# Patent

## Title
Diffraction-based diagnostic devices

## Abstract

A biosensor includes a substrate with a layer of receptive material disposed thereon. The receptive material is specific for an analyte of interest. A pattern of active and inactive areas of the receptive material are defined in the receptive material layer by a masking process.

## Background
TECHNICAL FIELD OF THE INVENTION
The present invention relates generally to the field of detecting analytes in a medium, and more particularly to a process for preparing analyte-specific diffraction based diagnostic sensors to indicate the presence of the analyte in a medium.
BACKGROUND
There are many systems and devices available for detecting a wide variety of analytes in various media. Many of the prior systems and devices are, however, relatively expensive and require a trained technician to perform the test. A need has been recognized in the art for biosensor systems that are easy and inexpensive to manufacture, and capable of reliable and sensitive detection of analytes. Reference is made, for example, to U.S. Pat. Nos. 5,922,550; 6,060,256; and 6,221,579 B1.
Various advances have been made in the industry for producing biosensors. For example, U.S. Pat. No. 5,512,131 to Kumar, et al., describes a device that includes a polymer substrate having a metal coating. An analyte specific receptor layer is stamped onto the coated substrate. A diffraction pattern is generated when an analyte binds to the device. A visualization device, such as a spectrometer, is then used to determine the presence of the diffraction pattern. A drawback to this type of device is, however, the fact that the diffraction pattern is not discernible by the naked eye and, thus, a complex visualization device is needed to view the diffraction pattern. Also, the device is generally not able to detect smaller analytes that do not produce a noticeable diffraction pattern.
U.S. Pat. No. 5,482,830 to Bogart, et al., describes a device that includes a substrate which has an optically active surface exhibiting a first color in response to light impinging thereon. This first color is defined as a spectral distribution of the emanating light. The substrate also exhibits a second color which is different from the first color. The second color is exhibited in response to the same light when the analyte is present on the surface. The change from one color to another can be measured either by use of an instrument, or by the naked eye. A drawback with the device is, however, the relatively high cost of the device and problems associated with controlling the various layers that are placed on the wafer substrate.
Contact printing techniques have been explored for producing biosensors having a self-assembling monolayer. U.S. Pat. No. 5,922,550 describes a biosensor having a metalized film upon which is printed (contact printed) a specific predetermined pattern of an analyte-specific receptor. The receptor materials are bound to the self-assembling monolayer and are specific for a particular analyte or class of analytes. Attachment of a target analyte that is capable of scattering light to select areas of the metalized plastic film upon which the receptor is printed causes diffraction of transmitted and/or reflected light. A diffraction image is produced that can be easily seen with the eye or, optionally, with a sensing device. U.S. Pat. No. 6,060,256 describes a similar device having a metalized film upon which is printed a specific predetermined pattern of analyte-specific receptor. The '256 patent is not limited to self-assembling monolayers, but teaches that any receptor which can be chemically coupled to a surface can be used. The invention of the '256 patent uses methods of contact printing of patterned monolayers utilizing derivatives of binders for microorganisms. One example of such a derivative is a thiol. The desired binding agent can be thiolated antibodies or antibody fragments, proteins, nucleic acids, sugars, carbohydrates, or any other functionality capable of binding an analyte. The derivatives are chemically bonded to metal surfaces such as metalized polymer films, for example via a thiol.
A potential issue of the contact printing techniques described above for producing diffraction-based biosensors is the possibility of contamination from the print surface (i.e., stamp) during the printing process. Also, there is the possibility of uneven application or inking of the substances due to pressure and contact variations inherent in the process, as well as surface energy variations.
The present invention relates to a biosensor system that is easy and inexpensive to manufacture, is capable of reliable and sensitive detection of analytes, and avoids possible drawbacks of conventional microcontact printing techniques.
SUMMARY OF THE INVENTION
Objects and advantages of the invention will be set forth in part in the following description, or may be obvious from the description, or may be learned through practice of the invention.
The present invention provides a relatively inexpensive yet sensitive biosensor device, a method for producing such biosensor devices, and a method for detecting analytes of interest present in a medium.
The biosensor includes a substrate member upon which a layer containing a receptive material (i.e., biomolecules) has been applied generally uniformly over an entire surface of the substrate member. The substrate may be any one of a wide variety of suitable materials, including plastics, metal coated plastics and glass, functionalized plastics and glass, silicon wafers, foils, glass, etc. Desirably, the substrate is flexible, such as a polymeric film, in order to facilitate the manufacturing process. The receptive material layer may be applied by any number of known techniques, including dipping, spraying, rolling, spin coating and any other technique wherein the receptive material layer can be applied generally uniformly over the entire test surface of the substrate. The invention also includes contact printing methods of applying the coating, as long as such methods are conducted in a manner to prevent inconsistent inking and contamination from contact during the initial coating process.
The receptive material layer is defined into a pattern of active and inactive areas of receptive material in a masking process. The masking process is based generally on the principle that the receptive material biomolecules depend on shape and conformational flexibility for function. If the shape or flexibility characteristics are altered substantially, the biomolecules lose their ability to interact or bind with ligands or analytes having a particular affinity for the molecules (i.e., an antibody-antigen interaction). This principle can be exploited to form a pattern of active and inactive biomolecules in the receptive material layer by a masking process wherein a crosslinking agent is activated to disrupt or alter the shape and/or flexibility characteristics of the biomolecules in areas exposed by the mask thereby rendering the biomolecules in such areas incapable of interacting with an analyte of interest.
A generally uniform layer or coating of the cross-linking agent is applied over the receptive material layer. A mask having any desired pattern of shielded areas and exposed areas (blank, transparent or translucent areas) is then placed over the substrate member. The mask and substrate combination are then exposed to a particular stimulus (e.g., light) that activates the crosslinking agent under the exposed areas of the mask. The activated crosslinking agent attaches to the receptive material, which renders the receptive material inactive in the exposed areas. After the mask is removed, the exposed areas thus define a pattern of inactive areas of the receptive material. The residual unreacted crosslinking agent in the areas that were under the shielded regions of the mask is removed (e.g., in a washing or rinsing step).
It should be appreciated that the invention is not limited to any particular pattern defined by the mask. Virtually any number and combination of exposed (active) shapes are possible. In one particular embodiment, the active area pattern is defined by about 10 micron diameter pixels at a spacing of about 5 microns apart over the test surface of the substrate.
Upon subsequent exposure of the biosensor to a medium containing an analyte of interest, the analyte binds to the receptive material in the active areas. The biosensor will then diffract transmitted light in a diffraction pattern corresponding to the active areas. The diffraction pattern may be visible to the naked eye or, optionally, viewed with a sensing device. In the case where an analyte does not scatter visible light because the analyte is too small or does not have an appreciable refractive index difference compared to the surrounding medium, a diffraction-enhancing element, such as polymer microparticles, may be used. These microparticles are coated with a binder or receptive material that also specifically binds to the analyte. Upon subsequent coupling of the analyte to both the patterned biomolecules in the receptive material layer as well as the microparticles, a diffraction image is produced which can be easily seen with the eye or, optionally, with a sensing device.
By “diffraction” it is meant the phenomenon, observed when waves are obstructed by obstacles, of the disturbance spreading beyond the limits of the geometrical shadow of the object. The effect is marked when the size of the object is of the same order as the wavelength of the waves. In the present invention, the obstacles are analytes (with or without or attached microparticles) and the waves are light waves.
In another embodiment of the present invention, nutrients for a specific class of microorganisms can be incorporated into the receptive material layer. In this way, very low concentrations of microorganisms can be detected by first contacting the biosensor of the present invention with the nutrients incorporated therein and then incubating the biosensor under conditions appropriate for the growth of the bound microorganism. The microorganism is allowed to grow until there are enough organisms to form a diffraction pattern.
The present invention provides a low-cost, disposable biosensor which can be mass produced. The biosensors of the present invention can be produced as a single test for detecting an analyte or it can be formatted as a multiple test device. The uses for the biosensors of the present invention include, but are not limited to, detection of chemical or biological contamination in garments, such as diapers, the detection of contamination by microorganisms in prepacked foods such as meats, fruit juices or other beverages, and the use of the biosensors of the present invention in health diagnostic applications such as diagnostic kits for the detection of antigens, microorganisms, and blood constituents. It should be appreciated that the present invention is not limited to any particular use or application.
These and other features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a schematic representation of a method for producing biosensors according to the invention in a masking process.



DETAILED DESCRIPTION
The invention will now be described in detail with reference to particular embodiments thereof. The embodiments are provided by way of explanation of the invention, and not meant as a limitation of the invention. For example, features described or illustrated as part of one embodiment may be used with another embodiment to yield still a further embodiment. It is intended that the present invention include these and other modifications and variations as come within the scope and spirit of the invention.
The present invention features improved biosensing devices, and methods for using such biosensing devices, for detecting and quantifying the presence or amount of an analyte of interest within a medium. The analytes that can be detected by the present invention include, but are not limited to, microorganisms such as bacteria, yeasts, fungi and viruses. The biosensing devices according to the invention are relatively inexpensive and have advantages over conventional micro-printed biosensors.
The present invention comprises, in broad terms, a process of defining an active pattern of analyte-specific receptor material on a substrate surface by photo-masking the substrate. A generally uniform coating or layer of the receptive material is applied to the substrate surface. A generally uniform coating or layer of a crosslinking agent is then applied over the receptive material layer. A mask is placed over the substrate, and the mask and substrate combination is exposed to a stimulus, such as a light or energy source. In its basic form, the “mask” serves to shield or “protect” at least one area of the receptive material from the stimulus or energy source and to expose at least one adjacent area to the stimulus or energy source so as to activate the crosslinking agent in the exposed area. For example, the mask may be a generally transparent or translucent blank (e.g., a strip of material) having any pattern of shielded regions printed or otherwise defined thereon. The exposed or unshielded regions of the mask correspond to the inactive areas of the receptive material layer. Alternatively, the mask may simply be a single object placed upon the substrate. The area under the object would be protected and thus define an active area of the receptive material and the area around the object would be exposed to the energy source and thus define an area of inactive receptive material. The object may alternately have any pattern of openings defined therethrough corresponding to the exposed areas.
The crosslinking agent is “activated” in the exposed areas to the extent that the agent attaches to the biomolecules of the receptive material and changes the shape and/or flexibility of the biomolecule receptor sites to the extent that the biomolecules are thereafter prevented from binding with the analyte of interest in a test medium. The un-activated crosslinking agent in the active receptive material areas is removed or disassociated from the receptive material by any suitable process, for example by washing or rinsing the substrate member with a solution sufficient to remove the crosslinking agent. Thus, upon removal of the mask and disassociation of the un-activated crosslinking agent, a pattern of active and inactive receptive material areas are defined. It should be understood that “pattern” includes as few as one active area and one inactive area.
Upon subsequent exposure of the biosensor to a medium containing the analyte of interest, such analyte will bind to the receptors in the active receptive material areas. The analyte facilitates diffraction of transmitted and/or reflected light in a visible diffraction pattern corresponding to the active areas. As discussed in greater detail below, an enhancer may be used for enhancing diffraction from extremely small analytes.
The crosslinking agent may be any one of a number of commercially available homobifunctional agents wherein both ends of the agent are photosensitive, one example of which is (bis-[β-(4-azidosalicylamido)ethyl]disulfide). Such crosslinking agents are typically available in powder form and, in order to coat the receptive material layer, it may be necessary to put the agent in solution with water or a buffer. The solution is applied over the receptive material by any appropriate method, such as dipping, spraying, coating, etc. The solution is then dried leaving a relatively thin dry coat of the agent over the receptive material. It should be ensured that the concentration of the agent is sufficient to essentially cover the receptive material. This is particularly important because the crosslinking agent is non-specific with respect to the receptive material. In other words, there is no particular attraction or affinity between the crosslinking agent and the receptive material. Alternatively, it may be desired to maintain the crosslinking agent in solvent form during the masking process. For example, a small amount of solution containing an adequate concentration of the crosslinking agent may be “sandwiched” between the substrate and a clear film or slide prior to placing the mask against the substrate.
Upon being exposed to a light source through the exposed regions of the mask, the ends of the cross-linking agent affix to the biomolecules in the receptive material layer and block or distort the receptor sites to the extent that the biomolecules are rendered incapable of subsequently interacting with the analyte of interest.
It should be appreciated that, due to the photosensitive nature of the crosslinking agent, it may be desired to conduct the processes of applying and masking the agent are done in a light protected environment.
The mask is then removed from the substrate member, desirably in a light protected environment, and the unactivated crosslinking agent is disassociated from the receptive material in the active areas. This may be done in various ways, including rinsing the substrate with a buffer or water.
The analytes that are contemplated as being detected using the present invention include, but are not limited to, bacteria; yeasts; fungi; viruses; rheumatoid factor; antibodies, including, but not limited to IgG, IgM, IgA, IgD, and IgE antibodies; carcinoembryonic antigen; streptococcus Group A antigen; viral antigens; antigens associated with autoimmune disease, allergens; tumor antigens; streptococcus Group B antigen; HIV I or HIV II antigen; or host response (antibodies) to these and other viruses; antigens specific to RSV or host response (antibodies) to the virus; antigen; enzyme; hormone; polysaccharide; protein; lipid; carbohydrate; drug or nucleic acid; Salmonella species; Candida species, including, but not limited to Candida albicans and Candida tropicalis; Neisseria meningitides groups A, B, C, Y and W sub 135; Streptococcus pneumoniae; E. coli; Haemophilus influenza type A/B; an antigen derived from microorganisms; prostrate specific antigen (PSA) and C-reactive protein (CRP) antigen; a hapten; a drug of abuse; a therapeutic drug; an environmental agent; and antigens specific to Hepatitis. In broad terms, the “analyte of interest” may be thought of as any agent whose presence or absence from a biological sample is indicative of a particular health state or condition.
It is also contemplated that nutrients for a specific class of microorganism can be incorporated into the receptive material layer. In this way, very low concentrations of microorganisms can be detected by exposing the biosensor of the present invention with the nutrients incorporated therein to the suspect medium and then incubating the biosensor under conditions appropriate for the growth of the bound microorganism. The microorganisms are allowed to grow until there are enough organisms to form a diffraction pattern. Of course, in some cases, the microorganism is present or can multiply enough to form a diffraction pattern without the presence of a nutrient in the active receptive material areas.
The receptive material is characterized by an ability to specifically bind the analyte or analytes of interest. The variety of materials that can be used as receptive material is limited only by the types of material which will combine selectively (with respect to any chosen sample) with a secondary partner. Subclasses of materials which fall in the overall class of receptive materials include toxins, antibodies, antibody fragments, antigens, hormone receptors, parasites, cells, haptens, metabolites, allergens, nucleic acids, nuclear materials, autoantibodies, blood proteins, cellular debris, enzymes, tissue proteins, enzyme substrates, coenzymes, neuron transmitters, viruses, viral particles, microorganisms, proteins, polysaccharides, chelators, drugs, aptamers, peptides, and any other member of a specific binding pair. This list only incorporates some of the many different materials that can be coated onto the substrate surface to produce a thin film assay system. Whatever the selected analyte of interest is, the receptive material is designed to bind specifically with the analyte of interest.
The matrix or medium containing the analyte of interest may be a liquid, a solid, or a gas, and can include a bodily fluid such as mucous, saliva, urine, fecal material, tissue, marrow, cerebral spinal fluid, serum, plasma, whole blood, sputum, buffered solutions, extracted solutions, semen, vaginal secretions, pericardial, gastric, peritoneal, pleural, or other washes and the like. The analyte of interest may be an antigen, an antibody, an enzyme, a DNA fragment, an intact gene, a RNA fragment, a small molecule, a metal, a toxin, an environmental agent, a nucleic acid, a cytoplasm component, pili or flagella component, protein, polysaccharide, drug, or any other material. For example, receptive material for bacteria may specifically bind a surface membrane component, protein or lipid, a polysaccharide, a nucleic acid, or an enzyme. The analyte which is specific to the bacteria may be a polysaccharide, an enzyme, a nucleic acid, a membrane component, or an antibody produced by the host in response to the bacteria. The presence or absence of the analyte may indicate an infectious disease (bacterial or viral), cancer or other metabolic disorder or condition. The presence or absence of the analyte may be an indication of food poisoning or other toxic exposure. The analyte may indicate drug abuse or may monitor levels of therapeutic agents.
One of the most commonly encountered assay protocols for which this technology can be utilized is an immunoassay. However, the general considerations apply to nucleic acid probes, enzyme/substrate, and other ligand/receptor assay formats. For immunoassays, an antibody may serve as the receptive material or it may be the analyte of interest. The receptive material, for example an antibody or an antigen, should form a stable, reactive layer on the substrate surface of the test device. If an antigen is to be detected and an antibody is the receptive material, the antibody must be specific to the antigen of interest; and the antibody (receptive material) must bind the antigen (analyte) with sufficient avidity that the antigen is retained at the test surface. In some cases, the analyte may not simply bind the receptive material, but may cause a detectable modification of the receptive material to occur. This interaction could cause an increase in mass at the test surface or a decrease in the amount of receptive material on the test surface. An example of the latter is the interaction of a degradative enzyme or material with a specific, immobilized substrate. In this case, one would see a diffraction pattern before interaction with the analyte of interest, but the diffraction pattern would disappear if the analyte were present. The specific mechanism through which binding, hybridization, or interaction of the analyte with the receptive material occurs is not important to this invention, but may impact the reaction conditions used in the final assay protocol.
In addition to producing a simple diffraction image, patterns of analytes can be such as to allow for the development of a holographic sensing image and/or a change in visible color. Thus, the appearance of a hologram or a change in an existing hologram will indicate a positive response. The pattern made by the diffraction of the transmitted light can be any shape including, but not limited to, the transformation of a pattern from one pattern to another upon binding of the analyte to the receptive material. In particularly preferred embodiments, the diffraction pattern becomes discernible in less than one hour after contact of the analyte with the biosensing device of the present invention.
The diffraction grating which produces the diffraction of light upon interaction with the analyte must have a minimum periodicity of about 1⁄2 the wavelength and a refractive index different from that of the surrounding medium. Very small analytes, such as viruses or molecules, can be detected indirectly by using a larger, “diffraction-enhancing element,” such as a microparticle, that is specific for the small analyte. One embodiment in which the small analyte can be detected comprises coating the enhancing particle, such as a latex bead or polystyrene bead, with a receptive material, such as an antibody, that specifically binds to the analyte of interest. Particles that can be used in the present invention include, but are not limited to, glass, cellulose, synthetic polymers or plastics, latex, polystyrene, polycarbonate, proteins, bacterial or fungal cells, silica, cellulose acetate, carbon, and the like. The particles are desirably spherical in shape, but the structural and spatial configuration of the particles is not critical to the present invention. For instance, the particles could be slivers, ellipsoids, cubes, random shape and the like. A desirable particle size ranges from a diameter of approximately 0.1 micron to 50 microns, desirably between approximately 0.1 micron and 2.0 microns. The composition of the particle is not critical to the present invention.
Desirably, the receptive material layer on the substrate will specifically bind to an epitope on the analyte that is different from the epitope used in the binding to the enhancing particle. Thus, for detecting a small analyte, such as viral particles, in a medium, the medium is first exposed to the latex particles having the virus-specific receptive material thereon. The small analytes of interest in the medium will bind to the latex particles. Then, the latex particles are optionally washed and exposed to the biosensor film with the pattern of active receptive material areas containing the virus-specific antibodies. The antibodies then bind to the viral particles on the latex bead thereby immobilizing the latex beads in the same pattern as the active areas on the film. Because the bound latex beads will cause diffraction of the visible light, a diffraction pattern is formed, indicating the presence of the viral particle in the liquid. Other combinations using diffraction enhancing particles are described, for example, in U.S. Pat. No. 6,221,579 incorporated herein for all purposes.
Any one of a wide variety of materials may serve as the substrate to which the receptive material is applied. Such materials are well known to those skilled in the art. For example, the substrate may be formed of any one of a number of suitable plastics, metal coated plastics and glass, functionalized plastics and glass, silicon wafers, foils, glass, etc. Rather than requiring a rigid substrate for the photopatterning process described herein, it has been found that thermoplastic films are quite suitable. Such films include, but are not limited to, polymers such as: polyethylene-terephthalate (MYLAR®), acrylonitrile-butadiene-styrene, acrylonitrile-methyl acrylate copolymer, cellophane, cellulosic polymers such as ethyl cellulose, cellulose acetate, cellulose acetate butyrate, cellulose propionate, cellulose triacetate, cellulose triacetate, polyethylene, polyethylene-vinyl acetate copolymers, ionomers (ethylene polymers) polyethylene-nylon copolymers, polypropylene, methyl pentene polymers, polyvinyl fluoride, and aromatic polysulfones. Preferably, the plastic film has an optical transparency of greater than 80 percent. Other suitable thermoplastics and suppliers may be found, for example, in reference works such as the Modern Plastics Encyclopedia (McGraw-Hill Publishing Co., New York 1923–1996).
In one embodiment of the invention, the thermoplastic film may have a metal coating. The film with metal coating thereon may have an optical transparency of between approximately 5 percent and 95 percent. A more desired optical transparency for the thermoplastic film used in the present invention is between approximately 20 percent and 80 percent. In a desired embodiment of the present invention, the thermoplastic film has at least an approximately 80 percent optical transparency, and the thickness of the metal coating is such as to maintain an optical transparency greater than about 20 percent, so that diffraction patterns can be produced by either reflected or transmitted light. This corresponds to a metal coating thickness of about 20 nanometers. However, in other embodiments of the invention, the metal thickness may be between approximately 1 nanometer and 1000 nanometers.
The preferred metal for deposition on the film is gold. However, silver, aluminum, chromium, copper, iron, zirconium, platinum, titanium, and nickel, as well as oxides of these metals, may be used. Chromium oxide can be used to make metalized layers.
The receptive material and crosslinking agent may be applied to the substrate by any conventional method. The material is applied so that it generally uniformly covers an entire (for example, upper) surface of the substrate. Non-contact methods for applying the receptive material may be desired so as to eliminate the possibility of contamination by contact during application. Suitable application methods include, but are not limited to, dipping, spraying, rolling, spin coating, and any other technique wherein the receptive material layer and blocking agent can be applied generally uniformly over the entire test surface of the substrate. Simple physisorption can occur on many materials, such as polystryene, glass, nylon, or other materials well known to those skilled in the art. One particular embodiment of immobilizing the analyte-specific receptive material layer involves molecular attachment, such as that possible between thiol or disulfide-containing compounds and gold. Typically, a gold coating of about 5 to about 2000 nanometers thick is supported on a silicon wafer, glass, or polymer film (such as a MYLAR® film). The analyte-specific receptor attaches to the gold surface upon exposure or spraying of a solution of the receptive material.
Although not preferred, the invention also includes contact printing methods of applying the receptive material and crosslinking agent.
The technique selected should minimize the amount of receptive material required for coating a large number of test surfaces and maintain the stability/functionality of the receptive material during application. The technique should also apply or adhere the receptive material to the substrate in a uniform and reproducible fashion.
It is also contemplated that the receptive material layer may be formed on the substrate as a self-assembling monolayer of alkanethiolates, carboxylic acids, hydroxamic acids, and phosphonic acids on metalized thermoplastic films. The self-assembling monolayers have receptive material bound thereto. Reference is made to U.S. Pat. No. 5,922,550 for a more detailed description of such self-assembling monolayers and methods for producing the monolayers. The '550 patent is incorporated herein in its entirety for all purposes.
The mask may be formed of any suitable material that protects or blocks portions of the underlying substrate from the irradiating stimulus or energy source. A material that has proven useful for defining patterns of active and inactive receptive material regions on a gold-plated MYLAR® film coated with an antibody solution is a transparent or translucent polymer film (such as MYLAR®) having a pattern of shielded or protected regions printed thereon. This type of mask is useful for light sources with a wavelength equal to or greater than about 330 nanometers. For light sources having a wavelength below about 330 nanometers, a quartz or fused silica mask having chrome plated shielded regions defined thereon may be used. The mask may define any pattern of active and inactive regions of the receptive material. The active regions creating the visible diffraction pattern may be of virtually any size, shape, and pattern. It may be desired to select a size and pattern so as to maximize the visible diffraction contrast between the active and inactive regions. As one example, it has been found suitable if the active regions are defined as generally circular with a diameter of about 10 microns and spaced from each other by about 5 microns.
FIG. 1 is a schematic representation of one method for producing biosensors according to the invention. Step A represents a receptive material layer 2 applied to a substrate member 4. Step B represents a crosslinking agent 6 as described above applied to the substrate member 4 such that the crosslinking agent 6 generally uniformly covers the receptive material 2. Step C represents a mask 8 disposed over the substrate member 4. The mask 8 includes exposed or open regions 12 and shielded or protected regions 10 defined thereon. Step D represents the mask 8 and substrate member 4 combination being irradiated with a light source 14. It can be seen that the areas of the substrate member 4 underlying the shielded regions 10 of the mask 8 are protected from the light source 14. The crosslinking agent 6 exposed to the light source 14 through the open regions 12 of the mask 8 is activated by the energy source 14 so that the ends of the cross-linking agent 6 affix to the individual biomolecules of the receptive material 2. Activated or crosslinked areas 16 are defined in a pattern corresponding to the pattern of the exposed regions 12 of the mask 8, and unactivated or uncrosslinked areas 18 are defined in a pattern corresponding to the pattern of the shielded regions 10 of the mask 8. Step E represents the biosensor after the mask 8 is removed and prior to the step of removing or disassociating the unactivated crosslinking agent 6 from the regions 18. Step F represents the biosensor in its final form after the unactivated crosslinking agent 6 has been removed (for example, by washing). The biosensor includes active areas 20 of the receptive material and inactive areas 22.
The biosensors according to the invention have a wide range of uses in any number of fields. The uses for the biosensors of the present invention include, but are not limited to, detection of chemical or biological contamination in garments, such as diapers, generally the detection of contamination by microorganisms in prepacked foods such as meats, fruit juices or other beverages, and the use of the biosensors of the present invention in health diagnostic applications such as diagnostic kits for the detection of proteins, hormones, antigens, nucleic acids, DNA, microorganisms, and blood constituents. The present invention can also be used on contact lenses, eyeglasses, window panes, pharmaceutical vials, solvent containers, water bottles, band-aids, wipes, and the like to detect contamination. In one embodiment, the present invention is contemplated in a dipstick form in which the patterned substrate is mounted at the end of the dipstick. In use the dipstick is dipped into the liquid in which the suspected analyte may be present and allowed to remain for several minutes. The dipstick is then removed and then, either a light is projected through the substrate or the substrate is observed with a light reflected from the substrate. If a diffraction pattern is observed, then the analyte is present in the liquid.
In another embodiment of the present invention, a multiple analyte test is constructed on the same support. A strip may be provided with several patterned substrate sections. Each section has a different receptive material that is different for different analytes. It can be seen that the present invention can be formatted in any array with a variety of patterned substrates thereby allowing the user of the biosensor device of the present invention to detect the presence of multiple analytes in a medium using a single test.
In yet another embodiment of the present invention, the biosensor can be attached to an adhesively backed sticker or decal which can then be placed on a hard surface or container wall. The biosensor can be placed on the inside surface of a container such as a food package or a glass vial. The biosensor can then be visualized to determine whether there is microbial contamination.
It should be understood that resort may be had to various other embodiments, modifications, and equivalents of the embodiments of the invention described herein, which, after reading this description of the invention, may suggest themselves to those skilled in the art without departing from the scope and spirit of the present invention.

## Claims

1. A biosensor, comprising:
a substrate member;
a receptive material layer covering a side of said substrate member, said receptive material being specific for an analyte of interest;
a pattern of at least one active area comprising said receptive material and optionally a crosslinking agent and at least one inactive area comprising said receptive material and crosslinking agent, said receptive material in said inactive area rendered inactive by said crosslinking agent;
wherein said active and inactive areas are defined in a masking process wherein the crosslinking agent is applied over said receptive material layer and a mask is then placed over said substrate member prior to exposing said substrate member to a stimulus for activating said crosslinking agent, areas underlying shielded regions of the mask defining said active areas and areas exposed by the mask having the crosslinking agent activated by the stimulus and defining said inactive areas, said crosslinking agent only crosslinked in said inactive areas to form an inactivating crosslinking agent; and
wherein when said biosensor is exposed to a medium containing said analyte of interest, the analyte binds to said receptive material in said active areas and facilitates subsequent diffraction of transmitted or reflected light in a diffraction pattern corresponding to said active areas.

2. The biosensor as in claim 1, wherein said pattern of active and inactive areas comprises a plurality of said active areas and a plurality of said inactive areas defined by said masking process.

3. The biosensor as in claim 1, wherein said active areas lack a portion of said crosslinking agent removed in a washing process.

4. The biosensor as in claim 1, wherein said crosslinking comprises a homobifunctional agent in which both ends of said agent are photosensitive, said stimulus being a light source at a wavelength sufficient to activate said ends of said cross-linking agent.

5. The biosensor as in claim 4, wherein said crosslinking agent comprises (bis-[β-(4-azidosalicylamido)ethyl]disulfide).

6. The biosensor as in claim 1, wherein said substrate comprises a material from the list of materials consisting of plastics, metal coated plastics and glass, functionalized plastics and glass, silicon wafers, glass, and foils.

7. The biosensor as in claim 1, wherein said substrate member comprises a polymer film coated with a metal.

8. The biosensor as in claim 7, wherein said polymer film comprises polyethylene-terephthalate.

9. The biosensor as in claim 7, wherein said metal is selected from the group consisting of gold, silver, chromium, nickel, platinum, aluminum, iron, copper, gold oxide, titanium, titanium oxide, chromium oxide or zirconium.

10. The biosensor as in claim 7, wherein said metal is gold.

11. The biosensor as in claim 1, wherein said diffraction pattern is visible to the naked eye.

12. The biosensor as in claim 1, wherein said receptive material is at least one of antigens, antibodies, nucleotides, chelators, enzymes, bacteria, yeasts, fungi, viruses, bacterial pili, bacterial flagellar materials, nucleic acids, polysaccharides, lipids, proteins, carbohydrates, metals, hormones, aptamers, peptides, and respective receptors for said materials.

13. The biosensor as in claim 1, wherein said analyte of interest is at least one of a bacteria, yeast, fungus, virus, rheumatoid factor, IgG, IgM, IgA, IgD, and IgE antibodies, carcinoembryonic antigen, streptococcus Group A antigen, viral antigens, antigens associated with autoimmune disease, allergens, tumor antigens, streptococcus group B antigen, HIV I or HIV II antigen, antibodies viruses, antigens specific to RSV, an antibody, antigen, enzyme, hormone, polysaccharide, protein, lipid, carbohydrate, drug, nucleic acid, Neisseria meningitides groups A, B, C, Y and W sub 135, Streptococcus pneumoniae, E. coli K1, Haemophilus influenza type A/B, an antigen derived from microorganisms, PZA and CRP antigens, a hapten, a drug of abuse, a therapeutic drug, an environmental agents, or antigens specific to Hepatitis.