with the chemical and biological agents of interest can provide additional information about adsorbed agents, thereby aiding in molecular identification. This may be accomplished by identifying spectral signatures of plasmatic resonances and their polarization dependent reflectance through extinction spectra studies. As described in International Application No. PCT/US2008/083283, which is hereby incorporated by reference in its entirety, it has been observed that wrinkled metallic surfaces having wrinkles that are oriented substantially parallel across the surface of a thermoplastic substrate exhibit a polarization-dependent shift in the surface plasmon resonance. Finally, to increase accuracy and sensitivity of the sensors, any of the disclosed sensors can be made to have overlapping extinction spectra with the extinction spectra of the chemical and biological agent of interest.

[0054] The disclosed methods involve exposing any of the disclosed sensors to a chemical or biological agent; exposing the sensor to light, and measuring the backscattered light from the sensor to detect the chemical or biological agent. Any chemical and biological agents may be detected using the disclosed sensors. A variety of light sources and a variety of wavelengths of light may be used. Techniques for measuring the backscattered light are known. Similarly, experimental set-ups for backscattering spectrometry are known.

Kits

[0055] This invention further provides a kit comprising, or alternatively consisting essentially of, or yet further consisting of the materials necessary to perform the methods described above. In one aspect, the kit comprises, or alternatively consists essentially of, or yet further consists of a thermoplastic material and instructions for carrying out the method. In one aspect, the kits further comprise one or more metals for forming wrinkles and instructions. In another aspect, the kit provides instructions for making and using the apparatus described above and incorporated herein by reference.

[0056] In another aspect, this invention provides a method for assaying or screening for new materials and methods having the same function of the inventions as described herein. In this aspect, the new materials and/or methods are used in the methods as described herein and compared to the performance of the devices of this invention.

EXAMPLES

[0057] The methods of the present disclosure will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present methods.

Example 1. Scattering of light by molecules over a rough surface [0058] This example demonstrates that rough surfaces provided by the metal wrinkles of the present technology are useful in application in biological and chemical sensing. A theory for multiple scattering of light by obstacles situated over a rough surface is also provided.

[0059] The applicants have herein developed low-cost and nanostructured metallic substrate which can be readily and ro-bustly integrated into micro fluidic devices. These self-assembled nano-structures are due to the stiffness mismatch between retracting shape memory polymers and a thin film of metal. Because the metal cannot retract, it buckles in a predictable manner with controllable predominant wavelengths. A diagram showing this process appears in FIG. 3. These complex non-periodic structures demonstrate strong and tunable plasmon resonances.

[0060] An important step in enabling this new fabrication technology for optical sensors lies in understanding optical signals emanating from molecules situated over nanoscale rough metal surfaces. These signals are inherently complicated due to the multiple scattering from the molecules and the rough metal surface. This problem is challenging because one must take into account accurately all of the interactions between the small obstacle and the rough surface. Scattering by the obstacle and the rough surface constitutes challenging problems by themselves. The objective here is to develop a multiple scattering theory that takes into account interactions between the obstacle and a rough surface. There are several studies that address obstacle scattering over flat planar surfaces in a variety of contexts [13-15]. For scattering by an obstacle over a rough surface, there are fewer results. In particular, Chiu and Sarabandi [16] studied the special case in which the obstacle is a dielectric cylinder and the surface is only slightly rough. Using the angular correlation function, Jin and Li [17] described a method to detect a scatter target over a randomly rough surface. Johnson [18] studied this problem numerically by taking into account up to fourth- order interactions between the obstacle and the rough surface. Recently, Guo et al. [19] used a parallel implementation of the fϊnite-difference/time-domain method to study this problem.

[0061] This example further presents a systematic method for studying the multiple scattering due to an obstacle situated over a rough surface. This theory requires knowledge of the scattering properties of the obstacle and the rough surface separately. This example combines these two operations in a self-consistent way. This theory is simply an extension to the Foldy-Lax theory for multiple scattering [20-23]. This example shows explicitly that this theory takes into account infinitely many interactions between an obstacle and the rough surface. Thus, this theory provides a foundation for studying carefully the multiple scattering by obstacles over rough surfaces provided that scattering by the obstacle and the rough surface themselves is sufficiently accurate.

[0062] It is noted that the rough surface here is not considered a random rough surface. Although one may not know the exact spatial properties of the surface for these applications, the surface is fixed. Thus, one may perform several calibration steps, if necessary. In particular, this example works under the assumption that one can first measure the light scattered by the rough surface without the presence of the obstacles. Then this example measures the light scattered by the obstacles over the rough surface. For this reason, this examples does not compute any statistical quantities. One may consider computing statistical quantities using this method to make statements about an ensemble of sensors.

[0063] This example seeks to develop a theoretical framework to study the interactions of light scattered by obstacles over a rough surface. To study this problem in a simple setting, this example studies time-harmonic (monochromatic light), scalar wave propagation, and scattering. In particular, this example considers a wave incident on several obstacles situated over a rough surface. A sketch of this problem appears in FIG. 4. In FIG. 4 the rough surface is given by the function z=βx,y). This example considers time-harmonic wave propagation with time dependence e ^ιωt and circular frequency ω. For this scattering problem, this example needs to solve the following reduced wave or Helmholtz equation:

M

- k^'12 V)₁₁U_* in z > fix.y) m⁼¹ , (2.1) with V =d χ+δ _y +d _z denoting the Laplacian. Here, Vm for m=\, . . . ,M denotes the M scattering "potentials" for each of the M scattering obstacles situated over the rough surface. To solve Eq. (2.1), one must prescribe boundary conditions. In particular, this example studies two different boundary conditions: Dirichlet and Neumann. The Dirichlet boundary condition is given by u = 0 on z =fix, y), (2.2) and the Neumann boundary condition is given by

V-VM = 0 on z =βx,y), (2.3) with

14M,/> 1)

* (2.4) denoting the unit normal to the rough surface.

[0064] The solution of Eq. (2.1) subject to Eq. (2.2) or Eq. (2.3) are written as the sum of the incident and scattered fields: u=Ui+u_s. The incident field Ui is an incoming solution of homogeneous problem,

V²M + k²u = 0, (2.5) near the bounding surface z=βx,y). This example assumes that u_{ is known explicitly. The scattered field u_s is an outgoing solution of Eq. (2.1) and is to be found. For this scattered field, this example prescribes also Sommerfeld radiation conditions far away from the boundary surface and scattering obstacles.

Self-consistent multiple scattering theory for obstacles over a rough surface

[0065] In what follows, this example develops a self-consistent theory for the multiple scattering of light by M obstacles situated over a rough surface. This theory requires knowledge of the scattering operator or the t-matrix for each of the obstacles and the reflection operator for the rough surface. Once those operators are established, this example combines them in a self-consistent manner to obtain a multiple scattering theory.

[0066] The scattering operator S_m gives the field scattered by the mth obstacle due to an exciting field. When Sm is known, the scattered field produced by the field uE exciting the obstacle is given by SmuE. The scattering operator Sm (otherwise known as the t-matrix or transition operator) with kernel t_m(r,r') for the mth obstacle is given by

S_mu^E(r) = I t_m{r_yr')u^E(r')dr^f λ^>« . (3.1)

Here, X_1n corresponds to the support of the mth obstacle.

[0067] The reflection operator R gives the field reflected by the rough surface due to an exciting field. When R is known, the reflected field produced by the field u^E exciting the rough surface is given by Ru^E. In general, R is defined by the solution of a surface integral equation derived from the Kirchhoff theory [22-24]. For the special case of a slightly rough surface, this example obtains an asymptotic result for R which will be used later.

[0068] For the problem corresponding to M obstacles situated over a rough surface, this example represents the total field as the following sum:

M

m = ^l . (3.2) Here, Φ_m denotes the field exciting the mth obstacle and ^represents the field exciting the rough surface. These fields are to be determined. Once they are determined, one can compute u(r) through the evaluation of Eq. (3.2).

[0069] This example represents the exciting fields as

M

&„ = {*, + 2_J S „</>„ + Rψ in ^₁₁, /H = I. . , . M,

" ^{~ m} (3.3)

M

w=i _{(3 4)}

[0070] Equations (3.3) and (3.4) comprise a self-consistent system for the exciting fields Φ_m and Ψ. This self-consistent system is an extension of the so-called Foldy-Lax theory for multiple scattering [20-23]. This extension incorporates scattering by the rough surface. In the same way that the Foldy-Lax theory includes infinitely many interactions, Eqs. (3.3) and (3.4) include infinitely many interactions between the obstacles and the rough surface.

[0071] Through substituting Eq. (3.4) into Eq. (3.3), one can construct an MxM system of equations for exciting fields at the obstacles: Φ_m for m=\, . . . , M. When those exciting fields are known, this example computes ^through the evaluation of Eq. (3.4). In what follows, this example will show this computation explicitly for the special case of point obstacles over a slightly rough surface.

Point obstacles over a slightly rough surface

[0072] This example specializes the general theory given in the previous section to point obstacles situated over a slightly rough surface. For this specific case, the problem reduces to a linear system of algebraic equations. Nonetheless, these simplifications lead to a model problem that allows one to study the complicated interactions between the obstacles and the rough surface.

[0073] Say an obstacle, with a characteristic length scale a, is small when ka«\. For that case, one may use the point scatterer approximation [25] in which the scattering operator for a point obstacle at position r_m is given by S₁, u,[r) = σ_tl,Gj τ;r_tn )d_t(r,_l} ⁾

with <j_m denoting the scattering cross-section for the point obstacle and

o

* * t,ϊ

Λ X.τ ilΛl & 1 -3 i- ( \ f hi,tUi. ) I" Hy i* — J wL _n

^L (4.2) is the free-space Green's function regularized to remove the singularity at r=r_m [25]. For M point obstacles at positions r_m with scattering cross-sections σ_m for m =1, . . . , M, Eq. (3.3) reduces to

φ_rn - !t,ir_n ) + V

r_f,, ', ;/ι - 1 , , ,3/.

(4.3)

[0074] Furthermore, Eq. (3.4) reduces to

M

Φ r⁾ - (f_ti r) + ^ ^G,,(i';r_Λ i(i_ni on r - ^ YΛ i ;^>'⁼¹ . (4.4)

[0075] The exciting fields σ_m for the point obstacles are just complex scalars.

[0076] The example now considers the case in which the roughness of the surface z=βx,y) is small compared to the wavelength. Moreover, the example assumes that the gradient of the rough surface is small compared to the wavelength. To make this assumption explicit, the example introduces the small dimensionless parameter 0<ε«l so that the rough surface is given by z=εβx,y). The example calls this rough surface a "slightly" rough surface. The example assumes that the functionary) and the parameter ε are known.

[0077] In the limit as ε→0⁺, one can compute an asymptotic approximation of the reflection operator R as a perturbation expansion [26,27]. This example considers an incident field of the form

with A denoting the angular spectrum of the incident field and

The field reflected by the slightly rough surface, Ru₁ can then be represented as

[0078] with R denoting a linear operator that takes into account scattering due to the surface roughness. In Appendix A of Long et al. (201O) J. Opt. Soc. Am. A 27(5): 1001- 11 , the applicants derive an asymptotic approximation for R_D, the operator for the Dirichlet problem. In Appendix B of Long et al. (2010), the applicants derive an asymptotic approximation for R_N, the operator for the Neumann problem. In what follows, the example proceeds as if R is known explicitly.

[0079] Evaluating Eq. (4.4) at z=0, the example finds that

}i

"^{< ]} . (4.8)

[0080] Fourier transforming this result with respect to x and y, the example obtains

Af

with ^(ξ,η) defined in Eq. (4.5),

Ψi ξ, ψ

S JT A- (_{4 1 1}) [0081] In Eq. (4.9), the example has made use of the Weyl representation for Go given by

+ iη(y -y^f ) + iκ ]άξd_v

(4.12)

[0082] Equations (4.11) and (4.12) correspond to the free-space Green's function rather than Eq. (4.2) since the example is not evaluating them near the singularity. Now, the example introduces the quantities

*0l - π _'' ^'Rg^ξ. ηyr^^^^άξd η,

(4.13)

[0083] Notice that RGo(x,'x_n) is the field reflected by the slightly rough surface due to a point source at position X_n. Similarly, RuIx) is the incident field reflected by the slightly rough surface evaluated at position r. By applying the reflection operator to Eq. (4.8) and evaluating that result at position x_m, the example obtains

RiHr₁J (T_nRG₀[V_1n I T_n)J*;

«=i (4.15)

[0084] Thus, substituting Eq. (4.15) into Eq. (4.3) and rearranging terms yields the following MxM linear system:

M

(4.16) with

1 ™ σ_mRGMr_m :r_m) m ~ n.

_*'-^r«[Go(^r;H ; ^r») + A⁵O₀I r_m ;r_Λ)] , m ≠ n .

(4.17) [0085] Upon the solution of Eq. (4.16), the example obtains the M complex numbers Φ\ , 0₂, . . . , Φ_M- With those complex numbers known, one can compute ^through the evaluation of Eq. (4.4). Thus, the field scattered by the point obstacles and the slightly rough surface is given by

M

U^r) = RUi(T) + 2 σ_m[G₀< r;r_m) + -RG₀< r;r_m)]<&_ϊj .

,'«=1 (4.18)

[0086] To summarize these results, the example give the following procedure to compute the field scattered by M point obstacles situated over a slightly rough surface.

1. Prescribe the slightly rough surface z=ε/(x,y).

2. With that slightly rough surface, compute the asymptotic approximation to R using Eq. (A12) for a Dirichlet surface (Appendix A of Long et al. (2010)) or Eq. (B13) for a Neumann surface (Appendix B of Long et al. (2010)).

3. Set the positions xm and scattering cross-sections m for the M point obstacles.

4. Solve Eq. (4.16) to obtain Φ\, Φι, . . . , Φ_M. 5. Evaluate Eq. (4.18) to obtain u_s(r).

Experimental Examples

[0087] In what follows, this example considers two particular examples. The first one is for a single point obstacle situated over a slightly rough surface. The second one is for two point obstacles situated over a slightly rough surface. These two examples are relevant for applications of optical sensors for point-of-care diagnostics. The ability to detect extremely low concentrations of analytes in a solution is important for this application, but remains a persistent challenge. For example, the limit of detection for the ELISA, the gold standard, is typically in the picomolar range. To be able to detect molecules at much more dilute concentrations would enable earlier stage detection with a less invasive sampling. Thus, this example assumes only a few obstacles in a site specific region to test the ability to detect extremely low concentrations.

[0088] For both of these examples, this example is able to obtain analytical results that the example interprets physically. Using those analytical results, this example computes asymptotic results for the scattered field u_s(r) evaluated in the far- field. A. One Point Obstacle

[0089] When there is only one point obstacle with scattering cross-section 1 situated over a slightly rough surface at position rl, Eq. (4.15) reduces to

[0090] The solution is given by

[0091] Expanding Eq. (5.2) formally, the example finds that

'^>=° (5.3)

[0092] One can interpret this result in the following way. The first term corresponds to the incident field ui and the incident field reflected by the slightly rough surface, Ru₁, exciting the point obstacle. The next term corresponds to the scattering of that exciting field down to the slightly rough surface and reflected back up to excite the point scatterer, and so on. A diagram showing these interactions appears in FIG. 5. Thus, Eq. (5.3) shows that this theory takes into account infinitely many interactions between the point obstacle and the slightly rough surface. Now that Φ_\ is known explicitly, the example computes the scattered field through the evaluation of

U_fSv) = Ru₁W^ + σ_s [G_n( I-F₁) + Λ^JG₀(r; r _i)]^₁ ,

[0093] This example has computed numerically the results given by Eq. (5.4). All quantities that are given below are in units of the wavelength λ. For these numerical calculations, the example considers a uni-axial slightly rough surface of the form z=εβx) with ε=0.05. For this example, it generated one realization of a Gaussian correlated random rough surface with a correlation length of 1.5 and a RMS height of 1 [23]. The point obstacle has scattering cross-section set to σι=\ . It is located at position T₁=(11.7,0.0,0.1). The location of the point obstacle in relation to this rough surface is shown in FIG. 6. [0094] A plane wave propagating in the xz-plane of the form

is incident on the point obstacle and rough surface. With these considerations, the symmetry with respect to the xz-plane is broken only due to scattering by the point obstacle. To compute these fields, this example replaced the Fourier transforms in the results from the previous section with the discrete Fourier transforms computed on a 512x512 grid of the computational domain: [-25.6,25.6] x [-25.6,25.6]. Figure 7 shows contour plots of the image /(x,y) defined as

for both the Dirichlet (top) and Neumann (bottom) cases. Here, the plane zo=5.O corresponds to the plane on which the light is detected. This difference image /(x,y) corresponds to the subtraction of the direct image without the point obstacles taken at the detector plane from the direct image with the point obstacles taken at the detector plane. It is normalized to the maximum absolute value of 7(x,y). This difference image shows the complicated interactions between the rough surface and the point obstacle. In FIG. 7, the example sees distortions of the scattering by the point obstacle by the uni-axial rough surface as faint vertical bands. The Dirichlet surface produces a more localized image about the point obstacle than does the Neumann surface. However, the example has observed widely varying results depending on the location of the point obstacle to the rough surface.

[0095] Using the standard expression for the far- field Green's function and the method of stationary phase [28], the example finds that eikR u_s — F₁ is ) ~~~— , kE — ∞ ,

^R (5.6) with

F₁(S) = - i2πks;RA{ks_x,ks_y) e -ikh~r

+ (T ₁ φi l2πks_s ^"R.gQ(ks_x,ks_v; r-ι)

(5.7)

Here, s =(s_x ,s_y ,s_z)=(sin #cos φ, sin #sin φ, cos θ), with # denoting the polar angle and φ denoting the azimuthal angle. B. Two Point Obstacles

[0096] When there are two point obstacles with scattering crosssections <j\ and <j\ situated over a slightly rough surface at positions T₁ and r₂, respectively, Eq. (4.15) reduces to

with A_mn defined in Eq. (4.16) and

(5.9)

[0097] This linear system is solved easily and it is found that 1

Ψi {[1 - <j3if?Go(r₂; r2)]6t - ^[G₀Cr₁; r₂) det(A

+ i?G₀(r₁;r₂)]δy] , (5.10)

+ i?G₀(iy,r_l)]6₁}, (5.11) with

- <τισ2[0(){c\ ] TI}G,){ F2 ; F-)) + Go(fι;r\}RGo(r2 ^'.i'2^ + EG o{r _\ ;r-] }Gr₍{r₂;i-j)] . (5.12)

[0098] Now that φ_\ and φ_\ are known explicitly, the example computes the scattered field u_s(x) through the evaluation of

«,(!^•}

J FO)

+ i?G₀ ⁽ r;r₂ ⁾]^₂- (5.13)

[0099] This example has computed numerically the results given by Eq. (5.13). The example uses the same rough surface that this example used for the numerical example above. The two point obstacles have scattering cross-section set to

One of the point obstacles is located at position T₁=(11.7,0.0,0.1). The other point obstacle is located at position r₂ =(9.7,0.0,0.1). Thus, the two point obstacles are two wavelengths apart from one another. The location of the two point obstacles in relation to this rough surface is shown in FIG. 8.

[0100] In FIG. 9 the example plots the image I(x,y) defined in Eq. (5.5) for both the Dirichlet (top) and Neumann (bottom) cases. Just as with FIG. 7, the detector plane is zo=5.O. These results are similar qualitatively to those in FIG. 7. However, one can observe a distorted dipole pattern resulting from the scattering by the two point obstacles.

[0101] Just as the example has done for the one point obstacle, one can evaluate u_s(r) in the far-field limit. In doing so, the example finds that

t_s ~" r %\t .—-, OO

(5.14) with

F₂(S) = - i2πks_tVΛ(ks_χiks_v)

+ CF_] φ-^ i2πks_zΕgQ(ks_x>ks_v:V] )

'1--7 eiH-r₂

+ O"<> < i2πks;R,g_Q ⁽ks_x,ks_y;r₂)

4 π

(5.15)

[0102] This example has developed a theoretical framework to study obstacle scattering over a rough surface. This theory involves combining each of the scattering operators for each of the obstacles and the reflection operator for the rough surface in a self-consistent way. For the simple case of point obstacles over a slightly rough Dirichlet or Neumann surface, this example is able to obtain analytical results. The example has shown analytical and numerical results for the cases involving one and two point obstacles. This theoretical framework provides, to the applicants' knowledge, a critical first step in studying the multiple scattering of light by nano -structured metallic substrates for sensor applications. It takes into account the interactions made between a single molecule and a rough surface. Here, the example has addressed this problem in an idealized setting. The obstacles are point scatterers and the rough surface is a small perturbation from a plane. Moreover, the surface is assumed to be a perfect electric conductor.

Example 2. Scattering of light by molecules over a rough surface [0103] This example shows preparation of tunable nano wrinkles suitable for detection of biological or chemical agents.

[0104] Researchers have long been fascinated by wrinkles as a pervasive natural phenomenon. [29-32] Recently, there has been a resurgence of interest in emulating and leveraging wrinkles for various applications. Polymeric wrinkles are finding increased utility as complex quasiperiodic structures important in applications, including cell-fate studies. [33,34] Flexible integrated circuits promising many new applications, such as wearable systems, have been demonstrated using thin buckled films of single-crystalline silicon based on elastomeric substrates. [35,36] Metal wrinkles, thin films of metal on polymer substrates, have promise for applications in molecular detection, optical devices, filters and sorters, high- surface-area conductors and actuators, and even metrology. [37-40] The applicants have developed a rapid approach to create metal nano wrinkles of tunable size and demonstrable utility on a shape memory polymer, [41,42] pre-stressed polystyrene (PS) sheets commercially available as the children's toy Shrinky-Dinks.

[0105] Previous demonstrations of wrinkles exhibited relatively large wrinkle wavelengths. For instance, Bowden et al. deposited metal onto a thermally expanded polydimethylsiloxane (PDMS) polymer; the cooling of the PDMS causes a compressive stress, which buckles the deposited metal film to achieve ca. 30μm structures. [37] Huck et al. augmented this approach with photochemically patterned areas that differ in stiffness and thermal expansion. [43] Watanabe and Hirai more recently developed the very simple approach of simply pre-stretching the PDMS sheet to achieve 6-20μm striped patterns. [44] Lacour et al. used this approach to create stretchable gold conductors. [39] Yoo et al. imposed order on the buckling of polystyrene by applying a physical mold during the buckling process. [45] While this group was able to achieve higher resolution wrinkles than previously reported (down to 2 μm periodicity) as well as directionality, the process required a micro fabricated mold and took several hours. [0106] In this example, by just leveraging the stiffness mismatch of materials, the applicants present a simple and ultra-rapid two-step (metal deposition and subsequent heating) method to controllably create nanometer- scale metal wrinkles.

Methods and Materials [0107] Fabrication of Nanowrinkles and Discrete Wrinkled Flowers: For biaxial wrinkles, gold of varying nanometer thicknesses was deposited on a Shrinky-Dink sheet (or KSF50-C; Grafϊx, 2 cm x 1 cm) using sputtering (SEM coating system; Polaron). To avoid preheating of the substrate, this step was divided into four cycles, and each cycle (including 10 s of sputtering and 20 s of cooling) deposited 2.5 nm of gold. After deposition, heating at 160 ⁰C for 6 min in an oven induced retraction of the substrate and caused the nonshrinkable gold film to form biaxial wrinkles. For uniaxial wrinkles, before heating two short edges of a gold-coated sheet were clamped by clips (2 inch (ca. 5 cm) binder clips; OfficeMax) to ensure it could only retract in the perpendicular direction. For discrete wrinkled flowers, the same approach was taken with the exception that an ad hoc shadow mask, a transmission electron microscopy (TEM) grid with 50 μm grid spacing, was placed over the PS sheet prior to sputtering. After sputtering, the physical shadow mask was simply removed and the patterned flowers left behind.

[0108] Integrating Wrinkles into Microchannels: First a Shrinky-Dink sheet (10 cm x 15 cm) was channel-patterned by manual scribing with a syringe tip (20 gauge luer stub syringe tip) to remove PS. Next, the sample is constrained by clips in one direction and allowed to shrink by heating it to 150 ⁰C. After cooling, the center (2.5 cm x 5 cm) of the sample was cut out using a diamond saw. The chip was then masked by placing adhesive tape (3M brand) on the surface of the sample adjacent to the channel previously scribed into the surface. The tape was aligned under a dissection microscope until the entire surface was covered and only the channel remained exposed. The sample was then coated with 45 nm of silver by sputter deposition. After coating, the tape was removed from the surface of the chip; what remained was only the silver that was deposited on the surface of the channel. The sample was then shrunk, without being constrained, at 150 ⁰C to create uniaxial wrinkles inside the channel.

[0109] Materials and Methods for MEF Experiments: Dye molecules (Red CMTPX dyes,

CellTracker; Invitrogen) with absorption peaking at 577 nm and emission peaking at 602 nm, were used. They were first dissolved in dimethylsulfoxide (Sigma) to a concentration of 10 niM and then diluted with poly( vinyl alcohol) solution (PVA, 1 wt% in water, MW ca. 13000-23000; Sigma) to ca. 10μM. Sample (10μL) was dropped onto the uniaxial wrinkles (50nm thick gold) and a glass plate and then spin-coated at 3000 rpm for 2 min to form a sample layer with tens of nanometers thickness [51]. PVA polymers are used to form a buffer layer between the metal surface and fluorophores to avoid metal-induced quenching by direct contact [51]. The fluorescence images were acquired using a wide-field epifluorescence microscope (TE 2000-U; Nikon) equipped with a illumination system (X- Cite Series 120; EXFO) and a green color excitation filter (D540/25X; Chroma Tech). The emission was collected by a 40 ^χ, numerical aperture (NA) 0.75 objective (Plan Fluor;

Nikon) with a 590-650 nm band-pass filter (D620/60 M; Chroma Tech) and recorded with a charge-coupled device (CoolSNAP EZ; Photometries). For fluorescence lifetime measurement, samples were mounted on a homemade confocal optical microscope for inspection. Excitation of the samples was performed through a 100^χ, NA 0.7 objective (Nikon) using a frequency-doubled mode-locked ultrafast Ti:Sapphire laser (MIRA 900; Coherent) operating at 410 nm with 76 MHz repetition rate. Emission passing through a 410 nm notch filter (CVI) and a 590-650 nm band-pass filter (D620/60 M; Chroma Tech) was detected by an avalanche photodiode (PDM 50ct; MPD). The corresponding fluorescence decays were measured with a time-correlated single-photon counting module (PH300; Picoquant).

[0110] First, a 10 nm thick gold film is deposited on the PS sheets. Heating at 160 ⁰C causes the substrates to retract to less than half of its original size and therefore induces the stiffer, nonshrinkable metal film to buckle (FIG. 10a, left). [46-48] Scanning electron microscopy (SEM) images (FIGS. 10b and 1 Ia) show that large areas of uniform biaxial nanowrinkles can be produced. To determine the resulting wrinkle wavelengths, the example took the two-dimensional fast Fourier transform (2D FFT, shown in the inset of FIG. 1 Ia) of the SEM images. The resulting disc-shaped power spectral densities indicate a broad distribution of wrinkle wavelength in £-space. From this, one can determine the distribution of wavelengths as a probability function. As shown by the black line in Figure 1 Ib, the prevailing wavelengths peak near 400 nm and range from ca. 200 nm to ca. 1 μm. This range is smaller but more heterogeneous than those reported from other approaches, where the wrinkles had periodicities ranging from 20 μm to 50 μm. [37] As discussed below, one can adjust this broader range to its advantage for sensing applications.

[0111] In order to tune the wavelength of the wrinkles, it is important to understand how the length scales of the wrinkles depends on the thickness of the metal film, the material properties of the film and substrate, and the overall shrinking strain produced. Wrinkles arise from competition between the elastic bending energy of a stiff skin and the elastic energy of deformation of the soft substrate on which it is supported. [30,31 ,49] For a skin of thickness h and Young's modulus 7_skin supported on a substrate of Young's modulus Y_sub, minimization of the overall elastic energy yields an equilibrium wrinkle wavelength of λ oc η^lβh, where η ∞ /YskiJYsub- [30,31,49] For large compressive stresses, it is known that hierarchical wrinkling can occur because the amplitude of the smaller, first generation wrinkles saturate, forming an effective skin that can undergo a similar wrinkling process with wavelengths λ oc η_eff ^V3h_eff, where η_es and /z_efr are the parameters corresponding to the new effective skin. [29] For biaxial strains, another critical length scale is the distance ξ, over which wrinkles lose orientational coherence. It scales as ξ ∞ η^2l3h. [50] In this case, a gold film (Fgoid ~ 78 GPa) on a PS substrate (Fps ~ 3.5GPa), the example anticipates η = Ygoid/Yps ~ 22.3. [51 ,52] To verify the theoretical predictions, this example varied the thickness of deposited gold from 10 nm to 50 nm. This caused a shift of hundreds of nanometers in the wavelength distributions (FIG. 1 Ib). The experimental scaling of the peak wavelength with film thickness (the black triangles of FIG. 1 Ie) has a slope of 8.4, which agrees well with the theorized slope for the scaling of the coherence length η²¹³ ~ 7.9 (the black dashed line of FIG. 1 Ie). This suggests that a loss of coherence is the dominant effect in determining the morphology in the biaxial case. It should be noted, however, that there is an offset, indicating a modified skin approximately 30 nm thicker than the bare metal film and with a comparable modulus. The occurrence of such modified films has been reported previously [37] and is a likely consequence of the penetration of the metal into the substrate during the heat-shrinking process. Heating at 160 ⁰C, above the glass transition temperature (95 ⁰C) and below the melting temperature (240 ⁰C) of PS, induces the substrate to become soft enough to be integrated into the wrinkling metal. [52] This phenomenon dramatically increases the binding strength between gold and PS and thus changes the durability of the wrinkles, as confirmed by the empirical measurements and SEM analyses. Compared with a flat gold film (film deposited after shrinking process but heated), which can be easily removed, wrinkles are considerably stronger and can even bear ultrasonic cleaning over 5 min (data not shown).

[0112] Uniaxial wrinkles can be easily created as well (FIG. 10a, right). This example modifies the fabrication process by introducing boundary conditions by clamping two edges of a gold-coated PS sheet during the heating process. This gives rise to constrained shrinking along one direction. Large areas of well-aligned linear wrinkles can be produced (FIG. 10c). For the 10 nm thick sample, these wrinkles exhibit two distinct populations with peaks at 300 nm and 800 nm (FIGS. 1 lc,d). Their cross-sectional SEM image (FIG. l ie, bottom) demonstrates that the two populations correspond to two hierarchical generations of wrinkles. [37] Similar to what was found for the biaxial case, the peak wavelengths of both populations are proportional to the thickness of deposited gold and can thus be controllably tuned by adjusting the thickness of deposited gold (FIG. l id). The scaling of the dominant wavelengths with film thickness for both first and second generations is linear, with slopes of 2.1 and 2.4, respectively (blue squares and red circles of FIG. l ie). The consistency between experimental results and the anticipated value for the bare metal film (η^lβ ~ 2.8, blue and red dashed lines in FIG. 1 Ie) indicates that the loss of coherence is not the dominant issue, allowing the sample to clearly see features of the underlying wrinkle distribution. Thus, the first population (smaller wrinkles) can be regarded as arising from a modified skin of Young's modulus comparable to the bare metal but with a thickness about 130 nm greater than the metal film. As shown in the bottom panel of Figure l ie, the second population (larger wrinkles) arises from the saturation of the previous generation of wrinkles (first population), leading to the formation of an effective skin, which can still be regarded as having a modulus comparable to the bare metal but with a thickness that is about 300 nm greater than the metal film thickness. Since hierarchical wrinkling is a nonlinear process, it is difficult a priori to predict the effective skin thicknesses of successive generations. As a rough approximation, one can take the wrinkle amplitude (ca. A^mλ, for overall strain Δ) of one generation to be the effective skin thickness for the next generation. The effective thicknesses for the first and second populations above are then consistent with second- and third-generation wrinkles for an overall strain of order unity. [0113] One can also estimate an upper bound for the peak compressive stresses in the film if one assumes all of the strain is relieved by the smallest wrinkles. This is an upper bound because some of the stress will be relieved by the higher order larger wrinkles. The stresses are then of the order of Y_goi_dΔh/λ, which yields values ranging from several hundred magapascals to IGPa over the range of thicknesses. While this is significantly larger than the yield point for bulk gold, it is known that the yield strength of materials at the nanometer scale shows a dramatic increase, of several orders of magnitude, compared to the bulk values. [53] For example, yield strengths of gold nano wires (3-6GPa) are much greater than that of the bulk (55-200 MPa). [51] In particular, for metallic nano films on polymer substrates, most groups have found that the yield stress scales inversely with the film thickness, with the most recently measured yield stress value for a 50 nm gold film (which happens to be the highest film thickness) on PS substrates being as high as 30 GPa. [54] Thus, for the film thicknesses used, the peak stresses are clearly well below the yield point — which means that predictions from linear elasticity theory are appropriate. This is further validated by the fact that the experimental data agrees well with predictions using this simple theory.

[0114] Other metals, such as silver, can also be coated on PS sheets. Here it is demonstrated that silver wrinkles can be fabricated and easily integrated into Shrinky-Dinks microfluidic-based devices, which were developed by the applicants. [46-48] Besides the simple biaxial wrinkles (data not shown), uniaxial wrinkles can be aligned inside the microchannel. As shown in Figure 12a, first, a microchannel was patterned on a Shrinky- Dink sheet by manual scribing with a syringe tip. Second, this example constrained it to shrink in only one direction, perpendicular to the direction of the channel. Third, a piece of channel-patterned adhesive tape was placed on its top as a mask during the silver-sputtering. Finally, after deposition of 40 nm thick silver, the tape was removed; heating without constraints makes the sheet shrink in the perpendicular direction and form uniaxial wrinkles. Figure 12b shows that these wrinkles are perpendicularly aligned within a channel with width of 280 μm. Thermal bonding can be achieved easily as described in applicants' previous papers. [47,48]

[0115] Notably, the ability to achieve nanometer-scale wrinkles enables us to use these self-organized structures for surface plasmon resonance (SPR)-based sensing applications, such as metal-enhanced fluorescence (MEF). This technique utilizes metallic nanostructures in which the plasmons resonate with the fluorophores to reduce their excited state lifetimes and simultaneously increase their fluorescence emission intensities. [55] Silver island films are often used to meet such a requirement, and the typically ca. 10-fold enhancement has been applied to improve detection of DNA hybridization [56] and immunoassay. [57] However, detrimental chemical properties of silver, such as low stability and easy oxidization, inevitably restrict its potential in biomedical applications. Although the enhancement factor of gold is smaller, biocompatible gold in discrete nanostructures is, in contrast, free of these limitations and holds promise to enhance fluorescence intensity of fluorophores by a factor of ca. 2-7. [55,58] Recently, utilization of "continuous" gold nanometer-scale features to improve the density of hotspots has been studied; typically, a ca. 0.5- to 5-fold increase of the fluorescence intensity can be achieved. [59,60]

[0116] Here, this example demonstrates that the continuous gold wrinkled substrate is useful for MEF as well. Figures 12c and d (top) show fluorescence images along with the corresponding intensity profiles of dyes, dissolved in polymer solution and then spin-coated on either a bare glass plate or uniaxial gold wrinkles. [61] The average fluorescence intensity increased approximately threefold over a relatively large area when the dyes were deposited on the wrinkles rather than the glass. Many bright lines parallel to the direction of the wrinkles indicates that there are many continuous hotspots along the wrinkles, with 5- to 7-fold enhancements. To confirm that the enhancement resulted from plasmon effects rather than aggregation of dyes, fluorescence lifetime measurements were performed with a homemade confocal microscope with a time-correlated single-photon counting module. Figure 12d (bottom) shows that the average fluorescence lifetime of dyes on a glass plate is 3.5 ns, but on wrinkles their average lifetime decreases dramatically to 0.4 ns (with a shorter lifetime of 0.3 ns and a longer lifetime of 1.7 ns). This ca. 9-fold decrease in average lifetime suggests that enhancement results from strong interactions between the fluorophore and surface plasmons. [55] In addition, the shorter and longer lifetimes may be attributed respectively to the strongly (bright) and weakly (dim) enhanced regions (FIG. 12c, bottom). Since the intensity of hotspots highly depends on the spacing between nanostructures, it is suggested that the heterogeneous enhancement is correlated to the wavelength distribution of wrinkles. [62] [0117] Compared with results from other continuous gold film nanostructures, a 7-fold enhancement is a significant achievement. [59,60] If the geometry of wrinkles is optimized, the critical spacing between the fluorophore and substrate controlled, and the density of intense hotspots increased, an average fluorescence intensity increase by at least one order of magnitude can be achieved. One potential MEF application is to improve the sensitivity of DNA and protein microarrays, in which high density probes with spot sizes from 10 to 500 μm are immobilized on substrates with areas of several square centimeters. [63] Fluorescence is widely adopted for detection owing to its ultrahigh sensitivity and capabilities for multiple-probe labeling. Recently, use of SPR to lower the detection limit has become desirable to broaden its applications. [63] To integrate the plasmonic material into commercial products, it is demonstrated that the wrinkles can be easily patterned. In Figure 12e, discrete "wrinkled flowers" with diameters around 50 μm are shown to be well aligned. The sizes of spots and spaces depend on the resolution of the shadow mask and thus can be well controlled to be compatible with commercial detection systems.

[0118] In summary, the approach can create tunable nano wrinkles with broad yet tunable wavelength distributions. Such flexibility and heterogeneity hold a number of advantages over single, homogeneous, wavelength wrinkles. For instance, for further studies on multiple-probe MEF, these properties make it possible to adjust broad SPR bands to overlap well with various absorption bands of fluorophores. [55] In addition, this approach is considerably faster and significantly less expensive and more robust than other means of achieving such size-controllable nanometer-scale structures (including nanosphere lithography, focused ion beam lithography, and electron-beam lithography). [55] A significant enhancement of fluorescence intensity, together with the high throughput and fine microfluidic control over reagents with lab-on-chip techniques, makes the nanowrinkles promising low-cost substrates for ultrasensitive and ultrafast detection for biomedical applications. [64] In addition, such nanoscale features, along with ease of surface functionalization, make the gold wrinkles a potentially useful substrate for studying cell membrane dynamics. [65]

[0119] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non- limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as "up to," "at least," "greater than," "less than," and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above.

[0120] All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document were specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

[0121] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non- limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as "up to," "at least," "greater than," "less than," and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above.

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Claims

WHAT IS CLAIMED IS:

1. A method of detecting a chemical or biological agent, the method comprising: exposing a sensor to the chemical or biological agent; exposing the sensor to light; and measuring the backscattered light from the sensor to detect the chemical or biological agent, wherein the sensor comprises a heat-shrunk thermoplastic substrate, and a film of metal disposed over the surface of the substrate, wherein the film of metal comprises a microstructure characterized by wrinkle-like features.

2. The method of claim 1 , wherein the wrinkle-like features are folded wrinkle-like features.

3. The method of claim 1 , wherein the wrinkle-like features are cracked wrinkle-like features.

4. The method of claim 1 , wherein the wrinkle-like features are randomly oriented across the surface of the substrate.

5. The method of claim 1, wherein the wrinkle-like features are oriented substantially parallel across the surface of the substrate.

6. The method of claim 1, wherein the wrinkle-like features are localized to regions on the surface of the substrate and the regions are separated by areas in which the film of metal is substantially flat.

7. The method of claim 3, wherein the cracked wrinkle-like features are randomly oriented across the surface of the substrate to provide petal-like features.

8. The method of claim 3, wherein the cracked wrinkle-like features are oriented substantially parallel across the surface of the substrate to provide ribbon-like features.

9. The method of claim 1 , wherein the average height of the wrinkle-like features ranges from about 2 nm to about 100 nm.

10. The method of claim 1, wherein the average spacing between wrinkles ranges from about 100 nm to about 3 μm.

11. The method of claim 1 , wherein the thermoplastic substrate is one or more high molecular weight polymer selected from the group of acrylonitrile butadiene styrene (ABS), acrylic, celluloid, cellulose acetate, ethylene-vinyl acetate (EVA), ethylene vinyl alcohol (EVAL), fluoroplastics (PTFEs, including FEP, PFA, CTFE, ECTFE, ETFE), ionomers kydex, a trademarked acrylic/PVC alloy, liquid crystal polymer (LCP), polyacetal (POM or Acetal), polyacrylates (Acrylic), polyacrylonitrile (PAN or Acrylonitrile), polyamide (PA or Nylon), polyamide-imide (PAI), polyaryletherketone (PAEK or Ketone), polybutadiene (PBD), polybutylene (PB), polybutylene terephthalate (PBT), polyethylene terephthalate (PET), Polycyclohexylene Dimethylene Terephthalate (PCT), polycarbonate (PC), polyhydroxyalkanoates (PHAs), polyketone (PK), polyester polyethylene (PE), polyetheretherketone (PEEK), polyetherimide (PEI), polyethersulfone (PES), polysulfone polyethylenechlorinates (PEC), polyimide (PI), polylactic acid (PLA), polymethylpentene (PMP), polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polyphthalamide (PPA), polypropylene (PP), polystyrene (PS), polysulfone (PSU), polyvinyl chloride (PVC), polyvinylidene chloride (PVDC) and spectralon.

12. The method of claim 1, wherein the film of metal comprises silicon dioxide, platinum, titanium, gold, silver, copper, or combinations thereof.

13. The method of claim 1 , wherein the thickness of the film of metal ranges from about 1 nm to about 200 nm.

14. The method of claim 1 , wherein the film of metal comprises two or more adjacent layers of metal.

15. The method of claim 14, wherein the film of metal comprises a first layer of a first metal and a second layer of a second metal adjacent to the first layer.

16. The method of claim 14, wherein each layer of metal has a thickness ranging from about 30 nm to about 50 nm.

17. The method of claim 1, wherein the extinction spectrum of the sensor overlaps with the extinction spectrum of the chemical or biological agent.