MXPA01006264A - Patterned deposition of antibody binding proteins for optical diffraction-based biosensors - Google Patents

Abstract

The present invention provides an inexpensive and sensitive device and method for detecting and quantifying analytes present in a medium. The device comprises a metalized film upon which is printed a specific, predetermined pattern of an antibody-binding protein. Upon attachment of a target analyte to select areas of the plastic film upon which the protein is printed, diffraction of transmitted and/or reflected light occurs via the physical dimensions and defined, precise placement of the analyte. A diffractionimage is produced which can be easily seen with the eye or, optionally, with a sensing device.

Description

DEPOSIT WITH PATTERN OF ANTIBODY BINDING PROTEINS FOR DIFFACTORY-BASED BIOSENSORS TECHNICAL FIELD The present invention relates generally to the field of detecting analytes in a medium and, more particularly, the present invention relates to the microcontact printing of antibody binding proteins on a substrate for the development of single use disposable sensors to indicate the presence of the analyte in a medium.

BACKGROUND OF THE INVENTION There are many systems and devices available to detect a wide variety of analytes in a varied medium. Most of these systems and devices are relatively inexpensive and require a trained technician to carry out the test. There are many cases where it would be advantageous to be able to quickly and cheaply determine whether an analyte is present. What is required is a biosensor system that is easy and inexpensive to manufacture and that is capable of reliable and sensitive detection of analytes, including the smallest analytes. Additionally, what is required is a flexible and easy method of preparing the biosensors which will allow optimal and improved processing.

Sandstrom et al., In the work 24 Optical Applied 472, 1985, describes the use of silicon optical substrate with a layer of silicon monoxide and a silicon layer formed of dielectric films. These indicate that a change in a 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 observed colo of a film provided on the upper part of an optical substrate that can produce a visible colo change. The authors indicate that a mathematical model can be used to quantify the color change, and that "the calculations carried out using the computer model show that very little can be gained in optical performance by the use of a multilayer structure. ... but a biolayer on the surface changes the reflection of such structures very little and that the optical properties are determined mainly by the interlayers within the multi-layer structure.The most sensitive system for the stopping of the biofilms is the coating of Unique layers, while in most other applications the performance can be by means of additional dielectric layer. " Sandstrom et al. Will indicate that the plates formed of metal-on-metal oxides have certain disadvantages, and that the presence of the metal ions can also be detrimental in many biochemical applications. This indicates that the ideal upper dielectric film is a thickness of 2-3 nm of silicon dioxide which can be formed spontaneously when the layer of silicon monoxide is deposited in the ambient atmosphere, and that a layer of 70-95 n of silicon dioxide on a 40-60 nm layer of silicon monoxide can be used on a glass or plastic substrate. These also describe the formation of a wedge of silicon monoxide by the selective pickling of silicon monoxide, the treatment of the surface of silicon dioxide with dichlorodimethylsilane and the application of a biofilm of an antigen and an antibody. From this wedge construction they were able to determine the film thickness with an ellipsometer, and note that the "maximum contrast was found in the region around 65 nm where the color of interference changed from purple to blue." These indicated that the sensitida of such a system is sufficiently high for the detection of protein antigen by the immobilized antibodies. These conclude "the given designs are sufficiently sensitive for a wide range of applications". The materials, such as glass, silicon and silicon oxides, are chemically inert and do not affect the biochemical reaction studied. The computations mentioned above is possible to design plates that are optimized for different applications. Plates can be manufactured and their quality assured by industrial methods, and are now commercially available designs.

U.S. Patent No. 5,512,131 issued to Kumar et al. Describes a device which includes a polymer substrate having a d metal coating. A layer of antibody-binding protein and 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 agglutinates the device. A display device such as a spectrometer is then used to determine the presence of the diffraction pattern.

However, the device described by Kumar others, has several disadvantages. A disadvantage is that an extra display device is necessary to see any diffraction pattern. By requiring a visualization device, the Kumar and other devices allow a large number of samples to be tested since it is not possible to determine the presence of an analyte by the use of the oj without help.

U.S. Patent No. 5,482,830 issued to Bogart et al. Describes a device that includes a substrate which has an optically active surface that exhibits a first color in response to lumping on it. The 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 colo (by having a combination of wavelengths of lu which differ from the combination present in the prime color, or having a different spectral distribution by having an intensity of one or more of those wavelengths different from those present in the prime color). The second color is displayed in response to the same light when the analyte is present on the surface. E change from one color to another can be measured either by the use of an instrument or by the eye. Such sensitive detection is an advance over the devices described by Sandstrom and Nygren, mentioned above, and allows the use of devices in a commercially viable and competitive manner, however, the method and device described in the Bogart patent and another 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 oble so that one has a reliable reading. What it requires is a biosensor device that is easy and inexpensive to manufacture and that is capable of reliable and sensitive detection of analyte that is to be detected.

SYNTHESIS OF THE INVENTION The present invention provides a cheap and sensitive device and a method for detecting analytes present in a medium. The device comprises a biosensor device having a substrate, preferably a metallized polymer film, on which a specific predetermined pattern of antibody binding proteins such as protein A or protein G is printed. Subsequent exposure to specific antibody for the desired analyte results in a patterned deposit of this antibody. Above all, this allows a modular production format so that large patterned protein rollers can be made for the use of different analytes. Then as needed, the final product can be made by exposure to the necessary antibody.

With the attachment of a target analyte the quad is capable of scattering the light, to selected areas of the polymer film on which the protein and the antibody are patterned, the diffraction of the light transmitted and / or reflected occurs at through the physical dimensions and the precise and defined placement of the analyte. A diffraction image produced which can be easily seen with the eye optionally, with a sensor device.

The present invention utilizes pattern contact methods of antibody-binding proteins. These proteins agglutinate antibodies to place them on the surface, as well as to maintain optimal orientation for receptor antibodies. The receptor antibodies are specific for a particular analyte or an analyte class, depending on the protein used. The contact printing methods which will be useful in general the sensor devices used in the present invention are fully described in the patent applications of the United States of America numbers 08 / 707,456 and 08 / 769,594, both of which are incorporated herein. by reference in its entirety. However, since these methods refer to self-assembly of monolayers, the methods require that they be slightly altered, as discussed below, to print the antibody-binder material because this material is not self-assembling.

Antibody-antibody agglutinant protein layers with bound antibodies make the placement with patter or the agglutination of the analytes thereon. The biosensing devices of the present invention produced in this way can be used in one or two ways, depending on the size of the analyte. For analytes which are capable of causing diffraction by themselves, such as micr organisms, the system is used by first exposing the biosensor device to a medium containing an analyte of choice and then, starting from a period of time. appropriate incubació, transmit a light, such as a laser, through the film or reflect it out of the film. If the analyte is present in the medium and is bound to the antibodies on the standard antibody-binder protein layer, the light is diffracted in such a manner as to produce a visible image.

Optionally, for very small analytes such as proteins, the system can use "diffraction enhancing elements" which are capable of agglutinating the target analyte and the biosensor and are capable of producing a substantial change in height and / or the refractive index, thereby increasing the biosensor diffraction efficiency and allowing the detection of smaller analytes. In use, an objective analyte binds either to the diffraction enhancement element, which is then subjected to biosensor, or directly to selected areas of the polymer film on which the protein and antibody are imprinted. Then the diffraction of the transmitted and / reflected light occurs through the physical dimensions and the defined and precise placement of the analyte. A diffraction image is produced which can be easily seen with the eye and optionally with a sensor device.

Another option for the use of this senso device involves the detection of analytes, which are antibodies. The sensing device may comprise only the antibody-agglutinating proteins with standard and then exposed to the medi plus the particular diffraction enhancement which has an antibody specific for the antibody to be detected. The selection of the antibody on the particle s preferably makes it so that it does not bind specifically to the standard antibody-binding binder protein, but instead binds only when the analyte antibody is also bound. In this manner, the diffraction enhancing elements will cause a substantial change in height and / or refractive index if the analyte antibody is present, thereby causing a diffraction image to be formed. It is anticipated that the same format will be used with other immunoassay formats, such as microtiter plates or lateral flow assays. " in other words, the antibody agglutination pattern and the antibody layers with the analyte bound thereto can produce optical diffraction patterns to indicate the presence of the analyte. The light may be in the visible spectr and may be either reflected from the film transmitted through it, and the analyte may be any compound or particle that reacts with the antibody-binding protein layer. The light can be a white light or an electromagnetic or monochromatic radiation in the visible region. The present invention also provides a flexible support for an antibody-binding protein layer on gold, another suitable metal or a metal alloy.

The present invention provides a low cost disposable biosense which can be mass produced. This includes the use of patterned surfaces with an antibody-binding protein. Typically, these proteins agglutinate an antibody by its constant region (Fc) so that the antigen-agglutination regions of the antibody (F ^) are free for optimal binder activity. The preparation of patterned protein surfaces also allows for maximum flexibility in sensor production. The fine step in the production, capturing the desired antibody in the patterned areas as may be done as necessary for the desired analyte (for example at the time of manufacture).

The biosensors of the present invention can be produced as a single test to detect an analyte these can be formatted as a multiple test device. The biosensors of the present invention can be used to detect contamination in garments, such as diapers and to detect contamination by microorganisms The present invention can also be used for contact lenses, glass eyes, window walls, pharmaceutical containers, solvent containers, water bottles, adhesive bandages and the like to detect contamination.

These and other features and advantages of the present invention will become apparent upon review of the following detailed description of the embodiments described.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a biosensor capable of simultaneously measuring several different analytes in a medium.

Figure 2 is a schematic of a contact print of the antibody-binder protein layers followed by patterned deposit of an antibody.

DETAILED DESCRIPTION The present invention relates to improved biosensing devices, and methods for using such biosensing devices, to detect and quantify the presence or amount of an analyte of interest within a medium. Analytes that can be detected by the present invention include, but are not limited to, microorganisms such as bacteria, yeast, fungi and viruses. In contrast to prior art devices, those of the present invention allow the detection of extremely small amounts of analyte in a medium in a rapid test lasting only a few minutes. In addition, no associated electronic or pointing components are required in the biosensing devices of the present invention.

The present invention comprises a microcontact print of antibody-binding proteins on a polymer film which may also have a metal coating. This allows a modular and easy method of production in the sense that subsequent exposure to an antibody causes a patterned agglutination. In addition, these proteins will increase the activity of the immobilized antibody. The present invention allows the development of single-use disposable biosensors based on diffraction of the lu to indicate the presence of the analyte. With the attachment of an objective analyte to selected areas of the plastic film which contain the protein, the diffraction of the transmitted and / or reflected light occurs through the physical dimensions and the precise and defined placement of the analyte. For example, yeast, fungi or bacteria are large enough to act as diffraction elements for visible light when placed in patterns organized on a surface. Additionally, the present invention may include diffraction enhancing elements which increase the diffraction efficiency of the biosensor, by which it becomes possible to detect any number of different analytes. In addition to producing a simple diffraction image, the analyte patterns can be such as to allow the development of a holographic sensor image and / or a change in visible color. Therefore, the appearance of a hologram or change in an existing hologram will indicate a positive response. The pattern made by the diffraction of transmitted light can be of any shape including, but not limited to, the transformation of a pattern from one to another with the agglutination of the analyte to the receptive material. In the particularly preferred embodiments, the diffraction pattern is discernible in less than one hour after contact of the analyte with the biosensor device of the present invention.

The diffraction grating which produces the diffraction of the light with the interaction with the analyte preferably has a minimum periodicity of the wavelength and a refractive index different from that of the surrounding medium. Very few analytes, such as viruses or molecule can be detected indirectly by using a larger particle that is specific for the small analyte. In an embodiment in which the small analyte can be detected comprises coating the particle, such as a latex cuent, with a protein material that binds specifically to the analyte of interest. The 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 or the like. The particles are preferably spherical in shape, but the structural and spatial configuration of the particles is not critical to the present invention. For example, the particles may be sliced, ellipsoids, cubes and the like. A desirable particle size varies from a diameter of about 0.2 μm to 50.0 μm, desirably within about 0.4 μm to μm. The composition of the particle is not critical to the present invention.

The antibody which is immobilized / patterned on the surface will agglutinate specifically to an epitope on the analyte that is different from the epitope used in the agglutination to the diffraction enhancement. Therefore, in order to detect a medium with a small analyte, such as viral particles, the medium is first exposed to the particles of the diffraction enhancing element, such as latex particles to which the viral particles agglutinate. Then, the particles of the diffraction enhancing element are optionally washed and exposed to the polymer film with the antibody-binding protein layers containing the virus-specific antibodies. The antibodies then agglutinate the viral particles on the element particle thereby immobilizing the element particles in the same pattern as the antibodies on the films. Because the particles of bound element will cause the. diffraction of the visible lu, a diffraction pattern is formed, indicating the presence of the viral particle in the liquid. Additionally, the polymer film may include a metacoating over it. The antibody binding protein layer will then be located on the metallized surface of the film.

Alternatively, the analyte can be detected by first exposing the substrate to the medium containing the analyte and causing the analyte to bind to the antibody-binding protein layer material containing the analyte-specific antibody. Then, a solution containing the diffraction enhancing element particles is contacted with the substrate having the analyte attached thereto. The particles then agglutinate the analyte. Because the particles of the binding element will cause the diffraction of visible light, a diffraction pattern is formed, indicating the presence of the analyte in the liquid.

Finally, in a preferred embodiment, the biosensor, the particles of the diffraction enhancing element the medium containing the analyte can be mixed simultaneously. This will result in a combination of the agglutinating procedures discussed above. Some of the analytes will first be bonded with a diffraction enhancer element particle prior to binding to the substrate.

Other analytes will be first bonded to the substrate and then joined with an element particle. When a light source is shown through the sensor, a diffraction pattern is formed indicating the presence of the analyte in the liquid.

Analytes that are contemplated as being detected using the present invention include but are not limited to bacteria; yeasts mushrooms; virus; rheumatoid factor antibodies, including, but not limited to, IgG IgM, Ig and IgE antigen, carcinoembryonic antigen; antigen streptococcus group A; viral antigens; antigens associated with autoimmunity disease; allergens; antigens of tumor antigen of streptococcus group B; human immunodeficiency virus I antigen or human II immunodeficiency virus; or the host response (antibodies) to these and another virus; antigens specific to RSV or host response (antibodies) to the virus; an antigen; enzyme; polysaccharide hormone; protein; lipid; carbohydrate; drug or nucleic acid; Salmonella species,, Candida species, including, but not limited to Candida albicans and Candida tropicalis; salmonella speci meningi tides Neisseria groups A, B, C, Y and W su 135, S treptococcus pneumoniae, E. coli Kl, Haemophilu influenza type B; an antigen derived from microorganisms; a haptén, a drug of abuse; a therapeutic drug; an environmental agent; antigens specific to hepatitis.

In another embodiment of the present invention, nutrients for a specific class of microorganism can be incorporated into the antibody binding protein layer. In this form, 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 appropriate conditions for the growth of the bound microorganism. The microorganism is allowed to grow until there are enough organisms to form a diffraction pattern. Of courseIn some cases, the microorganism 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 method used for patterned receptors such as antibodies on a polymer film or a metallized polymer film. The method involves printing with microcontact the proteins that agglutinate the antibodies. These proteins may include, but are not limited to protein A, protein G, protein L, as well as their recombinant forms. Commercial versions, such as KAPPALOCK "^ ™, by Zymed (of Sa Francisco, California) are also suitable, therefore, the protein material is defined as the base material to create a specific binding pair, with the other part comprising an antibody specific for the analyte d interest.

The receptor material that is bound to the standard protein is characterized by an ability to specifically agglutinate the analyte or analytes of interest. Whatever the selected analyte of interest, the protein is designed to specifically bind to the analyte of interest. In the preferred embodiments, the biosensor device is configured and arranged to provide a pattern detectable by the eye in response to polychromatic light when the analyte of interest is placed in the form of a sandwich between the body and a distraction enhancement element. In another embodiment where the analyte is large enough to diffract light, a diffraction enhancement element may not be necessary.

In many cases, a "blocker" may be necessary to avoid specific agglutination. The term "blocker" as used herein means a reagent that adheres to the surface of the sensor so that "blocking" prevents non-analyte materials from agglutinating specifically to the surface (either in the areas with or without Pattern) . The blocking step can be done as a post treatment to a surface which has already been printed with contact ("post-blocking") is the standard technique for filling the printed regions in contact with another thiol. However, the inventors have discovered that a "pro-block" technique is preferred over the "post-block" technique. In the pre-block technique the surface of the substrate is pretreated with a blocker that does not contain thiol and then printed with co-contact. Not wishing to be bound by any theory, it speculates that the printed contact material (usually containing sulfur) displaces the physisorbed blocker, thereby allowing the antibody binding protein material to be bound directly to the surface of the substrate. If desired, a subsequent post-blocking can be carried out. Blockers can include, but are not limited to.-Casein, albumins such as bovine serum albumin, Pluronic surfactant or others, polyethylene glycol, polyvinyl alcohol, sulfur derivatives of the above-mentioned compounds, any other blocking material known to those skilled in the art. in art.

The matrix containing the analyte of interest can be a solid, a gas or a body fluid such as an interstitial fluid, mucus, saliva, urine, fecal material, tissue, bone, hawthorn fluid, serum, plasma, whole blood, cinobial esput, buffered solutions, extracted solutions, semen, vaginal secretions, pericardial, gastric, peritoneal, pleural or cotton throat and other washes and the like. The analyte of interest may be an antigen, an antibody, an enzyme, a toxin, an environmental agent, a cytoplasm component, a flagella or pili component, protein, polysaccharide, drug or any other material that is capable of being recognized by a antibody. For example, the receptor material for the bacteria can specifically bind a surface membrane, protein or lipid component, or polysaccharide, a nucleic acid or an enzyme. The analyte which is indicative of the bacterium may be a polysaccharide saccharide, an enzyme, a nucleic acid, a membrane component, a gangliocide or an antibody produced by the host in response to the bacterium. The presence of the analyte may indicate an infectious disease (bacterial or viral), cancer, an allergy, or another medical condition or disorder. The presence of the analyte can be an indication of contamination of water or food or other harmful materials. The analyte may indicate drug abuse or may monitor therapeutic agent levels.

In some cases, the analyte may not simply agglutinate the receptor material, but may cause a detectable modification of the receptor material to occur. The interaction may cause an increase in the mass of the test surface a decrease in the amount of material receiving on the test surface. An example of the latter is the interaction of the degradative enzyme or the material with a specific immobilized substrate. In this case one will see a diffraction pattern before the interaction with the analyte d interest, but the diffraction pattern will disappear if the analyte is present. The specific mechanism through cua occurs when agglutination, hybridization or interaction of analyte with the receptor material is not important for this invention but may impact the reaction conditions used in the final assay protocol.

In general, the antibody-agglutinant protein can be passively adhered to the substrate layer. If the required functional groups can be conjugated on the antibody-binding protein to allow their covalent attachment on the test surface.

A wide range of techniques can be used to apply the protein material to the substrate. Test surfaces can be coated with binder-antibody protein by applying solution in discrete patterns or arrangement; spraying, inkjet or other printing methods, or by means of contact printing; The selected technique should minimize the amount of protein required to coat a large number of test surfaces and maintain the stability / functionality of the protein material during the application. The technique must also apply or adhere the protein material to the substrate in a very reproducible, uni-fold form.

The next step will involve exposing the patterned surface to the antibody specific for the desired analyte. This can be done by total immersion in a solution for a predetermined period of time, spraying or coating with rotation. In addition, a controlled placement of several antibodies can lead to a multianalyte system. Another advantage for this process is the modular format for production. In other words, a system with a binder-antibody protein with standard can be optimized and then provide detection of a wide variety of analytes, depending on the antibody found.

The medium in which the analyte can reside can be solid, gel, liquid or gas type. For the purpose of detecting an analyte in the body fluid, fluid is selected from but not limited to urine, serum, plasma, spinal fluid, sputum, whole blood, saliva, secretions urogenital, fecal, pericardial, gastric, peritoneal, pleural lavage, vaginal secretions, or throat cotton; and the method optionally includes using a spectrophotometer to measure the appearance of the refractive pattern. The most common gas that is contemplated as being used with the biosensing device of the present invention is air. The biosensor device of the present invention utilizes pattern contact printing methods, binding proteins-antibodies on substrates, desirably metallized polymer films, subsequent exposure to the antibody to achieve a patterned antibody, the compositions produced from this, and the use d these compositions. Antibody-bound antibody protein layers allow controlled placement of antibodies thereon which can bind an analyte. The term "binder protein-antibody patterned layers" as used herein means the binding protein-antibody plus the desired antibody layers in any pattern on the substrates including a solid standard.

When the film with the standard antibody-binding protein layers is exposed to an analyte which is capable of reacting with the antibody, the film produces optical diffraction patterns which differ depending on the reaction of the antibody with the analyte of interest. E liquid can be a high surface tension fluid such as water. The light may be in the visible spectrum, and may be either reflected from the film or transmitted through it and the analyte may be any compound that reacts with the antibody-binding protein layer.

In the preferred embodiments, the method involves contacting the substrate with a test sample potentially containing the analyte under conditions at which the substrate causes a change in the. Indic refringent. When light is transmitted through the film with the binder-antibody-co-standard protein layer, a visible diffraction pattern is formed and can be visualized by directing the light to a surface by looking directly through the substrate.

In one embodiment, the present invention is contemplated in an indicator rod form in which the printed microcontact film is mounted at the end of the indicator rod. In use the dipstick is immersed in the liquid in which the suspected analyte may be present and allowed to remain for several minutes. The indicator rod is then removed and then either a light is projected through the film or the film is observed with a light behind the film. If a 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. As shown in Figure 1, a strip 10 s provides with several microcontact printed films 20, 25, 30 and 35, each film having a pattern 40 printed thereon. Each of the metallic films printed with microcontact 15, 20, 25 and 30 have a different antibody which is specific for different analytes. It can be seen that the present invention can be formatted in any array with a variety of metallized films printed with microcontact thereby allowing the user of the biosensing device of the present invention to detect the presence of multiple analytes in a medium using a single test.

In some other embodiment of the present invention, the biosensor can be attached to a decal sticker which can then be placed on a hard surface or container wall. the biosensor may be placed on the inner surface of a container such as a food pack or a glass container. Biosensing can be visualized to determine the presence of the analyte Typically, a gold film, of 5 to 200 nm d thickness, is supported on a titanium printed polyethylene terephthalate film, a Si / SiO2 wafer or a glass sheet. Titanium serves as an adhesion promoter between gold and support. The antibody binding protein s attached to the gold surface during contact printing Figure 2 outlines the procedure used to print the microcontact. An elastomeric stamp is used to transfer the antibody-binding protein ink to a surface by contact; if the stamp has a pattern the binder protein-antibody patterned layer is formed The stamp is manufactured by setting polydimethylsiloxane (PDMS) on a pattern having the desired pattern. The patterns are prepared using standard photolithographic techniques, they are constructed of existing materials that have microscale surface characteristics.

In a preferred embodiment of a typical experimental procedure, a pattern produced photolithographically and placed in a plastic or glass Petri dish and a 10: 1 ratio mixture (w: w) of SYLGARDMARC REGISTERED 184 silicone elastomer and the agent. e Closure of silicone elastomers 18 SYLGARD ^^ REGISTERED (from Dow Corning Corporation) is poured over it. The elastomer is allowed to settle for about 3 minutes at room temperature and the reduced pressure to degas, then cure for at least 4 hours at 60 ° C is gently peeled off the standard. The inking of the elastomeric stamp is achieved by exposing the stamp to an aqueous solution of 0.1 to 10 μM of the binder antibody protein derived from the disulphide typically by placing the stamp face down the solution for 10 seconds at 1 minute. The stamp is allowed to dry either under ambient conditions or typically by exposure to a stream of air or nitrogen gas. After the inking, the stamp is applied to a gold surface. The light pressure is used to ensure full contact between the stamp and the surface. After 1 second to 5 minutes, the stamp e then gently peeled off the surface. After the removal of the stamp, the surface is rinsed and dried. Alternatively, additional derivation of stamped areas can be achieved, 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.-Casein, any other agent well known in the art can also be done. After the pattern is placed, the standard surface will be exposed to an antibody specific to the desired analyte either by immersion in a solution or by spraying or spinning the solution on the patterned surface.

The elastomeric nature of the stamp and important for the success of the process. Polydimethylsiloxane (PDMS) when cured, it is sufficiently elastomeric to allow a good conformal contact of the stamp and the surface, to a for surfaces with a significant relief this contact is essential for efficient contact transfer of the protein to the gold film. The elastomeric properties of polydimethylsiloxane are also important when the stamp is removed from the pattern; If the stamp were rigid (as is the pattern) it would be difficult to separate the stamp and the pattern after curing without damaging one of the two substrates. The polydimethylsiloxane is also sufficiently rigid to retain its shape, even with characteristics submicron dimensions. The surface of the polydimethylsiloxane has a low interfacial free energy (y = 22 l / s), and the stamp does not adhere to the gold film. The stamp is durable in the sense that the same stamp can be used up to 100 times over a period of several months without significant degradation in performance. The polymeric nature of polydimethylsiloxane also plays a critical role in the inking process by enabling a stamp to absorb the protein ink by swelling A more detailed description of the compositions methods of the present invention follows. All publications cited here 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 polymer such as polyethylene terephthalate (MYLARMARCA REGISTERED), acrylonitrile-butadiene-styrene, acrylonitrile methyl acrylate copolymer, cellophane, cellulosic polymers such as eti cellulose, cellulose acetate, cellulose acetate butyrate, propionate cellulose, cellulose triacetate, cellulose triacetate, polyethylene, polyethylene-vinyl acetate copolymers, ionomers (ethylene polymer) polyethylene-nylon copolymers, polypropylene, methylpentene polymers, aromatic polyvinyl fluoride polysulfones. Preferably, the plastic film has an optical transparency of more than 80%. Other suitable thermoplastics and suppliers can be found for example in reference works such as Encyclopedia of Modern Plastics (McGraw-Hill Publishing Co., New York 1923-1996).

In an embodiment of the invention, the polymer film has a metal coating on it having an optical appearance of between about 5% and 95% A more desired optical transparency for the thermoplastic film used in the present invention is about 20% and 80% by weight. In a desired embodiment of the present invention the polymer film has at least an optical transparency of about 80%, and the coating thickness of the metal is such as to maintain an optical transparency greater than about 20%, so that the diffraction patterns can be produced by already reflected or transmitted light. This corresponds to a metal coating thickness of around 10 nm. However, in other embodiments of the invention, the gold thickness may be approximately between 1 nm and 1000 nm.

The preferred metal for deposition on the film is gold. However, silver, aluminum, chromium, copper, iron, zirconium, platinum and nickel as well as the oxides of these metals can be used.

In principle any surface corrugations of appropriate size can be used as patterns. The microcontact printing process begins with an appropriate relief structure, from which an elastomeric stamp is forged. This pattern template can be generated photolithographically or by any other procedures such as commercially available diffraction gratings. In an embodiment, the stamp can be made from polydimethylsiloxane.

In another embodiment, the invention relates to an optical assay device having an optically active receptive surface configured and arranged to allow simultaneous testing of a plurality of samples on the surface for an analyte of interest, and an automated liquid handling apparatus. (for example a pipette device configured and arranged to supply sample solutions and reactive to the surface.

An indication of the methodology is provided by which the optimum materials and methods useful for the construction of the optical test surfaces of this invention can be made. Generally, the present invention involves novel optically active test surfaces for the direct detection of an analyte. These test surfaces have an analyte-specific antibody bound to the test surface by the use of a clamp layer-to know the antibody-binding proteins. Therefore, the present invention provides a detection device which includes selecting an optical substrate, patterning it with a binder-antibody protein, and then exposing this to antibody to the desired analyte. The detection method involves contacting that device with the sample fluid containing the analyte of interest and then examining the change in diffraction or reflected or transmitted light by observing whether the formed a diffraction pattern.

The present invention has a broad range of applications and can be used in a variety of specific binder-pair testing methods. For example, the devices of this invention can be used in immunoassay methods for either antigen or antibody detection. The devices can be adapted for use in direct, indirect or competitive detection schemes.

In an embodiment of the present invention, the binder-antibody protein layer has the following general formula: X-P-Ab X is optional as means for allowing the absorption of a metal or metal oxide. For example, X may be asymmetric or symmetrical disulfide (R'SSY ', -RSSY), sulfide (R'SY'. -RSY), diselenide (-R 'Se-SeY'), selenide (-R'SeY ', -RSeY) thiol (-SH), nitrile (-CN), isonitrile, nitro (-N02), selenol (SeH), trivalent phosphorus compounds, isothiocyanate, sodium thiocarbamate, phosphine, thioacid or dithioacid, carboxylic acids, hydroxylic acids and acids hydroxamics P represents the binding proteins-antibodies which can be derived with X. Ab represents the specific antibody for the desired analyte.

The stamp may be applied in air or under fluid such as water to avoid excess diffusion of protein material. For a continuous or large-scale printing process, it is more desirable to print in air, since the shortest contact times are desirable for those processes In one embodiment of the present invention, the pattern is formed on the metallized thermoplastic polymer with the binder-antibody layer. In another embodiment of the present invention, the pattern relief is formed with the antibody-binding protein layer. After the stamping process, the metallized areas on plastics can optionally become passive or blocked, for example, with a reagent such as / -casein. Preferably this is done before exposure to the antibody.

This invention is further illustrated by the following examples, which should or should not be considered in any way as imposing limitations on the scope thereof. On the contrary, it is clearly understood that several other additions must be made, modifying equivalents of the same, which, after reading the description given here, may suggest themselves to those experts in the art without departing from the spirit of the present. invention.

EXAMPLES EXAMPLE 1 The conjugated polystyrene antibody particles were produced by coupling carboxyid co-ethyldimethylaminodicarbodimide (EDAC), bottle number 3 of the Prolysciences kit, catalog number 19539). For this example, 0.125 mL of a 10% suspension of blue carboxylated particles of 0.5 microns in diameter (Bang Laboratories; from Fishers, Indiana; catalog number D0005070CB) s activated with an aqueous solution of EDAC for 1-4 hours, rinsed and then exposed. to 300 micrograms d monoclonal antibody to leutinize the hormone, its unit alf (Fitzgerald Industries, catalog number 10 -LIO, Clone number M94136). The particles were again rinsed, blocked with bovine serum albumin and stored at a concentration of 2.5% in a salt water buffered with phosphate.

A 10 mM aqueous solution of sulfo-LC-SPDP (from Pierce Chemical Co., of Rockford, Illinois) was prepared by dissolving 1.3 mg of the sulfo-LC-SPDP in 2.07 m of deionized water. The conjugation reaction was carried out in phosphate buffered salt water (PBS) containing 20 mM sodium phosphate buffer, 150 mM NaCl, 1 mM EDTA, 0.02% sodium acid at a pH of 7.5. One milligram of protein A or lyophilized protein G was dissolved in 450 microlitres PB and 50 microliters of Sulfo-LC-SPDP delivery solution and added to the antibody solution. The mixture was left to react at room temperature for sixty minutes. The sample was applied to 5 milliliters of unbalanced polyacrylamide column previously equilibrated with 5 bed volumes (25 milliliters) of PBS. The fractions were eluted using PBS as the elution buffer and the protein in the fractions was monitored using the protein plus assay COOMASSIEMARCA REGISTRAD (from Pierce Chemical Co.). Typically, 50 μL of the RECOMMENDED COOMASSIEMARK reagent were mixed with 50 μL of each fraction in a micro purification plate. The COOM SSIE ^ 0 REAGENT reagent reacted with the protein, producing a blue color, the intensity of which depends on the amount of protein present in the fraction. The fractions which produced the most intense blue colo were those containing the majority of the eluted protein. These fractions were stagnant together to form the disulfide of the final derivatized product. This was typically the form used for contact printing.

Optionally, the disulfide-pyridyl group present in the disulfide form of the thiolatad binder can be produced to a thiol group in a reduction reaction. Instead of scrubbing on a column balanced with PBS, the derivatized protein was disheveled on a column equilibrated with acetate buffer (100mM sodium acetate buffer, 100mM NaCl, pH 4.5). The acidic pH of this acetate buffer acts to protect any disulfide bonds present on the undesired native reduction protein. In the reduction reaction, 12 mg. d-dithiothirol (DTT) were dissolved in 500 mL of acetate buffer and added to 1 mL of the SPDP derivatized protein. The reaction mixture was incubated for 30 minutes at room temperature and scrubbed on 5 mL of slovenly balanced column with 5 volumes. of bed (25 mL) d acetate buffer. The protein content of the eluted fractions was again monitored by a COMMASSIE ^ 0 * 1 RECORD protein assay reagent as described above and the fractions containing the highest amount of protein were stalled for a subsequent contact print.

Both disulfide and reduced forms of the thiolated binders were stored in aqueous solution at 4 ° C until they were used for contact printing.

A recorded gold / MYL ^ ™ film was pretreated (or blocked) with 5mg / mL of casein beta solution for 1 minute, then thoroughly rinsed and dried under a stream of air. A PDMS stamp of 1-micron circles was coated with a thiolated antibody-binding protein by placing the stamp face down on a 0.5 mg / mL thiolated protein A (or protein G) solution soaking for 10 minutes. A strong air stream was used to completely dry the surface of the stamp. The coated stamp is placed in contact with the gold film / MY ^^^^ S RADA for 5 minutes? after removed. The printed gold film / MYLARMARCA REGISTERED was rinsed in distilled water and dried.

The sensor was then exposed to an antibody solution (e.g., a 2 μg / mL solution of PBS monoclonal antibody to gluteinize hormone-beta, catalog number 10-L15, clone number M94187, from Firzgerald Industrie International, Inc .; , Massachusetts) for 30 minutes followed by rinsing and air drying.

The sensor sample was exposed to an aqueous solution of 2 μg / mL (with 1% bovine serum albumin) of HR (labeled horseradish peroxidase) antirathon cabr by placing a drop of the solution on the upper part of the sensor surface for 30-60 minutes at room temperature. The sample was rinsed with 0.02% d Tween 20 solution and then with distilled water. A subsequent exposure to a TMB membrane enhancing solution (eg a mixture of 10: 1 v / v from Kirkegaard and Perry Laboratory reagents catalog number 50-76-18 and catalog number 50-77-01) s made by placing the solution_of TMB on the sensor sample for 10 minutes. This caused the development of a blue precipitate in circles or characteristics, as well as a diffraction image to form with the irradiation with a point light source.

Optionally, the analyte solution (for example, leutinizing hormone, for this example) is mixed with microparticles (typically 50.70 microliters of analyte solution with 10-20 microliters of 1.5-2.5% of a suspension of conjugated particles-antibodies), and placed on the upper part of the sensor (a sensor sample of 1 square centimeter is typically used). After 5-10 minutes, a nitrocellulose disk (pore size of 5 and 8 microns, Sigma number N377 or N4146) with a small hole drilled in the center was placed on top of the sensor. This acted to transmit out any excess fluid and n-linked microparticles. At this time, a point light source is transmitted through the sensor sample (using the small orifice in the nitrocellulose). If the analyte was present then a diffraction image is seen on the other side of the ray of light.

EXAMPLE 2 Polystyrene particles conjugated with antibody produced by carbodimide coupling with ethyldimethylaminodicarbodimine (EDAC, bottle number 3 of the Pilysciences study, catalog number 19539). For this example, 0.125 mL of a suspension of 10% blue carboxylated particles of 0.5 microns in diameter (Bangs Laboratories, of Fishers, Indiana, catalog number D0005070CB) were activated with an aqueous solution of EDAC for 1-4 hours, rinsed and then d exposed 300 micrograms of a polyclonal antibody to Ig (such as an anti-chicken IgE so as not to be reactive to protein A patterned). The particles were rinsed again, blocked with bovine serum albumin and stored at a concentration of 2.5% in salt water buffered with phosphate.

A 10 M aqueous solution of Sulfo-LC-SPDP (from Pierce Chemical Co., of Rockford, Illinois) was prepared by dissolving 1.3 mg of Sulfo-LC-SPDP in 2.07 m of deionized water. The conjugation reaction was carried out in phosphate buffered salt water (PBS) containing 20 mM sodium phosphate buffer, 150 mM NaCl, 1 mM EDTA, and 0.02% sodium acid with a pH of 7.5. One milligram of freeze-dried protein A was dissolved in 450 mL of phosphate-buffered salted water, and 50 microliters of Sulfo-LC-SPDP supply solution were added to the antibody solution. The sample was allowed to react at room temperature for 60 minutes. The sample was applied to a 5-mL column of disbanding polyacrylamide previously balanced with 5 bed volumes (25 mL) of salt water buffered with phosphate. The fractions were eluted using the PBS as the elution buffer, and the protein in the fractions was monitored using the protein plus COOM SSIE1 ^ ™ REGISTER assay.

(Pierce Chemical Co.). Typically, 50 μL of the COOMASSIEMARCA REGISTRADA reagent was mixed with 50 μL of each fraction in a microconcentration plate. The COOMASSIE * ^^ REAGENT reagent reacted with the protein producing a blue color, whose intensity depended on the amount of protein present in the fraction. The fractions which produced the most intense blue were those containing the majority of the eluted protein. These fractions were staggered together as the disulfide form of the final byproduct. This was typically the form used for the print contact.

Optionally, the disulfide-pyridyl group present on the disulfide form of the thiolatad binder can be reduced to a thiol group in a reduction reaction. Instead of scrubbing on a column balanced with TBS, the derived protein can be scrubbed on a column balanced with an acetate buffer (10 mM buffer sodium acetate, 100 mM NaCl, pH 4.5) The acidic pH of this buffer acetate acts to protect any disulfide bonds present in the native protein from unwanted reduction. In the reduction reaction, 12 mg of dithiothreite (DTT) were dissolved in 500 mL of acetate buffer and added to 1 mL of the SPDP derivatized protein. The reaction mixture was incubated for 30 minutes at ambient temperature and scrubbed on 5 mL of unbalanced column balanced with 5 bed volumes (25 mL) of acetate buffer. The protein content of the eluted fractions was again monitored by the COOMASSIE protein test reagent as described above and the fractions containing the highest amount of protein were stagnant for a subsequent contact print.

Both disulfide and reduced forms of the thiolated binders were stored in an aqueous solution at 4 ° C until they were used for contact printing.

A gold / MYL film? RMARCA REGISTRADA was retratad (or blocked) with 5 mg / mL of beta casein solution for 1 minute then it was thoroughly rinsed and dried under a stream of air. A PDMS stamp of 1-micron circles was coated with thiolated antibody-binder protein by placing the stamp face down on a thiolated protein A solution of 0.5 mg / mL and soaked for 1 minute. A strong air stream was used to completely dry the surface of the stamp. The coated stamp was placed in contact with the gold / MYLAR ™ REGISTRE for 5 minutes and then removed. The resulting printed / printed MYLAR film was rinsed in distilled water and dried.

The analyte solution (typically 50-7 microliters at 2 μg / mL of IgE PBS solution (catalog num.

-AI05 of Fitzgerald Industries International, Inc .; of Concord Massachusetts) was placed on top of the senso (typically one square centimeter) per 0.20 minutes, followed by 10.20 microliters of 1.5-2.5% conjugate-antibody particle suspension for an additional 5-20 minutes. A nitrocellulose disk (5 or 8 micras of pore size, Sigma number N377 or N4146) with a small hole drilled in the center was placed on top of the sensor. This acted to transmit the excess fluid and unbound particles to the outside. At this time, a point light source is transmitted through the sensor sample (using the small hole in the nitrocellulose). If the analyte was present then the diffraction image is seen on the other side of the ray of light as illustrated.

Claims (56)

R E V I N D I C A C I O N S
1. A biosensor comprising: a polymer film; Y a layer of agglutination-antibody protein imprinted in a pattern on the polymer film wherein the antibody binding protein layer has an antibody thereon that is specific for an analyte.
2. 2. The biosensor as claimed in clause 1, characterized in that the antibody agglutinating protein layer is printed in a pattern such that when the biosensor agglutinates the analyte, the biosensor diffracts the transmitted lu to form a diffraction pattern.
3. 3. The biosensor as claimed in clause 2, characterized in that the diffraction pattern is visible with an unaided eye.
4. 4. The biosensor as claimed in clause 1, characterized in that the polymer film also comprises a metal coating.
5. 5. The biosensor as claimed in clause 4, characterized in that the metal is selected from silver, gold, chromium, nickel, platinum, aluminum, iron, zirconium copper, or oxides thereof.
6. 6. The biosensor as claimed in clause 4, characterized in that the metal is gold.
7. 7. The biosensor as claimed in clause 6, characterized in that the gold coating is d between about a thickness of nanometer and 1000 nanometers.
8. 8. The biosensor as claimed in clause 1, characterized in that the polymer film is selected from polyethylene terephthalate, acrylonitrile butadiene-styrene, acrylonityl-methylacrylate copolymer, cellophane, cellulose polymers such as ethyl cellulose, cellulose acetate, butyrate cellulose acetate, cellulose propionate, cellulose triacetate, cellulose triacetate, polyethylene, polyethylene-vinyl acetate copolymers, nylon copolymers, polyethylene ionomers (polymers of ethylene), polypropylene, polymers of methyl pentene, polyvinyl fluoride, or polysulfones aromatic
9. 9. The biosensor as claimed in clause 8, characterized in that the polymer film is polyethylene terephthalate.
10. 10. The biosensor as claimed in clause 1, characterized in that the polymer film is optically transparent.
11. 11. The biosensor as claimed in clause 1, characterized in that the polymer film has an optical transparency of between 5% and 95%.
12. 12. The biosensor as claimed in clause 1, characterized in that the polymer film has an optical transparency of approximately 20% and 80%.
13. 13. The biosensor as claimed in clause 1, characterized in that the b-protein-antibody layer is formed of compounds with the following general formula: X-P-Ab where : X is reactive with the metal or the meta oxide on the polymer film; P is a binder-antibody protein; Ab is an antibody specific to a desired analyte.
14. 14. The biosensor as claimed in clause 13, characterized in that: X is asymmetric or symmetrical disulfide (-SSY '.SSY), sulfide (-'SY', SY), diselenide (-'Se-SeY '), selenide (SeY', -Sey), thiol (-SH), nitrile (-CN), isonitrile, nitro (N02), selenol (-SeH), trivalent phosphorus compounds isothiocyanate, xanthate, thiocarbamate, phosphine, thioacid dithioacid, carboxylic acids, hydroxylic acids and hydroxamic acids.
15. 15. The biosensor as claimed in clause 1, characterized in that the analyte is selected from bacteria, yeast, fungi, virus, rheumatoid factor, IgG, IgM, IgA and IgE antibodies, carcinoembryonic antigen, Streptococcus antigen, group A, viral antigens, antigen associated with autoimmune diseases, allergens, tumor antigens, Streptococcus group B antigen, HIV II HIV I antigen, antibodies, viruses, antigen specific for RSV antigen, enzyme, hormone, polysaccharide, protein, lipid carbohydrate, drug, nucleic acid , groups of Neisseri meningi tides A, B, C, Y and W sub 135, Streptococcus pneumoniae, E coli Kl, Haemophillus influenza type B, an antigen derived from microorganisms, a hapten, a drug of abuse, a therapeutic drug, an agent environmental, or specific antigens for hepatitis.
16. 16. The biosensor as claimed in clause 15, characterized in that the analyte is bacterium yeast, fungi or virus.
17. 17. The biosensor as claimed in clause 16, characterized in that the fungus is Candida species.
18. 18. The biosensor as claimed in clause 16, characterized in that the bacterium is Salmonell species.
19. 19. The biosensor as claimed in clause 1, characterized in that the protein material is selected from protein A, protein G, protein L, or recombinant thereof.
20. 20. A method for making a biosensor comprises printing a pattern of a layer of antibody-binding protein with a subsequent layer of antibody on the polymer film.
21. 21. The method as claimed in clause 20, characterized in that the antibody b-coat protein layer is printed in a pattern so that the biosensor agglutinates an analyte, the biosensor diffracts the transmitted lu to form a diffraction pattern.
22. 22. The method as claimed in clause 20, characterized in that the polymer film also comprises a metal coating.
23. 23. The method as claimed in clause 22, characterized in that the metal is selected from gold, silver, chromium, nickel, platinum, aluminum, iron, zirconium copper or oxides thereof.
24. 24. The method as claimed in clause 22, characterized in that the metal is gold.
25. 25. The method as claimed in clause 24, characterized in that the gold coating is d between approximately one nanometer and 1000 nanometers thick
26. 26. The method as claimed in clause 20, characterized in that the polymer film is selected from polyethylene terephthalate, acrylonityl butadiene-styrene, acrylonityl-methylacrylate cellophane copolymer, cellulose polymers such as ethyl cellulose, cellulose acetate, acetate butyrate cellulose, cellulose propionate, cellulose triacetate, cellulose polyethylene triacetate, polyethylene-vinyl acetate copolymers of nylon copolymers, polyethylene ionomers (polymers of ethylene), polypropylene, polymers of methyl pentene, polyvinyl fluoride, or aromatic polysulfones.
27. 27. The method as claimed in clause 26, characterized in that the polymer film is polyethylene terephthalate.
28. 28. The method as claimed in clause 20, characterized in that the polymer film is optically transparent.
29. 29. The method as claimed in clause 28, characterized in that the polymer film has an optical transparency of between 5% and 95%.
30. 30. The method as claimed in clause 28, characterized in that the polymer film has an optical transparency of approximately 20% and 80%.
31. 31. The method as claimed in clause 20, characterized in that the antibody-binding protein layer is formed of compounds with the following general formula: X-P-Ab where: X is reactive with the metal or the meta oxide on the polymer film; P is a binder-antibody protein, - Ab is an antibody specific to a desired analyte.
32. 32. The method as claimed in clause 31, characterized in that: X is asymmetric or symmetrical disulfide (-SSY '. • SSY), sulfide (-'SY', SY), diselenide (-'Se-SeY '), selenide (- SeY', -Sey), thiol (-SH) , nitrile (-CN), isonitrile, nitro (-N02), selenol (-SeH), trivalent phosphorus compounds isothiocyanate, xanthate, thiocarbamate, phosphine, thioacid dithioacid, carboxylic acids, hydroxylic acids and hydroxamic acids.
33. 33. The method as claimed in clause 20, characterized in that the analyte is selected from bacteria, yeast, fungi, virus, rheumatoid factor, IgG, IgM, IgA and IgE antibodies, carcinoembryonic antigen, Streptococcus antigen, group A, viral antigens, antigen associated with autoimmune diseases, allergens, tumor antigens, Streptococcus group B antigen, HIV II HIV I antigen, antibodies, viruses, antigens specific to RSV antigen, enzyme, hormone, polysaccharide, protein, lipid carbohydrate, drug, nucleic acid, Neisseri groups meningi tides A, B, C, Y and W sub 135, Streptococcus pneumoniae, E coli Kl, Haemophillus influenza type B, an antigen derived from microorganisms, a hapten, a drug of abuse, a therapeutic drug, an environmental agent, or antigens specific for hepatitis.
34. 34. The method as claimed in clause 33, characterized in that the analyte is bacterium yeast, fungi or virus.
35. 35. The method as claimed in clause 20, characterized in that the protein material is selected from protein A, protein G, protein L, or a recombinant form thereof.
36. 36. A method for detecting an analyte in a medium comprising: coating the medium suspected of containing an analyte with a biosensor device, the biosensing device comprises: a polymer film; Y an antibody binding protein layer imprinted in a pattern on the polymer film wherein the antibody-binding protein layer has a protein material thereon which is specific for and analyte; Y transmit a light through the polymer film; Y detecting the presence of the analyte bound to protein material by detecting a pattern shape or diffraction of the transmitted light.
37. 37. The method as claimed in clause 36, characterized in that the diffraction pattern is visible with an unaided eye.
38. 38. The method as claimed in clause 36, characterized in that the polymer film also comprises a metal coating.
39. 39. The method as claimed in clause 38, characterized in that the metal is gold.
40. 40. A biosensor comprising: a polymer film; Y a layer of binder-antibody protein printed in a pattern on the polymer film wherein the binder-antibody protein layer is capable of acting as a receptor for an analyte.
41. 41. The biosensor as claimed in clause 40, characterized in that the antibody agglutinating protein layer is printed in a pattern such that when the biosensor binds the analyte, the biosensor diffracts the transmitted lu to form a diffraction pattern.
42. 42. The biosensor as claimed in clause 41, characterized in that the diffraction pattern is visible with the unaided eye.
43. 43. The biosensor as claimed in clause 40, characterized in that the polymer film also comprises a metal coating.
44. 44. The biosensor as claimed in clause 43, characterized in that the metal is selected from gold, silver, chromium, nickel, platinum, aluminum, iron, zirconium copper or oxides thereof.
45. 45. The biosensor as claimed in clause 43, characterized in that the metal is gold.
46. 46. The biosensor as claimed in clause 45, characterized in that the gold coating is d between approximately one nanometer and 1000 nanometers thick
47. 47. The biosensor as claimed in clause 40, characterized in that the polymer film is selected from polyethylene terephthalate, acrylonityl butadiene-styrene, acrylonityl-methylacrylate copolymer, cellophane, cellulose polymers such as ethyl cellulose, cellulose acetate, butyrate cellulose acetate, cellulose propylate d, cellulose triacetate, cellulose polyethylene triacetate, polyethylene-vinyl acetate copolymers of nylon copolymers, polyethylene ionomers (polymers of ethylene), polypropylene, polymers of methyl pentene, polyvinyl fluoride, or aromatic polysulfones.
48. 48. The biosensor as claimed in clause 47, characterized in that the polymer film is polyethylene terephthalate.
49. 49. The biosensor as claimed in clause 40, characterized in that the polymer film is optically transparent.
50. 50. The biosensor as claimed in clause 40, characterized in that the polymer film has an optical transparency of between 5% and 95%.
51. 51. The biosensor as claimed in clause 40, characterized in that the polymer film has an optical transparency of approximately 20% and 80%.
52. 52. The biosensor as claimed in clause 40, characterized in that the binder-antibody protein layer is formed of compounds with the following general formula: X-P where : X is reactive with the metal or the meta oxide on the polymer film; P is an antibody binding protein; Y
53. 53. The biosensor as claimed in clause 52, characterized in that: X is asymmetric or symmetrical disulfide (-SSY '.SSY), sulfide (-'SY', SY), diselenide (-'Se-SeY '), selenide (SeY', -Sey), thiol (-SH), nitrile (-CN), isonitrile, nitro (N02), selenol (-SeH), trivalent phosphorus compounds, isothiocyanate, xanthate, thiocarbamate, phosphine, thioacid dithioacid, carboxylic acids, hydroxylic acids and hydroxamic acids.
54. 54. The biosensor as claimed in clause 40, characterized in that the analyte is selected from antibodies such as IgG, IgM, IgA and IgE.
55. 55. The biosensor as claimed in clause 40, characterized in that the protein material is selected from protein A, protein G, protein L, or a recombinant form thereof.
56. 56. The biosensor as claimed in clause 40, further characterized in that it comprises a diffraction enhancing element wherein the diffraction enhancing element includes an antibody that is capable of agglutination to the analyte. SUMMARY The present invention provides a sensitive and inexpensive method and device for detecting and quantifying analytes present in a medium. The device comprises a metallized film on which a predetermined and specific pattern is printed d an antibody binding protein. With the attachment of target analyte to proportioned areas of the plastic film on which the protein is imprinted, the diffraction of light transmitted and / or reflected through the physical dimensions and the precise and defined placement of analyte occurs. . A diffraction image is produced which can be easily seen with the eye or optionally with a sensor device. 5 -3 i