Title of Invention

A METHOD OF DETECTING AN ANALYTE

Abstract

The present invention provides an inexpensive and sensitive system and method for detecting analytes present in a medium. The system comprises a diffraction enhancing element, such as functionalized micropsheres, which are modified such that they are capable of binding with a target analyte. Additionally, the system comprises a polymer film, which may include a metal coating, upon which is printed a specific, predetermined pattern of analyte-specific receptors. Finally, the system includes a wicking agent which permits the system to be a single step system which avoids the necessity of any additional rinsing steps. Upon attachment of a target analyte to select areas of the polymer film, either directly or with the diffraction enhancing element, diffraction of transmitted and/or reflected light occurs via the physical dimensions and defined, precise placement of the analyte. A diffraction image, such as a hologram, is produced which can be easily seen with the eye or optionally, with a sensing device.

Full Text

USE OF WICKING AGENT TO ELIMINATE WASH STEPS FOR OPTICAL DIFFRACTION-BASED BIOSENSORS TECHNICAL FIELD The present invention is generally in the field of detecting analytes in a medium and, more particularly, the present invention relates to the methods of making optical diffraction-based sensors which are capable of indicating the presence of the analyte in a medium. BACKGROUND OF THE INVENTION There are many systems and devices available for detecting a wide variety of analytes in various media. Most of these systems and devices are relatively expensive and require a trained technician to perform the test. There are many cases where it would be advantageous to be able to rapidly and inexpensively determine if an analyte were present. What is needed is a biosensor system that is easy and inexpensive to manufacture and is capable of reliable and sensitive detection of analytes, including smaller analytes. Sandstrom et al., 24 Applied Optics 472, 1985, describe use of an optical substrate of silicon with a layer of silicon monoxide and a layer of silicon formed as dielectric films. They indicate that a change in film thickness changes the properties of the optical substrate to produce different colors related to the thickness of the film. The thickness of the film is related to the color observed and a film provided on top of an opdcal substrate may produce a visible color change. The authors indicate that a mathematical model can be used to quantitate the color change, and that "[c]alculations performed using the computer model show that very little can be gained in optical performance from using a multilayer structure... but a biolayer on the surface changes the reflection of such structures very little since the optical properties are determined mainly by the interfaces inside the multilayer structure. The most sensitive system for detection of biolayers is a single layer coating, while in most other applications performance can be by additional dielectric layers." Sandstrom et al., go on to indicate that slides formed from metal oxides on metal have certain drawbacks, and that the presence of metal ions can also be harmful in many biochemical applications. They indicate that the ideal top dielectric film is a 2-3 nm thickness of silicon dioxide which is formed spontaneously when silicon monoxide layer is deposited in ambient atmosphere, and that a 70-95 nm layer silicon dioxide on a 40-60 nm layer of silicon monoxide can be used on a glass or plastic substrate. They also describe formation of a wedge of silicon monoxide by selective etching of the silicon monoxide, treatment of the silicon dioxide surface with dichlorodimethylsilane, and application of a biolayer of antigen and antibody. From this wedge construction they were able to determine film thickness with an ellipsometer, and note that the "maximum contrast was found in the region about 65 nm where the interference color changed from purple to blue." They indicate that the sensitivity of such a system is high enough for the detection of protein antigen by immobilized antibodies. They conclude "the designs given are sensitive enough for a wide range of applications. The materials, i.e., glass, silicon, and silicon oxides, are chemically inert and do not affect the biochemical reaction studied. Using the computations above it is possible to design slides that are optimized for different applications. The slides can be manufactured and their quality ensured by industrial methods, and two designs are now commercially available. U.S. Patent 5,512,131 issued to Kumar et al. describes a device that includes a polymer substrate having a metal coaling. An analyte-specific receptor layer is stamped on the coated substrate. The device is used in a process for stamping or as a switch. 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. However, the device described by Kumar et al. has several disadvantages. One disadvantage is that an extra visualization device is needed to view any diffraction pattern. By requiring a visualization device, the Kumar et al. device does not allow a large number of samples to be tested since it is not possible to determine the presence of an analyte by using the unaided eye. Additionally, this device is not able to detect smaller analytes as these analytes do not produce a noticeable diffraction pattern. U.S. Patent 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 spectra! distribution of the emanating light. The substrate also exhibits a second color which is different from the first color (by having a combination of wavelengths of light which differ from that combination present in the first color, or having a different spectral distribution, or by having an intensity of one or more of those wavelengths different from those present in 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 eye. Such sensitive detection is an advance over the devices described by Sandstrom and Nygren, supra, and allow use of the devices in commercially viable and competitive manner. However, the method and device described in the Bogart, et al. patent has several disadvantages. One disadvantage is the high cost of the device. Another problem with the device is the difficulty in controlling the various layers that are placed on the wafer so that one obtains a reliable reading. Additionally, biosensors having a self-assembling monolayer have been used to detect analytes and are set forth in U.S. Patent Application Nos. 08/768,449 and 08/991,844, both of which are incorporated herein by reference in their entirety. However, these biosensors currently do not have the requisi"te sensitivity required to detect smaller analytes since these smaller analytes do not produce a sufficient diffraction pattern to be visible. Some commercial lateral flow technologies have been used which employ latex bead technology. These technologies are currently employed in most of the commercially-available home diagnostic kits (e.g. pregnancy and ovulation kits). These kits use colored beads which accumulate in a defined "capture zone" until the amount of beads becomes visible to the unaided eye. However, these systems lack the requisite sensitivity to test for many analytes, since a much larger number of latex beads must bind in the capture zone to be visible to the naked eye than that required to cause diffraction in the same size zone. Theoretically, the number of beads needed is about 2 to 3 orders of magnitude higher than the sensors of the present invention. Biosensors having a self-assembling monolayer and using microparticle technology have been used to detect smaller analytes and are set forth in U.S. Patent Application No. 09/210,016, which is incorporated herein by reference in its entirety. However, these biosensors require multiple process steps to produce, thereby increasing the difficulty and cost for using these types of sensors.. What is needed is a biosensor system that is easy and inexpensive to manufacture and is capable of reliable and sensitive detection of analytes, including smaller analytes. SUMMARY OF THE INVENTION The present invention provides an inexpensive and sensitive system and method for detecting analytes present in a medium. The invention provides a new approach to reduce the number of steps involved by a user of diffraction diagnostic devices using "diffraction enhancing elements," such as microparticles. The approach involves the use of a wicking agent that is used to remove unbound labeled microparticles, as well as any residual liquid from the sample. The wicking agent then avoids the necessity of any additional rinsing, which may be cumbersome or more difficult for a user. Additionally, a small hole (e.g., 3/32 of an inch) can be punched out of the center of the wicking agent so that once the sample and excess panicles are wicked away, the hole allows the user to immediately check for a diffraction image without removing the wicking matenal. Examples of wicking agents include nitrocellulose membranes, cellulose acetate membranes, and glass microfiber structures In addition, the pore size of the membrane may be varied to control the rate and force of wicking. This can affect the accuracy of the diagnostic device, and can also be taken advantage of to create a one-step device. To achieve this the one-step device consists of the contact printed capture antibody on a substrate, such as the gold/MYLAR®, which then has labeled particles pre-dried onto its surface. Additionally, a small pore size membrane (e.g., 0.45 micron nitrocellulose) with a hole cut out is placed on top of the device to complete it. The user simply adds the sample (e.g., serum or blood) to be tested, and then views for a diffraction-image once wicking has occurred The small pore size delays wicking long enough to allow adequate incubation, such as that needed for antibody-antigen interactions to take place. Alternatively, wicking may be delayed by using an erodible reagent at the periphery of the wicking agent circle. The reagent would eventually dissolve or derivatize so that it would allow wicking after a specific time period The system of the present invention is much more sensitive than current inexpensive systems. The system of the present invention is able to detect low to high molecular weight analytes, microorgariisms, and DNA or RNA species in fluid samples. More specifically, the system is able to detect hormones, steroids, antibodies, drug metabolites, and even nucleic acids, among others. This is a significant expansion of the optical diffraction-based sensing technology set forth in U.S. Patent Application Nos. 08/768,449 and 08/991,844. The present invention utilizes diffraction enhancing elements, such as latex microspheres, which aid in the detection of smaller analytes. Normally, after an analyte binds to an analyte-specific receptor on a biosensor, the analyte will diffract or reflect transmitted light to produce a diffraction pattern. If the analyte is larger, the diffraction pattern is able to be seen with the unaided eye. However, some analytes are too small such that the diffraction pattern produced is not able to be seen. By using diffraction enhancing elements, the biosensor having the analyte-specific receptor material may be used to detect these smaller analytes. The diffraction enhancing elements used are capable of binding to the analyte, and then the element with bound analyte binds to the biosensor. Then, as the light is transmitted through or reflected from the biosensor, the element enhances the diffraction pattern generated by the analyte such that the resulting diffraction pattern may be seen by the unaided eye. The present invention also utilizes methods of contact printing of patterned, analyte-specific receptors. The analyte-specific receptors have receptive materials bound thereto. The receptive materials are specific for a particular analyte or class of analyte, depending upon the receptor used. Methods of contact printing which would be useful in generating the sensing devices used in the present system are disclosed fully in U.S. Patent Application Nos. 08/707,456 and 08/769,594, both of which are incorporated herein by reference in their entirery. However, since these methods relate to self-assemblin monolayers, the methods need to be altered slightly, as discussed below, to print the analyte-specific receptor material as this material is not self-assembling. Patterned analyte-specific receptor layers allow for the controlled placement of analytes with or without diffraction enhancing elements thereon via the patterns of analyte-specffic receptors. The biosensing devices of the present invention produced thereby are used by first exposing the biosensing device to the sample medium (that may or may not contain the analyte of choice) mixed with the diffraction enhancing element. Then, after an appropriate incubation period, a light, such as a laser or other point light source, is transmitted through or reflected from the film. If the analyte is present in the medium and is bound, either directly or in conjunction with the diffraction enhancing element, to the receptors on the patterned analyte-specific receptor layer, the light is diffracted in such a way as to produce a visible image. In other words, the analyte-specific receptor layers with the analyte and/or diffraction enhancing element bound thereto can produce optical diffraction patterns which differ depending on the reaction of the receptors on the analyte-specific receptor layer with the analyte of interest. The light can be in the visible spectrum, and be either reflected from the film, or transmitted through it, and the analyte can be any compound or particle reacting with the analyte-specific receptor layer. The light can be a point white light source or monochromatic electromagnetic radiation in the visible region. While visible light is the desired light source, the present invention may also be used with non-visible point light sources, such as near-infrared light, coupled with a detector. The thickness of the film and the size of the microparticle may be adjusted to compensate for the non-visible light source. Additionally, the present invention also provides a flexible support for an analyte-specific receptor layer either directly on the substrate or on gold or other suitable metal or metal alloy. The present invention provides an analyte-specific receptor layer on gold or other material which is suitable for mass production. The biosensors used in 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 biosensors of the present invention can be used to detect (1) antigens or antibodi"es associated with medical conditions, (2) contamination in garments, such as diapers, and (3) contamination by In another embodiment of the present invention, nutrients for a specific class of microorganisms can be incorporated into the analyte-specific receptor 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, if necessary, 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. 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 FGURES Figure 1 shows a biosensor capable of simultaneously measuring several different analytes in a medium. Figure 2 is a schematic of contact printing of analyte-specific receptor layers. Figure 3 is an atomic force microscopy image of evaporated gold on MYLAR®, purchased from Courtaulds Performance Films (Canoga Park, CA). The average roughness of the gold layer is 3-4 nanometers, with maximum roughness of 9 nanometers. Figure 4 is an SEM photomicrograph showing patterned attachment of diffraction enhancing elements in the presence of an analyte,. Figure 5 is a schematic of the present invention using the wicking agent to eliminate the need for rinsing steps. Figure 6 is a schematic of the one-step product design of the present invention. Figure 7 shows a light source illuminated through the hole of the wicking agent to check for a diffraction image. DETAILED DESCRIPTION The present invention features an improved method for making biosensing devices. The present invention may be used to make biosensing devices which are much more sensitive and can be used to detect smaller analytes which, until now, were not able to be detected without the use of expensive instruments. The analytes that can be detected include, but are not limited to, hormones, proteins such as antibodies, steroids, drug metabolites, nucleic acids, microorganisms such as bactena. yeasts, fungi and viruses. In contrast to prior devices, those made by the present invention allow detection of extremely small quantities and sizes of analytes in a medium in a rapid assay lasting only a few minutes. In addition, no signaling or associated electronic components are required. In the present invention, a biologically active material is deposited onto a metal surface, such as gold, in a defined pattern, such that a diffraction hologram is generated when the target binds to the surface. Typically, antibodies that specifically react with a target molecule are printed in a pattern on a metal-coated surface. In one embodiment, the invention covers a method to make a one-step device. The method involves pre-drying labeled diffraction enhancing elements or microparticles (e.g., labeled with the allergen of interest) on to the patterned surface, and then placing the wicking agent disk on top prior to use. This device is then exposed to the test fluid (e.g., serum or blood containing allergen-specific IgE) by placing it on top within the area of the hole cut from the wicking agent. The proper selection of pore size and wicking agent allows the time for wicking to be tailored to the desired incubation time. For example, a 0.45 micron pore size nitrocellulose has delayed wicking of diluted serum with 0.3 micron diameter microparticles for 8-12 minutes; this gives adequate time for the diagnostic device to work. In another embodiment, the invention includes the use of an erodible reagent at the periphery of the wicking agent circle to inidally prevent wicking from occurring. The reagent could be a hydrophobic material that prevents wicking, but that would eventually dissolve or derivatize so that it would allow wicking after a specific time period. This time period would correspond to the desired incubation period. The present invention modifies the process to use the biosensing device to make it easier for the end-user. It employs a wicking agent, such as nitrocellulose, to remove unbound diffraction enhancing elements and excess fluid, therebv eliminating the need for rinsing steps. This greatly simplifies an immunoassay approach, since rinsing is often the most cumbersome step. The detection system is also unique from commercial immunoassay systems in that the binding event creates a simple holographic image upon exposure to light. 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 fronn one pattern to another upon binding of the analyte to the receptive material. In particularly preferred embodiments, the diffraction pattern is 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 and/or element should have a minimum periodicity of the wavelength of incident light. Very small analytes can be detected indirectly by using diffraction enhancing element panicles that are specific for the small analyte. One embodiment in which the small analyte can be detected comprises coating the element particle, such as a latex bead, with a receptor material that specifically binds to the analyte of interest. A variety of methods may be used to attach the receptor material onto the diffraction enhancing particle. These methods include, but are not limited to, simple physisorption to a hydrophobic particle (e.g., binding a protein onto polystyrene particles); binding using a protein A or protein G linker; binding using a streptavidin or avidin-biotin linker; or binding using covalent attachment. A preferred embodiment of the present invention is to use carbodiimide coupling of a proteinaceous receptor to carboxylated particles. Other methods of coupling well-known to those of ordinary skill in the art may be used as well. Diffraction enhancing element particles that can be used in the present invention include, but are not limited to, glass, cellulose, synthetic polymers or plastics, latex, polystyrene, polycarbonate, bacterial or fungal cells and the like. The particles are preferably spherical in shape, but the structural and spatial configuration of the particle is not critical to the present invention. For instance, the particles could be slivers, ellipsoids, cubes, and the like. A desirable particle size ranges from a diameter of approximately 0.1 µm to 100.0 µm, desirably between approximately 0.1 µm to 1µm. The composition of the element particle is not critical to the present invention. Preferably, the difference in refractive index between the medium and the enhancing element is between 0.1 and 1.0. More preferably, the difference in refractive index between the medium and the enhancing element is between 0.2 and 0.7 The analyte-specific receptor layer on the polymer film contains a receptive material, such as an antibody, that will specifically bind to an epitope on the analyte that is different from the epitope used in the binding to the particle. Thus, for detecting a small analyte, such as viral panicles, the medium" is first exposed to the diffraction enhancing element particles, such as latex particles, to which the viral particles bind. Then, the diffraction enhancing element particles are optionally washed and exposed to the polymer film with the analyte-specific receptor layers containing the virus specific antibodies. The antibodies then bind to the viral particles on the element particle thereby immobilizing the element particles in the same pattern as the receptors on the film Because the bound element particles will cause diffraction of the visible light, a diffraction pattern is formed, indicating the presence of the viral particle in the liquid. Addidonally, the polymer film may include a metal coating thereon. The analyte-specific receptor layer would then be located on the metalized surface of the film. Alternatively, the analyte may be detected by first exposing the biosensor comprising the polymer film with the analyte-specific receptor layers containing the antibodies to the medium containing the analyte and causing the analyte to bind to the analyte-specific receptor layer material. Next, a suspension containing the diffracdon enhancing element particles is contacted with the sensing device having the analyte bound thereto. The particles then bind to the analyte. Because the bound element panicles will cause diffracdon of the visible light, a diffraction pattern is formed, indicating the presence of the anaiyte in the liquid. In another preferred embodiment, the biosensor, the diffraction enhancing element particles and the medium containing the anaiyte may be admixed simultaneously. This will result in a combination of the binding procedures discussed above. Some of the analytes will first bind with a diffraction enhancing element particle prior to binding to the substrate. Other analytes will first bind with the substrate and then bind with an element particle. When a point-light source is shone through the sensor, a diffraction pattern is formed, indicating the presence of the anaiyte in the liquid. Finally, in a simpler embodiment, the diffraction enhancing element particles are pre-dried on the biosensor as pan of the preparation. During use, the medium containing the anaiyte is placed on the biosensor surface. This causes re-suspension of the particles, which then bind in the patterned receptor areas of the film if the anaiyte is present. The analytes that are contemplated as being detected using the present invention include, but are not limited to, bacteria; yeasts; fungi; viruses; protozoa; or antigens specific to these microbes; rheumatoid factor; antibodies, including, but not limited to IgG, IgM, IgA and IgE antibodies: carcinoembryonic antigen; streptococcus Group A antigen; viral antigens; antigens associated with autoimmune disease; allergens: njmor antigens; streptococcus Group B antigen; HIV I or HTV II antigen; or host response (antibodies) to these and other viruses: andgens specific to RSV or host response (antibodies) to the virus; an 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; Salmonella species; Neisseria meningitides groups A, B, C, Y and W sub 135, Streptococcus pneumoniae, E. coli KL Haemophilus influenza type B; n antigen derived from microorganisms; a hapten, a drug of abuse; a therapeutic drug; an environmental agent; and antigens specific to Hepatitis. In another embodiment of the present invention, nutrients for a specific class of microorganisms can be incorporated into the analyte-specific receptor 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 unful 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 on the patterned monolayer. A part of the present invention is the analyte- specific receptor material that can be microprinted on the polymer film and will specifically bind to the analyte of interest. Thus, the receptor material is defined as one part of a specific binding pair and includes, but is not limited to, antigen/ antibody, enzyme/substrate, oligonucleoude/DNA, chelator/metal. enzyme/inhibitor, bacteria/receptor, virus/receptor, hormone/receptor, DNA/RNA, or RNA/RNA, oligonucleotide /RNA, and binding of these species to any other species, as well as the interaction of these species with inorganic species. Additionally, when a metalized polymer film is used, the analyte-specific receptor material can be microprinted on the metalized surface of the film. The receptor material that is bound to the attachment layer is characterized by an ability to specifically bind the analyte or analytes of interest. The variety of materials that can be used as receptor material are limited only by the types of material which will combine selectively (with respect to any chosen sample) with the analyte. Subclasses of materials which can be included in the overall class of receptor materials include toxins, antibodies, 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, 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 attachment layer to produce a thin film assay system. Whatever the selected analyte of interest is, the receptor material is designed to bind with the analyte of interest. In the preferred embodiments, the biosensing device is configured and arranged to provide a pattern detectable by eye in response to transmission of a point light source when the analyte of interest is sandwiched between the receptor material and a diffraction enhancing element. In many instances, a "blocker" may be necessary to prevent non-specific binding. The term "blocker" as used herein means a reagent that adheres to the sensor surface so that it "blocks" or prevents non-analyte materials from binding to the surface (either in the patterned or un-pattemed areas). The blocking step may be done as a post-treatment to a surface which has already been contact printed ("post-block"), and is the standard technique for filling in non-contact printed regions with another thiol. However, the inventors have discovered that a "pre-block" technique is preferred over the post-block technique. In the pre-block technique, the surface of the substrate is pre-treated with a non-thiol containing blocker and then contact printed. Not wishing to be bound to any theory, it is theonzed that the contact printed material (usually sulfur containing) displaces the physisorbed blocker, thereby permitting the analyte-specific receptor material to be bound directly to the surface of the substrate. A subsequent post-block may also be performed, if desired. Blockers can include, but are not Limited to, β-casein, albumins such as bovine serum albumin, pluronic or other surfactants, polyethylene glycol or its derivatives, polyvinyl alcohol, or derivatives of the above compounds, and any other blocking material known to those of ordinary skill in the art. The matrix containing the analyte of interest may be an interstidal fluid, a solid, a gas, or 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, a throat swab 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, dmg, or any other material. For example, receptor 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 indicative of the bacteria may be a saccharide or polysaccharide, an enzyme, a nucleic acid, a membrane component, a ganglioside or an antibody produced by the host in response to the bacteria. The presence of the analyte may indicate an infectious disease (bacterial or viral), cancer, an allergy, or other medical disorder or condition. The presence of the analyte may be an indication of water or food contamination or other harmful materials. 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 receptor material and/or it may be the analyte of interest. The receptor material, for example an antibody or an andgen, must form a stable, reactive layer on the attachment layer of the test device. If an antibody is the receptor material, the antibody must be specific to the antigen of interest; and the antibody (receptor 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 receptor material, but may cause a detectable modification of the receptor material to occur. This interaction could cause an increase in mass at the test surface or a decrease in the amount of receptor 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 receptor material occurs is not important to this invention, but may impact the reaction conditions used in the final assay protocol. In general, the receptor material may be passively applied to the substrate layer. If required, the free functional groups introduced onto the test surface by the attachment layer may be used for covalent attachment of receptor material to the lest surface. A wide range of techniques can be used to apply the receptor material to the substrate layer. Test surfaces may be coated with receptor material by application of solution in discrete arrays or patterns; spraying, ink jet, contact printing or other imprinting methods; or printing a blocker material in a pattern followed by total immersion or spin coating with the receptor material. The technique selected should minimize the amount of receptor material required for coating a large number of test surfaces and maintain the stability/functionality of receptor material during application. The technique must also apply or adhere the receptor material to the attachment layer in a very uniform and controlled fashion. The biosensing device of the present invention utilizes methods of contact printing of patterned, analyte-specific receptor layers on polymer or metalized polymer films, desirably transparent or semi-transparent, the compositions produced thereby, and the use of these compositions. Patterned analyte-specific receptor layers allow for the controlled attachment (or binding) placement of the analyte receptor. The term "patterned analyte-specific receptor layers thereon" as used herein means the analyte-specific receptor layers in any pattern on the polymer or metalized polymer films. The biosensing device also includes the wicking agent which removes any residual liquid from the analyte sample, thereby avoiding the necessity of any additional rinsing. When the film with the patterned analyte-specific receptor layers thereon is exposed to an analyte that is capable of reacting with the analyte-specific receptor layer, the film will produce optical diffraction patterns which differ depending on the reaction of the patterned analyte-specific receptor layer with the analyte of interest. The medium would contain the diffraction enhancing element particles. The medium may be a high surface tension fluid such as water. The light can be in the visible spectrum, and be either reflected from the film, or transmitted through it, and the analyte can be any compound reacdng with the analyte-specific receptor layer. In preferred embodiments, the method involves contacting the sensing device with a test sample containing the diffraction enhancing element particles and potentially containing the analyte. Then, the wicking agent is used to remove unbound labeled particles, as well as any residual liquid from the sample. If the analyte is present in the sample, then when light is transmitted through a metalized polymer film with the analyte-specific receptor layer, a visible diffraction image is formed. The medium in which the analyte may reside can be solid, gel-like, liquid or gas. For purposes of detecting an analyte in a body fluid, the fluid is selected from, but not limited to, urine, serum, plasma, spinal fluid, sputum, whole blood, saliva, uro-genital secretions, fecal extracts, pericardial, gastric, peritoneal, pleural washes, vaginal secretions, or a throat swab. The most common gas that is contemplated as being used with the biosensing device of the present invention is air. In one embodiment, the present invention is contemplated in a dipstick form in which a micro-contact printed metalized film 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. The liquid would also contain the diffraction enhancing element particles. The dipstick is allowed to remain for several minutes. Upon removing the dipstick, the wicking agent is then used to remove unbound labeled microparticles. as well as any residual liquid from the sample. A small hole may be punched out of the center of the wicking agent so that once the sample and excess particles are wicked away, the hole allows the user to immediately check for a diffraction image without removing the wicking material. The light is projected through the metalized film or the film is observed with a light behind the film. If a diffraction image 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. As shown in Figure 1, a strip 10 is provided with several micro-contact printed films 20, 25, 30 and 35, each film having a pattern 40 printed thereon. Each of the micro-contact printed films 15, 20, 25, and 30 have a different receptor material that is specific for different analytes. Each of the printed films 15, 20. 25 and 30 may include an array or strip of wicking agents to aid in the use of the strip 10. It can be seen that the present invention can be formatted in any array with a variety of micro-contact printed films 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 while the wicking agent avoids the necessity of any additional rinsing steps, which may be cumbersome or more difficult for the user. There are many possible supports for the analyte-specific receptor layers. Simple physisorption can occur on many materials, such as polystyrene, glass, nylon, or others well known to those of ordinary skill in the art. Preferred embodiments of immobilizing the analyte-specific receptor layers have involved covalent attachment, such as that possible between thiol or disulfide-containing compounds and gold. Typically, a gold coating, 5 to 2000 nm thick, is supported on a Si/SiCb wafer, glass, or a polymer film. Optionally, titanium can be used to serve as an adhesion promoter between gold and the suppon. The analyte-specific receptor attaches to the gold surface during contact printing or immersion from a solution. Preferably, the support comprises a gold coating on a MYLAR® film. Figure 2 outlines the procedure used for microcontact printing. An elastomeric stamp is used to transfer analyte-specific receptor "ink" to a gold surface by contact: il the stamp is patterned, a pattemed analyte-specific receptor layer forms. The stamp is fabricated by casting polydimethylsiloxane (PDMS) on a master having the inverse of the desired pattern. Masters are prepared using standard photolithographic techniques, or constructed from existing materials having microscale surface features. In a preferred embodiment of a typical experimental procedure, a photolithographically produced master is placed in a glass or plastic Petri dish, and a 10:1 ratio (w:w) mixture of SYLGARD® silicone elastomer 184 and SYLGARD® silicone elastomer 184 curing agent (Dow Coming Corporation) is poured over it. The elastomer is allowed to sit for approximately 30 minutes at room temperature and reduced pressure to degas, then cured for at least 4 hours at 60°C, and gently peeled from the master. "Inking" of the elastomeric stamp is accomplished by exposing the stamp to a 0.1 to 10 µM aqueous solution of disulfide-derivatized antibody typically by placing the stamp face down in the solution for 10 seconds to 10 minutes. The stamp is allowed to dry, either under ambient conditions, or typically by exposure to a stream of air or nitrogen gas.. Following inking, the stamp is applied to a gold surface. Light pressure is used to ensure complete contact between the stamp and the surface. After 1 second to 5 minutes, the stamp is then gently peeled from the surface. Following removal of the stamp, the surface is rinsed and dried. Alternatively, further derivatization of unstamped areas can be accomplished, either by using a second stamp or by exposing the entire surface with a different reagent. Subsequently, exposure to a protein-blocking agent, such as BSA or β-casein, or any other agent well known in the art, can also be done. The elastomeric character of the stamp is important to the success of the process. Polydimethylsiloxane (PDMS), when cured, is sufficiendy elastomeric to allow good conformal contact of the stamp and the surface, even for surfaces with significant relief; this contact is essential for efficient contact transfer of the receptor to a gold film. The elastomeric properties of PDMS are also important when the stamp is removed from the master: if the stamp were rigid (as is the master) it would be difficult to separate the stamp and master after curing without damaging one of the two substrates. PDMS is also sufficiently rigid to retain its shape, even for features with sub-micron dimension. The stamp is durable in that the same stamp can be used over 200 times over a period of a year without significant degradation in performance. Using a printing roll for the stamp could allow for a continuous printing operation. Alternatively, ink-jet printing of the desired pattern could also be done if capable of producing the feature sizes needed for diffraction, for example A more detailed description of the methods and compositions of the present invention follows. All publications cited herein are incorporated by reference in their entirety. Any plastic film is suitable for the present invention. Preferably, the plastic film is also capable of having a metal coating deposited thereon. These include, but are not limited to polymers such as: polyethylene-terephthalate (e.g., 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, nylon, polypropylene, methyl pentene polymers, polyvinyl fluoride, and aromatic polysulfones. Preferably, the plastic film has an optical transparency of greater than 80%. Other suitable plastics and suppliers may be found, for example, in reference works such as the Modem Plastics Encyclopedia (McGraw-Hill Publishing Co., New York 1923-1996). In one embodiment of the invention, the polymer film has a metal coating thereon and has an optical transparency of between approximately 5% and 95%. A more desired optical transparency for the plastic film used in the present invention is between approximately 20% and 80%. In a desired embodiment of the present invention, the polymer film has at least an approximately 80% optical transparency, and the thickness of the metal coating is such as to maintain an optical transparency greater than about 60%, so that diffraction images can be produced by transmitted light. This corresponds to a metal coating thickness of about 10 nm. However, in other embodiments of the invention, the gold thickness may be between approximately 1 nm and 1000 nm; for example, thicker gold coatings (>20 nm) would still be suitable for producing diffraction images by reflected light. The preferred metal for deposition on the film is gold. However, silver, aluminum, chromium, copper, iron, zirconium, platinum and nickel, as well as oxides of these metals, may be used. In principle, any surface with corrugations of appropriate size could be used as masters. The process of microcontact printing starts with an appropriate relief structure, from which an elastomeric stamp is cast. This "master" template may be generated photolithographicaily, or by other procedures, such as commercially available diffraction gratings. In one embodiment, the stamp may be made from polydimethylsiloxane. The stamp may be applied in air, or under a fluid capable of preventing excess diffusion of the receptor material. For large-scale or continuous printing processes, it is most desirable to print in air. In one embodiment of the present invention, the pattern is formed on the metalized plastic polymer with the analyte-specific receptor layer. After the stamping process, the metalized areas on the plastic may optionally be blocked, for example, with a protein-repelling agent such as β-casein. This invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof, which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention. EXAMPLES Example #1: Gold-coated MYLAR® was treated with a 5 mg/mL beta casein diluted in StabilGuard® (by SurModics, Inc.; Eden Prairie, MN) as a blocking agent. The casein treated film was then contact printed with a thiolated antibody to IgE (e.g., a monoclonal anti-IgE, such as one specific to the C3-C4 domains of IgE) to provide a patterned x,y array of antibody in 10-micron circles. Further preparation of this one-step device involved applying 11 microliters of mold-mix labeled particles (0.3 micron particles conjugated with ALK Cat # 512042, suspended in a 5 mg/mL beta casein diluted in StabilGuard®) to this surface, and drying them at ambient conditions. A 0.45-micron pore size nitrocellulose disk with 3/32-inch diameter hole was then placed over the dried particles to complete the diagnostic preparation. This device was tested with samples consisting of IgE-spiked serum at 10 ug/mL IgE which were diluted in 6 parts phosphate buffer solution (pH 8, ionic concentration of 0.1 M). A good source of IgE to use as a positive control was typically a polyclonal source, which showed reactivity to a wide range of allergens. Controls were tested with unspiked serum diluted in the same way as the samples. Testing involved placing 34 microliters of the diluted serum on top of the diagnostic device within the center of the nitrocellulose hole. Wicking is delayed due to the small 045-micron pore size of the material, and typically occurs between 5-15 minutes after adding the diluted serum. This time allows for adequate incubation to occur between the analyte and the diagnostic. If a shorter (or no) delay of wicking is desired, then a larger pore size material can be used as the wicking agent. After wicking has occurred, the sample was checked for a diffraction image using a point light source (e.g., laser) that is illuminated through the hole. A diffraction image indicates that the analyte (e.g., mold mix-specific IgE in this case) is present. Example #2: Gold-coated MYLAR® was treated with a 5 mg/mL buffered solution of beta casein as a blocking agent. The casein treated film was then contact printed with a thiolated antibody to luteinizing hormone (e.g., a monoclonal) to provide a patterned x,y array of antibody in 10-micron circles. The resulting sample was exposed to 60 microliters of LH-spiked buffered BSA solution. This was immediately followed by the addition of 20 microliters of a suspension containing 0.5 micron particles conjugated with another monoclonal to luteinizing hormone antibody (such as a monoclonal that recognizes a different epitope on the hormone than the patterned antibody) at a concentration of 10" or 10"° particles / mL. In some cases, samples were heated to 60C during incubation; incubation times typically range from 5-15 minutes. After incubation, a disk of nitrocellulose (e.g., 8 micron pore size) having a hole (e.g., 3/32 inch diameter) in the center is placed on top of the sample and liquid / particle mixture. This wicks away unbound particles and excess liquid, so that the sample can be checked for a diffraction image using a point light source (e.g., laser) aimed to shine through the hole. A diffraction image indicates that the analyte (luteinizing hormone in this case) is present. Example #3: Gold-coated MYLAR® was treated with a 5 mg/mL buffered solution of beta casein as a blocking agent. The casein-treated film was then contact printed with a thiolated antibody to IgE (e.g., a monoclonal having an affinity constant > 4x10"°) to provide a patterned x,y array of antibody in 10-micron circles. This patterned film was then exposed to 34 microliters of diluted human serum (e.g.. International Enzymes Cat#8005) that had been spiked with IgE. Typical dilution of serum was 1 part spiked serum to 2 parts phosphate buffer solution (pH 7.2). After 5 minutes, 11 microliters of a suspension containing 0.3-micron particles conjugated with another monoclonal antibody to IgE (e.g., a monoclonal anti-IgE, such as one specific to the C3-C4 domains of IgE) was added (typically at a concentration of 10" or 10"° particles / mL). After 10 minutes, a disk of nitrocellulose (e.g., 8 micron pore size) having a hole (e.g., 3/32 inch diameter) in the center was placed on top of the sample and liquid / particle mixture. This wicks away unbound particles and excess liquid, so that the sample can be checked for a diffraction image using a point light source (e.g., laser) aimed to shine through the hole. A diffraction image indicates that the analyte (total IgE in this case) is present. Detection down to at least 1000 ng/mL (initial concentration of IgE in serum) was achieved. Example #4: Gold-coated MYLAR® was treated with a 5 mg/mL buffered solution of beta casein as a blocking agent. The casein-treated film was then contact printed with a thiolated polyclonal antibody to Group B Strep to provide a patterned x,y array of antibody in 10-micron circles. This patterned film was then exposed to 34 microliters of a solution of Strep B antigen (Difco Cat#2979-50; Detroit, MI) for 5 minutes. This was followed by the addition of 11 microliters of a suspension containing 0.3-micron particles conjugated with an antibody to Strep B (typically at a concentration of 10" or 10"° particles / mL). After 10 minutes, a disk of nitrocellulose (e.g., 8 micron pore size) having a hole (e.g., 3/32 inch diameter) in the center was placed on top of the sample and liquid / particle mixture. This wicks away unbound particles and excess liquid, so that the sample can be checked for a diffraction image using a point light source (e.g., laser) aimed to shine through the hole. A diffraction image indicates that the analyte (Strep B antigen in this case) is present. Detection between 10 to 100 ng/mL was achieved. Example #5: Gold-coated MYLAR® was treated with a 5 mg/mL buffered solution of beta casein as a blocking agent. The casein-treated film was then contact printed with a thiolated polyclonal antibody to Group B Strep to provide a patterned x,y array of antibody in 10-micron circles. Strep B cell suspensions (at concentrations ranging from 9x10" to 9x10" cells/mL) were first treated with an enzyme such as achromopeptidase diluted to 710 units/mL in deionized water. This cell extraction step typically was done by mixing the enzyme solution with cell suspension (e.g., a 4:3 volume:volume ratio of enzyme solution to cells), and heating at 37C for 20 minutes. A 34 microliter aliquot of the resulting lysed cells were exposed to the patterned film for 5 minutes. This was followed by the addition of 11 microliters of a suspension containing 0.3-micron particles conjugated with an antibody to Strep B (typically at a concentration of 10" or 10"° particles / mL). After 10 minutes, a disk of nitrocellulose (e.g., 8 micron pore size) having a hole (e.g., 3/32 inch diameter) in the center was placed on top of the sample and liquid / particle mixture. This wicks away unbound particles and excess liquid, so that the sample can be checked for a diffraction image using a point light source (e.g., laser) aimed to shine through the hole. A diffraction image indicates that the analyte (Strep B cells in this case) is present. Detection down to at least 9x10 cells/mL was achieved. Example #6: Gold-coated MYLAR® was treated with a 5 mg/mL buffered solution of beta casein as a blocking agent. The casein-treated film was then contact printed with a thiolated antibody to IgE (e.g., a monoclonal) to provide a patterned x,y array of antibody in 10-micron circles. This patterned film was then exposed to 34 microliters of diluted human EDTA plasma (e.g., Interstate Blood Bank, Inc; Memphis, TN) that had been spiked with IgE. Typical dilution of plasma was 1 part spiked plasma to 3 parts phosphate buffer solution (pH 7.2). After 5 minutes, 11 microliters was added of a suspension containing 0.3-micron particles (typically at a concentration of 10" or 10"° particles / mL) which were conjugated with another monoclonal antibody to IgE (e.g., a monoclonal anti-IgE, such as one specific to the C3-C4 domains of IgE). After 10 minutes, a disk of nitrocellulose (e.g., 8 micron pore size) having a hole (e.g., 3/32 inch diameter) in the center was placed on top of the sample and liquid / particle mixture. This wicks away unbound particles and excess liquid, so that the sample can be checked for a diffraction image using a point light source (e.g., laser) aimed to shine through the hole. A diffraction image indicates that the analyte (total IgE in this case) is present. Detection between 1000-10,000 ng/mL (initial concentration of IgE in plasma) was achieved. Those skilled in the art will recognize that the present invention is capable of many modifications and variations without departing from the scope thereof. Accordingly, the detailed description and examples set forth above are meant to be illustrative only and are not intended to limit, in any manner, the scope of the invention as set forth in the appended claims. WE CLAIM: 1. A method of detecting an analyte in a medium comprising: contacting the medium with a sensing device, the sensing device comprising: a) a polymer film; b) an analyte-specific receptor layer printed in a pattern onto the polymer film wherein the analyte-specific receptor layer has a receptor material thereon that is specific for the analyte; and c) a wicking agent on the layer of the analyte- specific receptor layer; transmitting a light through the wicking agent and the polymer film; and detecting presence of the analyte by detecting a pattern formed by diffraction of the transmitted light. 2. The method as claimed in Claim 1, wherein the analyte-specific receptor layer is printed in a pattern such that when the sensing device binds an analyte, the sensing device diffracts transmitted light to form a diffraction pattern. 3. The method as claimed in Claim 1, comprising a metal coating on the polymer film and wherein the analyte-specific receptor layer is printed onto the metal coating. 4. The method as claimed in Claim 1, wherein the polymer film has an optical transparency between 5% and 95%. 5. The method as claimed in Claim 1, wherein the polymer film has an optical transparency between 20% and 80%). 6. The method as claimed in Claim 1, wherein the pattern formed is a holographic pattern. 7. The method as claimed in Claim 1, wherein the wicking agent is removed before transmitting the light through the polymer film to detect the presence of the analyte. 8. The method as claimed in Claim 1, wherein the wicking agent has a hole in its center and the light is transmitted through the hole in the wicking agent and through the polymer film to detect the presence of the analyte. 9. A method of detecting an analyte in a medium comprising: contacting the medium with a sensing device, the sensing device comprising: a) a polymer film; b) an analyte-specific receptor layer printed in a pattern onto the polymer film wherein the analyte-specific receptor layer has a receptor material thereon that is specific for the analyte; c) a wicking agent on the analyte-specific receptor layer; reflecting a light source through the wicking agent and off a surface of the metal-coated polymer film; and detecting presence of the analyte by detecting a pattern formed by diffraction of the reflected light. 10. The method as claimed in Claim 9, wherein the analyte-specific receptor layer is printed in a pattern such that when the sensing device binds an analyte, the sensing device diffracts reflected light to form a diffraction pattern. 11. The method as claimed in Claims 2 and 10, wherein the diffraction pattern is visible to an unaided eye. 12. The method as claimed in Claims 3 and 9, wherein the metal is selected from gold, silver, chromium, nickel, platinum, aluminum, iron, copper, gold oxide, chromium oxide or zirconium. 13. The method as claimed in Claim 12, wherein the metal is gold. 14. The method as claimed in Claim 13, wherein the gold coating is between 1 nanometer and 1000 nanometers in thickness. 15. The method as claimed in Claims 1 and 9, wherein the polymer film, is selected from polyethylene-terephthalate, acrylonitrile-butadiene-styrene, acrylonitrile-methyl acrylate copolymer, cellophane, cellulosic polymers such as ethyl cellulose, cellulose acetate, cellulose acetate butyrate, cellulose propionate, cellulose triacetate, polyethylene, polyethylene-vinyl acetate copolymers, ionomers (ethylene polymers) polyethylene-nylon copolymers, nylon, polypropylene, methyl pentene polymers, polyvinyl fluoride, or aromatic polysulfones. 16. The method as claimed in Claim 15, wherein the polymer film is polyethylene-terephthalate. 17. The method as claimed in Claims 1 and 9, wherein the wicking agent is selected from a nitrocellulose membrane, a cellulose acetate membrane, or a glass microfiber structure. 18. The method as claimed in Claims 1 and 9, wherein the analyte is selected from bacteria, yeast, fungus, virus, rheumatoid factor, IgG, IgM, IgA 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 or nucleic acid. Neisseria meningitides groups A, B, C, Y and W sub 135, Streptococcus pneumoniae, E. coli K1 Haemophilus influenza type B, an antigen derived from microorganisms, a hapten, a drug of abuse, a therapeutic drug, an environmental agent, or antigens specific to Hepatitis. 19. The method as claimed in Claim 18, wherein the analyte is bacteria, yeast, fungus or virus. 20. The method as claimed in Claims 1 and 9, wherein the receptor material is selected from antigens, antibodies, oligonucleotides, chelators, enzymes, bacteria, yeasts, fungi, viruses, bacterial pili, bacterial flagellar materials, nucleic acids, polysaccharides, lipids, proteins, carbohydrates, metals, hormones or receptors for said materials. 21. The method as claimed in Claims 1 and 9, a layer of diffraction enhancing elements on the analyte-specific receptor layer, wherein the diffraction enhancing element has a receptor material thereon that is specific for the analyte; wherein the wicking agent is placed on the layer of diffraction enhancing elements. 22. The method as claimed in Claim 21, wherein the diffraction enhancing element is selected from glass, cellulose, synthetic polymers or plastics, latex, polystyrene, polycarbonate, bacterial or fungal cells. 23. The method as claimed in Claim 21, wherein the diffraction enhancing element is polystyrene latex microspheres. 24. The method as claimed in Claims 1 and 9, comprising the step of applying a blocking material to the non-printed areas of the metal-coated polymer film. 25. The method as claimed in Claim 24, wherein the blocking material is selected from p-casein, an albumin, a surfactant, polyethylene glycol or polyvinyl alcohol. 26. The method as claimed in Claims 1 and 9, wherein the sensing device comprises a layer of blocking material on the metal-coated polymer film through which the analyte-specific receptor material is printed. 27. The method as claimed m Claim 26, wherein the blocking material is selected from P-casein, an albumin, a surfactant, polyethylene glycol or polyvinyl alcohol. 28. The method as claimed in Claim 9, wherein the pattern formed is a holographic pattern. 29. The method as claimed in Claim 9, wherein the wicking agent is removed before reflecting the light through the polymer film to detect the presence of the analyte. 30. The method as claimed in Claim 9, wherein the wicking agent has a hole in its center and the light is reflected through the hole in the wicking agent and through the polymer film to detect the presence of the analyte.

Documents:

in-pct-2002-0906-che abstract.pdf

in-pct-2002-0906-che assignment.pdf

in-pct-2002-0906-che claims-duplicate.pdf

in-pct-2002-0906-che claims.pdf

in-pct-2002-0906-che correspondence-others.pdf

in-pct-2002-0906-che correspondence-po.pdf

in-pct-2002-0906-che description (complete)-duplicate.pdf

in-pct-2002-0906-che description (complete).pdf

in-pct-2002-0906-che drawings.pdf

in-pct-2002-0906-che form-1.pdf

in-pct-2002-0906-che form-19.pdf

in-pct-2002-0906-che form-26.pdf

in-pct-2002-0906-che form-3.pdf

in-pct-2002-0906-che form-4.pdf

in-pct-2002-0906-che form-5.pdf

in-pct-2002-0906-che others.pdf

in-pct-2002-0906-che pct.pdf

in-pct-2002-0906-che petition.pdf