• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
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    • 专利摘要:
    The present invention provides a low cost and high sensitivity system and method for detecting and quantifying analytes present in a medium. The device comprises a metallized film with a particular pattern of binder, eg, an antibody, printed on its surface. After the target analyte is attached to a particular area of the plastic film on which the binder is printed, diffraction of transmitted and / or reflected light occurs through the physical dimensions and the exact placement of the analyte. Diffraction images are produced that are readily visible to the naked eye or optionally using a sensing device.
    公开号:KR20020097226A
    申请号:KR1020027014198
    申请日:2001-04-11
    公开日:2002-12-31
    发明作者:로잔 엠. 케일러；치부에쩨 오빈나 치데벨루-에쩨；아브라함 비. 최
    申请人:킴벌리-클라크 월드와이드, 인크.；
    IPC主号:
    专利说明:

    Use of Ink-Jet Printing to Produce Diffractometer Biosensors
    [2] There are many systems and devices for detecting different analytes in different media. Many of these systems and devices are relatively expensive and require skilled technicians to perform the tests. In many cases, it would be advantageous to measure the presence of an analyte quickly and inexpensively. There is a need for a biosensor system that is easy to manufacture, low in manufacturing cost, and capable of detecting analytes, including small analytes, with high reliability. There is also a need for an easy and flexible method of manufacturing biosensors with optimal scale-up processing.
    [3] Sandstrom et al. (Sandstrom et al., 24 Applied Optics 472, 1985) describe the use of silicon monoxide substrates with silicon monoxide and silicon layers formed as dielectric films. Sandstrom et al. Pointed out that as the film thickness changes, the characteristics of the optical substrate change, resulting in different colors depending on the thickness of the film. The thickness of the film is related to the color observed, and the film provided on the top surface of the optical substrate will cause a visible color change. The authors of this document pointed out that a mathematical model can be used to quantify color changes, and stated: "The calculations performed using a computer model show that very small optical properties are obtained with multilayer structures. However, the surface biolayer hardly changes the reflection of such a structure because its optical properties are largely determined by the interface inside the multilayer structure.The most sensitive biolayer detection system is a single layer coating, but most other applications The performance of the test system can be obtained with additional dielectric layers. "
    [4] Sandstrom et al. Pointed out that slides formed from metal oxides on metals have certain drawbacks, and the presence of metal ions can also be detrimental to many biochemical applications. Sandstrom et al. Is an ideal top dielectric film of 2 to 3 nm thick silicon dioxide spontaneously formed when a silicon monoxide layer is deposited in the ambient atmosphere, and a 70 to 95 nm silicon dioxide layer on a 40 to 60 nm silicon monoxide layer. It is pointed out that this can be used on glass or plastic substrates. They also describe the formation of wedges of silicon monoxide by selective etching of silicon monoxide, treatment of silicon dioxide surfaces with dichlorodimethylsilane, and biolayer application of antigens and antibodies. From this wedge structure, they were able to measure the thickness of the film with an ellipsometer and stated that "maximum contrast appears in the region of about 65 nm where the interference color changes from purple to blue." They pointed out that the sensitivity of such systems is high enough that protein antigens can be detected by immobilized antibodies. They concluded that the given design is sensitive enough to cover a wide range of applications. This material, glass, silicon and silicon oxide, is chemically inert and does not affect the biochemical reactions under study. The computer operation can be used to design slides that are optimal for other applications. Such slides are manufacturable, the quality of the slides is guaranteed by industrial methods, and two designs are currently commercially available.
    [5] U. S. Patent No. 5,512, 131 to Kumar et al. Describes a device comprising a polymer substrate coated with a metal. The antibody-binding protein layer is immobilized on the coated substrate. This device is used in the process for stamping or as a switch. Diffraction patterns are generated when the analyte binds to this device. Visualization devices such as spectrometers can be used to measure the presence of the diffraction pattern.
    [6] However, the apparatus described by Kumar et al. Has several disadvantages. One disadvantage is the need for a separate visualization device to see any diffraction pattern. Because of the need for a visualization device, Kumar et al. Cannot test a large number of samples because the device cannot visually measure the presence of an analyte.
    [7] US Pat. No. 5,482,830 to Bogart et al. Describes a device comprising a substrate having an optically active surface that exhibits a first color in response to light hit. This first color is defined as the spectral distribution of the emitted light. In addition, the substrate may be formed by having a combination of light wavelengths different from the combination appearing in the first color, or having a different spectral distribution, or having one or more intensities of light wavelengths different from those appearing in the first color. A second color different from one color is shown. This second color appears in response to the same light when the analyte is present on the surface. The change from one color to another can be detected with a particular instrument or with the naked eye. Such sensitive detection is superior to the device described by Sandstrom and Nygren, and allows the device to be used in a commercially viable and competitive manner.
    [8] However, the methods and apparatus described in the patents of Bogart et al. Have several disadvantages. One disadvantage is the high cost of the device. Another problem with this device is that it is difficult to control to reliably read the various layers lying on the wafer.
    [9] The prior art also includes the use of jetting techniques in certain applications. For example, US Pat. No. 4,877,745 to Hayes et al. Discloses a method and apparatus for analyzing an analyte. The method and apparatus include a jetting chamber used to disperse the correct amount of diagnostic reagent and fluid sample on a substrate. The device uses a means for placing either a diagnostic reagent or a fluid sample on a substrate. The other one of the diagnostic reagent or fluid sample is then jetted to react with the first placed fluid. The method alternately ink-jets the diagnostic fluid and the sample using electromechanical means until the desired amount of fluid is dispersed on the substrate. The method includes placing the substrate correspondingly to the jetting chamber and applying the diagnostic fluid to the substrate in a predetermined manner using electrical pulses. Place the diagnostic reagent in a collapsible container whose volume changes during use of the device. However, because the method prints both the analyte-specific receptor material and the sample to be tested, it is difficult to manipulate and requires complex electromechanical means.
    [10] Accordingly, there is a need for a biosensor device that is easy to manufacture, low in manufacturing cost, and capable of detecting an analyte to be detected with high reliability.
    [11] <Overview of invention>
    [12] The present invention provides an inexpensive and high sensitivity device and method for detecting analytes present in a medium. The device comprises a biosensing device having a substrate, such as an ink-jet printed metallized polymer film, on which a particular pattern of binder comprising an antibody or thiolated antibody or DNA is placed. Alternatively, antibody-binding proteins such as Protein A or Protein G can be ink-jet printed. Exposure to antibodies specific for the analyte of interest is then allowed to deposit the antibody in a pattern. Overall, this method allows for a modular production format and can be made with large rolls of patterned proteins for use in different analytes. The final product can then be prepared, if necessary, by exposing to the required antibody.
    [13] When a target analyte capable of scattering light is attached to a specific area of the polymer film in which the pattern of proteins and / or antibodies is formed, transmitted light and / or through the physical dimensions of the analyte and certain precise placement ) Diffraction of reflected light occurs. Diffraction images are produced that can be easily seen by the eye or optionally using a sensing device.
    [14] The present invention utilizes a method of ink-jet printing of patterned binders. These binders may be antibodies or proteins. When using proteins, these proteins bind to the antibodies to maintain optimal orientation for the receptor antibody as well as to pattern them on the surface. Receptor antibodies are specific for a particular analyte or analyte, depending on the protein used.
    [15] Patterned antibodies result in patterned placement of analytes or binding thereon. The biosensing device produced by the present invention can be used in one of two ways depending on the size of the analyte. For analytes that can cause diffraction by themselves, such as microorganisms, the system first exposes the biosensing device to a medium containing the selected analyte and then transmits light, such as a laser, through the film after an appropriate incubation period. Or by reflecting light from the film. If the analyte is present in the medium and bound to the patterned antibody layer, light is diffracted in a manner that forms a visible image.
    [16] Optionally, for very small analytes, such as proteins or DNA, the system can bind to the target analyte and biosensor and cause substantial changes in height and / or refractive index to increase the diffraction efficiency of the biosensors and A "diffractive enhancing component" can be used that can enable detection of the analyte. In use, the target analyte attaches to the diffractive enhancing component and then to the biosensor, or directly to a selected region of the polymer film in which the pattern of the antibody is formed. Subsequently, diffraction of the transmitted and / or reflected light occurs through the physical dimensions of the analyte and the defined exact placement. Diffraction images are produced that are easily visible using the eye or optionally a sensing device.
    [17] Another case of using such a sensor involves the detection of an analyte that is an antibody. The sensing device may comprise only the patterned antibody-binding protein and then may be exposed to the combination of the medium having the antibody specific for the antibody to be detected and the diffractive enhancing particles. The antibody on the particles is preferably selected to not bind non-specifically to the patterned antibody-binding protein but to bind only when the analyte antibody also binds. In this way, the diffractive enhancing component will cause a substantial change in height and / or refractive index in the presence of the analyte antibody, causing the diffraction image to form. It is contemplated that the same format can be used with other immunoassay formats such as lateral flow assays or microwell plates.
    [18] Thus, antibodies and antibody layers with analytes bound thereon can generate optical diffraction patterns to indicate the presence of analytes. Light may be in the visible spectrum and may be reflected from or transmitted through the film. The light may be a white point light source or monochromatic electromagnetic radiation. The present invention can be used to print directly on a flexible support. The support may comprise a layer of gold or other suitable metal or metal alloy on which the antibody is printed.
    [19] Another case of using this sensor is the detection of nucleotide based analytes such as DNA. In this case, a sensing device having a patterned oligonucleotide complementary to the target is used, which is exposed to the sum of the diffractive enhancing particles and the medium having the oligonucleotide specific to another segment of the target DNA. Nucleotides are then detected by examining the diffraction image in the manner discussed above.
    [20] The present invention provides a low cost disposable biosensor that can be mass produced. The present invention encompasses the use of patterned surfaces with antibodies or antibody-binding proteins. Typically, the antibody-binding protein binds to the antibody through the constant region (F c ), so that the antigen-binding portion (F ab ) of the antibody is free and its binding activity is optimal. Fabrication of patterned protein surfaces also allows for maximum flexibility in sensor fabrication. The final production step of capturing the desired antibody in the patterned region can be performed if necessary (ie, at the time of manufacture) depending on the analyte of interest.
    [21] The biosensor of the present invention may be manufactured for a single test to detect an analyte, or may be configured as multiple test devices. The biosensor of the present invention can be used to detect medical conditions or contamination of clothing such as diapers and to detect contamination by microorganisms.
    [22] As a preferred ink-jet printing method, a printer with a piezoelectric printer-head may be preferred for printing temperature-sensitive proteins such as antibodies.
    [23] The present invention can also be used to detect contamination of contact lenses, glasses, glass plates, vials, solvent containers, water bottles, adhesive bandages, and the like.
    [24] These and other features and advantages of the present invention will become more apparent with reference to the detailed description of the embodiments disclosed below.
    [1] The present invention generally belongs to the field of detecting analytes in a medium, and more particularly, to a sensing device capable of indicating the presence of an analyte in a medium.
    [25] 1 is a plan view of a biosensor capable of simultaneously measuring several different analytes in a medium.
    [26] 2 shows micrographs of patterned particulates in ink-jet printed features showing the presence of analytes.
    [27] The present invention features an improved biosensing device for detecting the presence or amount of an analyte of interest in the medium, and methods of making and using the biosensing device. Analytes that can be detected by the present invention include, but are not limited to, microorganisms such as bacteria, yeasts, fungi and viruses. Unlike the devices of the prior art, the devices of the present invention allow for the detection of very small amounts of analytes present in the medium with rapid assays in just minutes. In addition, the biosensing device can be manufactured at a much lower cost and at a higher speed than other biosensing devices.
    [28] The present invention discloses a method of ink-jet printing a binder on a polymer film, such as a plastic film. The polymer film may also have a metal coating. A "binding agent" may include an antibody, eg, a thiolated antibody, or an antibody-binding protein. The present invention provides an easy manufacturing method suitable for high speed manufacturing. The present invention enables the development of disposable biosensors based on diffraction of light indicating the presence of an analyte. When the target analyte is attached to a particular area of the polymer film containing the binder, diffraction of transmitted and / or reflected light occurs through the physical dimensions and the exact placement of the analyte. For example, yeast, fungi or bacteria are large enough to act as diffraction components for visible light when placed in a pattern formed on a surface. In addition, the present invention may include diffractive enhancement components that increase the diffraction efficiency of the biosensor to allow detection of any number of different analytes. In addition to forming a simple diffraction image, the pattern of the analyte may enable the formation of a holographic sensed image and / or a visible change in color. Thus, the appearance of holograms or alteration of existing holograms will mean a positive response. The pattern formed by diffraction of transmitted light may be in any form, including but not limited to, pattern modification from one pattern to another upon binding of the analyte to the recipient material. In a particularly preferred embodiment, the diffraction pattern can be recognized within 1 hour after contacting the analyte with the biosensing device of the present invention.
    [29] The diffraction grating, which can produce light diffraction upon interaction with the analyte, preferably has a different refractive index than the surrounding medium. Very small analytes, such as viruses or molecules, can be detected indirectly by using large particles that are specific for small analytes. One embodiment capable of detecting small analytes includes coating particles, such as latex beads, with a protein material that specifically binds to the analyte of interest. Particles that can be used in the present invention include, but are not limited to, glass, cellulose, synthetic polymers or plastics, latex, polystyrene, polycarbonates, proteins, bacterial or fungal cells, and the like. The shape of the particles is preferably spherical, but any structural and spatial shape of the particles can be used in the present invention. For example, the particles can be sliver-shaped, elliptical, cube-shaped, and the like. Preferred particle sizes range from about 0.2 μm to 50.0 μm in diameter, preferably from about 0.4 to 1 μm. The composition of the particles can also be varied.
    [30] Antibodies immobilized / patterned on the surface will specifically bind to epitopes that are different from the epitopes on the analytes used for binding with the diffractive enhancing component. Thus, in order to detect a medium with a small analyte, such as viral particles, the medium is first exposed to diffractive enhancing component particles, for example latex particles, to which the viral particles are bound. Thereafter, the diffractive enhancing component particles are optionally washed and exposed to a polymer film printed with virus specific antibodies. The antibody is then bound to the viral particles on the component particles to immobilize the component particles in the same pattern as the antibodies on the film. Since the bound component particles will cause diffraction of visible light, a diffraction pattern is formed, which indicates the presence of viral particles in the liquid. In addition, the polymer film may include a metal coating thereon. In this case, the printed antibody layer is placed on the metallized surface of the film.
    [31] Alternatively, the analyte can be detected by first exposing the substrate to an analyte containing medium and allowing the analyte to bind with the printed analyte-specific antibody. The diffractive enhancing component particle containing solution is then contacted with the substrate to which the analyte is bound. The particles then bind to the analyte. Since the bound component particles cause diffraction of visible light, a diffraction pattern is formed, which indicates the presence of the analyte in the liquid.
    [32] In another embodiment, the biosensor, diffractive enhancing component particles, and analyte containing medium can be mixed simultaneously. This results in a combination of the binding procedures discussed above. A portion of the analyte will be combined with the diffractive enhancing component first and then with the substrate. Other analytes will bind to the substrate first and then to the component particles. When the point light source shines through the sensor, a diffraction pattern is formed, indicating the presence of the analyte in the liquid.
    [33] Finally, in yet another embodiment, the number of steps that a user of a diffraction diagnostic apparatus using a diffractive enhancement component must perform can be reduced. This approach involves the use of wicking agents to remove unbound labeling particles and any residual liquid from the sample. The wicking agent avoids any additional cleaning steps that may be burdened and more difficult for the user. In addition, a small hole (e.g., 4/32 inch (0.32 cm)) is drilled in the center of the absorbent so that if the sample and excess particles are sucked up, the user can immediately see the diffraction image without removing the absorbent material. To confirm. Examples of wicking agents include nitrocellulose membranes, cellulose acetate membranes, and glass microfiber structures.
    [34] In addition, the pore size of the membrane can be varied to control the speed and force of the wicking. The pore size of the membrane can affect the precision of the diagnostic device and can be used to create a one-stage device. To achieve this, the one-stage device consists of capture antibodies ink-jet printed on a substrate, such as a gold-coated polyethylene-terephthalate substrate (Gold / Mylar®, MYLAR), which is the surface of the substrate Will have pre-dried labeling particles. In addition, a small pore size membrane (eg, 0.45 micron nitrocellulose) with cut out holes is placed on top of the device to complete the device. The user simply adds the sample to be tested (e.g., serum or blood) and then observes the diffraction image once absorption occurs. Small pore size delays uptake sufficiently to allow adequate incubation for the time required for antigen-antibody interaction to occur. Alternatively, wicking may be delayed by using erosive reagents around the wicking cavities. This reagent eventually dissolves or derivatizes after a certain time, causing wicking.
    [35] Analytes expected to be detected using the device of the present invention include bacteria; leaven; Fungi; virus; Rheumatoid factor; Antibodies such as IgG, IgM, IgA and IgE antibodies; Cancer-causing antigens; Streptococcus group A antigen; Viral antigens; Antigens associated with autoimmune diseases; Allergens; Tumor antigens; Streptococcus group B antigen; HIV I or HIV II antigens; Or such and other viral reactive hosts (antibodies); Antigen specific to RSV or to a viral reactive host (antibody); antigen; enzyme; hormone; Polysaccharides; protein; Lipids; carbohydrate; Drugs or nucleic acids; Salmonella spp .; Candida species, such as Candida albicans and Candida tropicalis; Salmonella spp .; Meningococcal group A, B, C, Y and W subgroup 135, pneumococcal streptococci, E. coli K1, Haemophilus influenzae type B; Antigens derived from microorganisms; Haftenes, drugs of abuse; Therapeutic drugs; Environmental agents; And hepatitis specific antigens, but are not limited thereto.
    [36] In other embodiments of the invention, nutrients for certain species of microorganisms may be incorporated into the binder layer. In this way, very low concentrations of microorganisms can be detected by first contacting the biosensor of the present invention with the nutrient to be incorporated and then culturing the biosensor under conditions suitable for the growth of the bound microorganisms. The microorganisms are grown until there is enough organic material to form a diffraction pattern. Of course, in some cases, microorganisms can proliferate to a degree sufficient to form a diffraction pattern even when no nutrient is present on the patterned binder.
    [37] Part of the invention is a method used to pattern receptors such as antibodies on a polymer film or metallized polymer film. In some, the method comprises an ink-jet printing protein that binds to the antibody. These proteins include, but are not limited to, Protein A, Protein G, Protein L, as well as their recombinant forms. Commercial forms of these proteins are also suitable, for example KAPPALOCK (tradename) from Zymed, San Francisco, CA. Thus, this protein material is limited to the base material which forms a binding pair specific with the antibody specific to the analyte of interest on the other side.
    [38] In the present invention, a biologically active material such as an antibody is printed on a metallized polymer film (eg gold / mylar substrate) in a predetermined pattern using an ink-jet printer. The resolution of 720 dpi provides an arrangement that can produce a diffraction image when the target analyte and labeled particles are combined. However, other resolutions may also be used. Lower resolution ink-jet printers also provide sufficiently small feature sizes (40-100 microns in diameter) to form diffraction images. The antibody may be thiolated and the resulting phosphate-buffered solution may also have glycerin (up to 40% by weight), which is added to the solution to prevent smearing of the features printed on the film. The resulting device can be used as discussed above. If a diffraction image is formed, it means that the analyte is present. Since the diffraction angle is inversely proportional to the feature spacing, it has been found that the distance from the light source must be further to obtain a recognizable diffraction image. Special observation instruments may be used for easier detection.
    [39] Receptor material bound to the patterned protein is defined as the ability to specifically bind to the analyte (s) of interest. Whatever the analyte of interest is chosen, the protein material is designed to specifically bind to the analyte of interest. In a preferred embodiment, the biosensing device is made and arranged to provide an eye identifiable pattern in response to transmission of multicolored light when the analyte of interest is placed between the antibody and the diffractive enhancing component. In another embodiment, if the analyte is large enough to diffract light, the diffractive enhancement component may not be needed.
    [40] In many cases, a "blocking agent" may be necessary to prevent nonspecific binding. As used herein, the term "blocker" refers to a reagent that is attached to a sensor surface that "blocks" or prevents non-specific binding of a substance other than the analyte to a surface (patterned or unpatterned region). The blocking step is done as a post-treatment (“post-blocking”) step of the already ink-jet printed surface, and is a standard technique for filling non-ink-jet print areas with other thiols. However, the inventors have found that the "pre-blocking" technique is preferred over the post-blocking technique. In preblocking techniques, the substrate surface is pretreated with a thiol free barrier and then ink-jet printed. While not wishing to be bound by any theory, in theory (usually sulfur containing) ink-jet printing materials replace the physisorption blockers, causing the printed materials to bind directly to the substrate surface. If necessary, subsequent post-blocking may also be performed. Blocking agents include β-casein, albumin such as bovine serum albumin, pluronic or other surfactants, polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, or sulfur derivatives of these compounds, and those known to those skilled in the art. And any other blocking material.
    [41] The analyte-containing matrix of interest may be solid, gas or body fluids, such as interstitial fluid, mucus, saliva, urine, feces, tissues, bone marrow, cerebrospinal fluid, serum, plasma, whole blood, synovial sputum, buffers, extracts, semen, Vaginal secretions, pericardium, stomach, peritoneum, pleura, throat swabs or other lavage fluids. The analyte of interest may be an antigen, an antibody, an enzyme, a toxin, an environmental agent, a cytoplasmic component, a hair or flagellar component, a protein, a polysaccharide, a drug, or any other substance recognizable by the antibody. For example, receptor material for bacteria can specifically bind surface membrane components, proteins or lipids, polysaccharides, nucleic acids or enzymes. The analyte that is indicative of bacteria may be an antibody produced by the host in response to a saccharide or polysaccharide, enzyme, nucleic acid, membrane component, ganglioside or bacteria. The presence of the analyte may indicate an infectious (bacterial or viral) disease, cancer, allergy, or other medical disease or condition. The presence of the analyte can be an indicator of water or food contamination or other harmful substances. The analyte may indicate drug abuse or monitor the level of the therapeutic agent.
    [42] In some cases, the analyte may not only bind the receptor material but also cause the deformation of the receptor material to an observable extent. Such interaction may cause an increase in mass at the test surface or a decrease in the amount of receptor material at the test surface. An example of reducing the amount of receptor material is the interaction of specific immobilized substrates with degradable enzymes or materials. In this case, the diffraction pattern may be observed before interaction with the analyte of interest, but if the analyte is present the diffraction pattern will disappear. The specific mechanism by which binding, hybridization, or interaction of the analyte with the receptor material occurs is not critical to the present invention but does not affect the reaction conditions used in the final assay protocol.
    [43] The medium in which the analyte may remain may be solid, gel, liquid or gaseous. For the purpose of detecting analytes in body fluids, the fluid may be from urine, serum, plasma, spinal fluid, sputum, whole blood, saliva, genital secretions, feces, pericardium, stomach, peritoneum, pleural lavage, vaginal secretions, or pharyngeal swabs. Although selected, but not limited to, the method optionally includes using a spectrophotometer to measure the appearance of a refractive pattern. The most common gas that can be used in the biosensing device of the present invention is air.
    [44] The biosensing device of the present invention provides a method for achieving a patterned antibody by ink-jet printing a patterned binder onto a substrate, preferably a metallized polymer film, followed by exposure to the antibody, a composition prepared therefrom and a composition of the composition Use usability. The patterned binder layer can control the placement of antibodies to which the analyte can bind. As used herein, the term "patterned binder layer" refers to the combination of a binder (eg, a protein or an antibody) with a desired antibody in any pattern on a substrate comprising a continuous pattern.
    [45] When a film with a patterned binder layer is exposed to an analyte that can react with the antibody, the film will form an optical diffraction pattern that depends on the reaction of the antibody with the analyte of interest. The liquid may be a high surface tension fluid such as water. Light may be present in the visible spectrum, reflected from or transmitted through the film, and the analyte may be any compound that reacts with the patterned binder layer.
    [46] In a preferred embodiment, the method comprises contacting the substrate with a test sample potentially containing the analyte under conditions that cause the substrate to change in refractive index. Light is transmitted through the film with the patterned binder layer to form a visible diffraction pattern that can be visualized by directing light to the surface or by looking directly through the substrate.
    [47] Alternatively, the diffraction image can be visualized using a simple viewing device consisting of a point light source and an optical component such as a lens and a mirror. Such observers can be specifically designed for samples obtained from ink-jet printing methods. If properly designed, an observer (eg, dimensions) from a hand-held device comparable to an image obtained from a much longer observation path length (eg, about 5 feet (152.4 cm)) without using an observer Is about 6 inches (15.24 cm). Longer observation path lengths will be needed in the absence of an observer, due to the wider spacing (> 50 micron period) usually obtained from ink-jet printers compared to prior art contact printing methods.
    [48] In one embodiment, it is contemplated that the present invention is a dipstick type in which an ink-jet printed metallized film is installed at the end of a dipstick. In use, the dipstick is immersed in a liquid in which the suspect analyte may be present and held for several minutes. The dipstick is then removed and then light is transmitted through the film or the film is observed by the light behind the film. If a pattern is observed, the analyte is in the liquid.
    [49] In another embodiment of the present invention, a single support strip can be used to test a plurality of analytes. As shown in FIG. 1, the strip 10 is provided with several ink-jet print films 20, 25, 30 and 35 each having a pattern 40 printed thereon. Each ink-jet print film 20, 25, 30 and 35 may be a metallized polymer film with a film having different antibodies thereon specific for the different analytes. It can be seen that the present invention can be in any arrangement with various ink-jet printing films, so that the user of the biosensor device of the present invention can detect the presence of a plurality of analytes using a single support by the wicking agent. Can be.
    [50] In another embodiment of the present invention, the biosensor can be placed on a hard surface or on the wall of the container after it is attached to a sticker or transfer paper with a backside adhesive. The biosensor may be placed on the inner surface of the container, such as a food package or glass bottle. The biosensor can then be visualized to determine the presence of the analyte.
    [51] Typically, 5 to 2000 nm thick gold films are supported on polyethylene-terephthalate films, Si / SiO 2 wafers or glass sheets. Adhesion promoters such as titanium may also be used as adhesion promoters between gold and the support. The binder adheres to the gold surface during ink-jet printing.
    [52] A more detailed description of the methods and compositions of the present invention follows. All publications cited herein are hereby incorporated by reference in their entirety throughout this disclosure.
    [53] Any polymer film is suitable for the present invention. Preferably, the polymer film is a plastic film. More preferably, the polymer film may also have a metal coating deposited thereon. Plastic films include polyethylene-terephthalate (e.g., Mylar®, MYLAR), acrylonitrile-butadiene-styrene, acrylonitrile-methyl acrylate copolymers, cellophane, cellulose polymers such as Ethyl cellulose, cellulose acetate, cellulose acetate butyrate, cellulose propionate, cellulose triacetate, cellulose triacetate, polyethylene, polyethylene-vinyl acetate copolymer, ionomer (ethylene polymer) polyethylene-nylon copolymer, polypropylene, methyl pentene polymer Polymers such as, but not limited to, polyvinyl fluoride and aromatic polysulfones. Preferably, the plastic film has an optical transparency of greater than 80%. Other suitable thermoplastics and sources are described in references such as, for example, Modern Plastics Encyclopedia (McGraw-Hill Publishing Co., New York 1923-1996).
    [54] In one embodiment of the invention, the polymer film has a metal coating thereon and the resulting metallized polymer film has an optical transparency of about 5% to 95%. More preferred optical transparency for the polymer film used in the present invention is about 20% to 80%. In a preferred embodiment of the present invention, the polymer film has an optical transparency of at least about 80% and the thickness of the metal coating is about 20 The diffraction pattern may be generated by reflected light or transmitted light since the thickness is such that the optical transparency can be maintained at least%. Optical transparency is based on the visible transmission of the wavelength of visible light passing through the film. This level of optical clarity corresponds to a metal coating thickness of about 10 nm. However, in another embodiment of the present invention, the gold thickness may be about 1 nm to 1000 nm.
    [55] Preferred metals deposited on the film are gold. However, other metals such as silver, aluminum, chromium, copper, iron, zirconium, platinum and nickel, as well as oxides of these metals may be used, but are not limited thereto.
    [56] In another embodiment, the present invention provides an optically active water soluble surface arranged and arranged to simultaneously analyze multiple samples on a surface for one analyte of interest, and dispersing the sample and reagent solutions on the surface. And an optical analysis device having an automated liquid handling device (e.g., a pipetting device) that is made and arranged to allow.
    [57] By the description provided below, optical materials and methods useful for the structure of the optical test surface of the present invention can be prepared. In general, the present invention includes novel optically active test surfaces for direct detection of analytes. This test surface has an analyte-specific antibody printed in a pattern specific to the test surface. The detection method includes testing the change in diffraction of transmitted or reflected light by contacting the device with a sample fluid containing the analyte of interest and seeing if a diffraction pattern is formed.
    [58] Alternatively, analyte-specific antibodies are bound using an adhesion layer (ie, antibody-binding protein). Accordingly, the present invention provides a detection device comprising selecting an optical substrate, printing a pattern of antibody-binding protein and then exposing it to an antibody for the desired analyte.
    [59] In one example, the analyte may be a nucleotide based analyte, such as DNA. The complementary oligonucleotides to the target DNA are ink-jet printed onto the substrate, and then the sensor is optionally blocked and rinsed with blocking materials such as β-casein, albumin, surfactants, polyethylene glycols, polyvinyl alcohol or sulfur derivatives thereof. And can be air-dried. The sensor is then placed in the analyte solution containing the DNA strand of interest. Next, diffractive enhancing component particles with oligonucleotides complementary to other regions of the analyte DNA are added to the sensor. The sensor and the particles can then be heated and then rinsed. Thereafter, the point light source may be transmitted or reflected through the sensor sample. Diffraction images may be seen on the other side of the light beam in the presence of the DNA analyte.
    [60] The invention has a wide range of applications and can be used in a variety of specific binding pair analysis methods. For example, the devices of the present invention can be used in immunoassay methods for detecting antigens or antibodies. The device may be suitable for use in direct, indirect or competition detection.
    [61] In one embodiment of the invention, the antibody-binding protein layer has the formula X-P-Ab.
    [62] X is optional as a means of enabling chemisorption on metals or metal oxides. For example, X is asymmetric or symmetric disulfide (-R'SSY ', -RSSY), sulfide (-R'SY', -RSY), diselenide (-R'Se-SeY '), sele Nide (-R'SeY ', -RSeY), thiol (-SH), nitrile (-CN), isonitrile, nitro (-NO 2 ), selenol (-SeH), trivalent phosphorus compound, isothiocyanate , Xanthate, thiocarbamate, phosphine, thioacid, dithioacid, carboxylic acid, hydroxy acid and hydroxamic acid.
    [63] P represents an antibody-binding protein that can be derived using X. Ab represents an antibody specific for the analyte of interest.
    [64] In other embodiments, the sensing device may include a substrate on which a binder material, such as an antibody or protein, is applied to the surface of the entire substrate (eg, by dipping or spraying the entire surface). The binder will cover the entire surface of the substrate, such as a polymer film or a metal-coated polymer film. An inactivating agent such as a protease or a strong anionic surfactant such as sodium dodecyl sulphate (SDS) is then printed onto the binder-coated substrate in a manner that results in inactivation of some of the binder material. The inactivated portion will be formed in a preselected pattern on the substrate. The device will then be used as discussed above, even if the patterned portion of the device is inactive. The analyte will only attach to the active portion of the binder material. The transmitted or reflected light will also cause the formation of a diffraction pattern, which can be seen by the naked eye, preferably using a specially designed observation device.
    [65] The invention is further illustrated by the following examples, but the scope of the present invention is not limited in any way by these examples. On the contrary, it will be apparent to those skilled in the art that various other embodiments, modifications, and equivalents are possible without departing from the subject matter of the present invention after reading the disclosure herein.
    [66] <Example 1>
    [67] This example relates to a thiolation process of a protein (eg, an antibody or antibody-binding protein, eg, Protein A).
    [68] To 1 g of protein was added 450 μl of phosphate buffered saline (PBS, pH 7.2 using 0.1 M sodium phosphate and 0.15 M sodium chloride according to the instructions in Pierce catalog # 28372). Then 50 μl of a 10 mM aqueous solution of sulfo-LC-SPDP (Pierce Catalog # 21650) was added. The mixture was allowed to react at room temperature for 60 minutes and then poured into a D-salt polyacrylamide desalting column (Pierce, 5 ml bed volume equilibrated with appropriate buffer). Thereafter, if reduction of disulfide bonds was performed, an acetate buffer prepared at pH 4.5 with 0.1 M sodium acetate and 0.1 M sodium chloride was used, and PBS buffer (pH 7.2) was used if the protein derivative remained in disulfide form. [Note: It is suitable to be prepared from PBS buffer because it does not require a subsequent reduction step, but both forms have been found to be suitable for preparing biosensing devices.] 500 μl of each fraction was collected from the column and the protein derivative Fractions were determined using Coomassie® protein analysis.
    [69] <Examples 2 to 7>
    [70] Examples 2-7 relate to sensing devices using ink-jet printing techniques.
    [71] <Example 2>
    [72] Gold-coated at 720 dpi using thiolated polyclonal antibodies against Streptococcus group B (eg, Cat M-C664-50L from Lee Laboratories, Graison, GA) Polyethylene-terephthalate mylar (MYLAR®) was printed. The gold-coated mylar was “Icl 453 / Au-20” / coating, with 19-20 kW / cm 2 (+ 20%) from Coutulds Performance Films, Canoga Park, CA. It was a 200 gauge polyethylene-terephthalate film with a gold coating that gave an average surface resistance. The printer was an ink jet printer (e.g., an Epson Stylus 640 or 740 printer) set up as follows: paper = photo paper; Ink = black ink only; Print quality = 720 dpi; Halftone = No halftone. An example is a 20 MB file (Adobe Photoshop) with an x, y array set at a resolution of 1500 pixels / inch. Upon printing, it produces a patterned x, y array of antibodies characterized in the range of 40 to 100 microns in diameter, which is suitable for diffraction upon analyte deposition. Place 34 microliters of Streptococcus Group B antigen-spiked buffer solution (e.g., phosphate buffered saline at pH 7.2; Antigen = Difco Cat No. 2979-50) onto the printed film Thereby, the obtained printed sample was exposed to the solution. After 5 minutes, 11 [mu] l of 0.5 micron particles bound to the polyclonal antibodies against Streptococcus group B were added by pipetting directly onto the antigen-containing droplets. Particles were suspended with about 10% solids in a beta casein containing saline / Tween 20 solution (eg, 0.005 to 0.01% Tween 20, 0.001 to 0.01 M NaCl and 5 mg / ml beta casein). After incubation for 10 minutes, a nitrocellulose disc (eg 0.8 micron pore size) with a hole in the center (eg, a diameter of 4/32 inch (0.32 cm)) was placed on top of the liquid / particle mixture. It was able to absorb unbound particles and excess liquid and irradiate the sample against a diffraction image using a point light source (eg a laser). The light was aimed to shine through the hole, showing a diffraction image when the analyte was present. Alternatively, miniature hand-held viewing devices using mirrors and microscope objective lenses can also be used to display diffraction images when the analyte is present.
    [73] <Example 3>
    [74] The gold-coated polyethylene-terephthalate film of Example 2 was printed at 720 dpi with a thiolated monoclonal antibody against IgE (eg, as specific for the C3 to C4 domains of IgE). The printer was an ink-jet printer (eg, an Epson Stylus 640 or 740 printer): paper = photo paper; Ink = black ink only; Print quality = 720 dpi; Halftone = No halftone. An example is a 20 MB file (Adobe Photoshop) in an x, y array set at a resolution of 1500 pixels / inch. Upon printing, it produces a patterned x, y array of antibodies characterized in the range of 40 to 100 microns in diameter, which is suitable for diffraction upon analyte deposition.
    [75] <Example 4>
    [76] The gold-coated polyethylene-terephthalate film of Example 2 was printed at 1440 dpi with a thiolated monoclonal antibody against IgE (eg, as specific for the C3 to C4 domains of IgE). The printer was an ink-jet printer (eg, an Epson Stylus 640 or 740 printer): paper = photo paper; Ink = black ink only; Print quality = 720 dpi; Halftone = No halftone. An example is a 20 MB file (Adobe Photoshop) in an x, y array set at a resolution of 1500 pixels / inch. Upon printing, it produces a patterned x, y array of antibodies characterized in the range of 40 to 100 microns in diameter, which is suitable for diffraction upon analyte deposition.
    [77] Example 5
    [78] The gold-coated polyethylene-terephthalate film of Example 2 was printed at 720 dpi with a thiolated monoclonal antibody against IgE (eg, as specific for the C3 to C4 domains of IgE). The printer was an ink-jet printer (eg, an Epson Stylus 640 or 740 printer): paper = photo paper; Ink = black ink only; Print quality = 720 dpi; Halftone = No halftone. An example is a 20 MB file (Adobe Photoshop) in an x, y array set at a resolution of 1500 pixels / inch. Upon printing, it produces a patterned x, y array of antibodies characterized in the range of 40 to 100 microns in diameter, which is suitable for diffraction upon analyte deposition. By placing 34 microliters of IgE-doping buffer solution (e.g. phosphate buffered saline at pH 7.2; IgE = human polyclonal IgE from Biodesign, Cat No. A10164H) as a droplet on top of the printed film The printed sample obtained was exposed to the solution. After 5 minutes, 11 μl of 0.5 micron particles bound to the yeast fungus ( Aspergillus fumigatus ) allergen extract (eg, Greer Laboratories) were added by pipetting directly onto the antigen-containing droplets. Particles were suspended with about 10% solids in a beta casein containing saline / twin 20 solution (eg, 0.005 to 0.01% Tween 20, 0.001 to 0.01 M NaCl and 5 mg / ml beta casein). After incubation for 10 minutes, a nitrocellulose disc (eg 0.8 micron pore size) with a hole in the center (eg, a diameter of 3/32 inch (0.24 cm)) was placed on top of the liquid / particle mixture. It was able to absorb unbound particles and excess liquid and irradiate the sample against a diffraction image using a point light source (eg a laser). Aim the light through the hole to see the diffraction image when the analyte is present. Alternatively, miniature hand-held viewing devices using mirrors and microscope objective lenses can also be used to display diffraction images when the analyte is present.
    [79] <Example 6>
    [80] The gold-coated polyethylene-terephthalate film of Example 2 was printed at 720 dpi with a thiolated monoclonal antibody against IgE (eg, as specific for the C3 to C4 domains of IgE). The printer was an ink-jet printer (eg, an Epson Stylus 640 or 740 printer): paper = photo paper; Ink = black ink only; Print quality = 720 dpi; Halftone = No halftone. An example is a 20 MB file (Adobe Photoshop) in an x, y array set at a resolution of 1500 pixels / inch. Upon printing, it produces a patterned x, y array of antibodies characterized in the range of 40 to 100 microns in diameter, which is suitable for diffraction upon analyte deposition. Prints obtained by placing 34 microliters of IgE-doping buffer solution (e.g., phosphate buffered saline at pH 7.2; IgE = human polyclonal IgE from Biodesign, Cat No. A10164H) as droplets on top of the printed film Sample was exposed to the solution. After 5 minutes, 11 μl of 0.5 micron particles bound to the yeast fungus allergen extract (eg, Greer Laboratories) were added by pipetting directly onto the antigen-containing droplets. Particles were suspended with about 10% solids in a beta casein containing saline / twin 20 solution (eg, 0.005 to 0.01% Tween 20, 0.001 to 0.01 M NaCl and 5 mg / ml beta casein). After incubation for 10 minutes, a nitrocellulose disc (eg 0.8 micron pore size) with a hole in the center (eg, a diameter of 3/32 inch (0.24 cm)) was placed on top of the liquid / particle mixture. It was able to absorb unbound particles and excess liquid and irradiate the sample against a diffraction image using a point light source (eg a laser). Aim the light through the hole to see the diffraction image when the analyte is present. Alternatively, miniature hand-held viewing devices using mirrors and microscope objective lenses can also be used to display diffraction images when the analyte is present.
    [81] <Example 7>
    [82] The gold-coated polyethylene-terephthalate film of Example 2 was preliminary for 10 minutes with a 1% polyvinylpyrrolidone (e.g. Cat PVP-10, Sigma, St. Louis, MO) distilled water solution After treatment, rinse with distilled water. Alternatively, the PVP solution may also contain 1% Triton (eg Triton X-100) surfactant. The pre-treated film was then loaded into a paper tray of an ink-jet printer (eg, Epson Stylus 640 or 740). Thiolated monoclonal antibodies against IgE containing 10 to 30% glycerin (such as those specific for the C3 to C4 domains of IgE, for example Cat Z86410 from Biodesign from Mainne Kennebunk) Was placed in an empty "black ink" cartridge and printed on gold / meyer at 720 dpi using the following parameters:
    [83] Paper-Photo Paper
    [84] Ink-use only black ink
    [85] Print Quality-Fine 720 dpi
    [86] Halftone = no halftone
    [87] A pattern of x, y arrays of pixels (resolution of 1500 pixels / inch, total size of 20 megabytes) as printed by Adobe Photoshop 3.0 was printed on gold / meyer. It has been found that glycerin helps to provide a small printed circle (eg, typically 40-100 um in size) by preventing bleeding of the droplets when printed on PVP-treated gold surfaces. Upon printing, it produces a patterned x, y array of antibodies characterized in the range of 40 to 100 microns in diameter, which is suitable for diffraction upon analyte deposition. Prints obtained by placing 34 microliters of IgE-doping buffer solution (e.g., phosphate buffered saline at pH 7.2; IgE = human polyclonal IgE from Biodesign, Cat No. A10164H) as droplets on top of the printed film Sample was exposed to the solution. After 5 minutes, 11 μl of 0.5 micron particles bound to the yeast fungus allergen extract (eg, Greer Laboratories) were added by pipetting directly onto the antigen-containing droplets. Particles were suspended with about 10% solids in a beta casein containing saline / twin 20 solution (eg, 0.005 to 0.01% Tween 20, 0.001 to 0.01 M NaCl and 5 mg / ml beta casein). After incubation for 10 minutes, a nitrocellulose disc (eg 0.8 micron pore size) with a hole in the center (eg, a diameter of 3/32 inch (0.24 cm)) was placed on top of the liquid / particle mixture. It was able to absorb unbound particles and excess liquid and irradiate the sample against a diffraction image using a point light source (eg a laser). Aim the light through the hole to see the diffraction image when the analyte is present. Alternatively, miniature hand-held viewing devices using mirrors and microscope objective lenses can also be used to display diffraction images when the analyte is present.
    [88] Those skilled in the art will appreciate that many modifications and variations are possible in the present invention without departing from its scope. The foregoing detailed description and examples are, therefore, for illustrative purposes only and are not intended to limit the scope of the invention as described in the appended claims in any way.
    权利要求:
    Claims (27)
    [1" claim-type="Currently amended] Polymer film, and
    Binder layer ink-jet printed in pattern on polymer film and comprising an antibody specific for the analyte
    Biosensor comprising a.
    [2" claim-type="Currently amended] The biosensor of claim 1, wherein the polymer film further comprises a metal coating.
    [3" claim-type="Currently amended] Polymer films; And
    Ink-jet printed binder layer produced throughout the polymer layer to inactivate the patterned portion of the binder layer
    Biosensor comprising a.
    [4" claim-type="Currently amended] The biosensor of claim 3, wherein the binder layer has an antibody thereon specific for the analyte.
    [5" claim-type="Currently amended] 4. The biosensor of claim 3, wherein the binder layer is disruptive to the pattern by ink-jet printing of a material that can disrupt the analyte-binding activity and is selected from proteases or strongly anionic surfactants.
    [6" claim-type="Currently amended] The biosensor of claim 3, wherein the polymer film further comprises a metal coating.
    [7" claim-type="Currently amended] Polymer films; And
    Antibody-binding protein layer capable of ink-jet printing in a pattern on the polymer film and binding to the antibody
    Biosensor comprising a.
    [8" claim-type="Currently amended] 8. The biosensor of claim 7, wherein the antibody is specific for the analyte.
    [9" claim-type="Currently amended] The biosensor of claim 7, wherein the polymer film further comprises a metal coating.
    [10" claim-type="Currently amended] A method of making a biosensor comprising ink-jet printing a pattern of a binder material layer comprising an antibody specific for an analyte on a polymer film.
    [11" claim-type="Currently amended] The method of claim 10, wherein the polymer film further comprises a metal coating.
    [12" claim-type="Currently amended] Coating a layer of binder material on the polymer film; And
    Ink-jetting an inactivating material to inactivate the patterned portion of the binder layer
    Method of manufacturing a biosensor comprising a.
    [13" claim-type="Currently amended] The method of claim 12, wherein the binder material layer has an antibody thereon specific for the analyte.
    [14" claim-type="Currently amended] 13. The method of claim 12, wherein the binder layer is pattern-inactivated by ink-jet printing a material that can disrupt analyte-binding activity and is selected from proteases or sodium dodecyl sulfate.
    [15" claim-type="Currently amended] The method of claim 12, wherein the polymer film further comprises a metal coating.
    [16" claim-type="Currently amended] A method of making a biosensor comprising ink-jet printing a pattern of an antibody-binding protein layer capable of binding to an antibody on a polymer film.
    [17" claim-type="Currently amended] The method of claim 16, wherein the antibody is specific for the analyte.
    [18" claim-type="Currently amended] The method of claim 16, wherein the polymer film further comprises a metal coating.
    [19" claim-type="Currently amended] Contacting a biosensing device comprising a polymer film and a binder layer printed in a pattern on the polymer film and comprising a binder layer comprising an antibody specific for the analyte, and a medium suspected of containing the analyte;
    Transmitting light through the polymer film or reflecting light from the polymer film; And
    Detecting the presence of an analyte bound to the antibody by detecting a pattern formed by diffraction of transmitted or reflected light
    Method of detecting the analyte in the medium comprising a.
    [20" claim-type="Currently amended] The method of claim 19, wherein the polymer film further comprises a metal coating.
    [21" claim-type="Currently amended] Contacting a biosensing device comprising a polymer film and an ink-jet printed binder layer formed throughout the polymer layer to inactivate the pattern portion of the binder layer, and a medium suspected of containing the analyte;
    Transmitting light through the polymer film or reflecting light from the polymer film; And
    Detecting the presence of an analyte bound to the antibody by detecting a pattern formed by diffraction of transmitted or reflected light
    Method of detecting the analyte in the medium comprising a.
    [22" claim-type="Currently amended] The method of claim 21, wherein the binder material layer has an antibody thereon specific for the analyte.
    [23" claim-type="Currently amended] The method of claim 21, wherein the binder layer is inactivated according to the pattern by ink-jet printing a material that can disrupt analyte-binding activity and is selected from protease or sodium dodecyl sulfate.
    [24" claim-type="Currently amended] The method of claim 21, wherein the polymer film further comprises a metal coating.
    [25" claim-type="Currently amended] Contacting a biosensing device comprising a polymer film and an antibody-binding protein layer ink-jet printed in a pattern on the polymer film and capable of binding to an antibody, and a medium suspected of containing the analyte;
    Transmitting light through the polymer film or reflecting light from the polymer film; And
    Detecting the presence of an analyte bound to the antibody by detecting a pattern formed by diffraction of transmitted or reflected light
    Method of detecting the analyte in the medium comprising a.
    [26" claim-type="Currently amended] The method of claim 25, wherein the antibody is specific for the analyte.
    [27" claim-type="Currently amended] The method of claim 25, wherein the polymer film further comprises a metal coating.
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    同族专利:
    公开号 | 公开日
    KR100761639B1|2007-10-04|
    AU2001251522B2|2006-09-07|
    CA2406511C|2010-07-06|
    WO2001081921A2|2001-11-01|
    DE60130879T2|2008-02-07|
    TWI239399B|2005-09-11|
    CN1437708A|2003-08-20|
    WO2001081921A3|2002-06-27|
    AT375513T|2007-10-15|
    CN1318850C|2007-05-30|
    MXPA02009829A|2003-03-27|
    DE60130879D1|2007-11-22|
    EP1277056A2|2003-01-22|
    EP1277056B1|2007-10-10|
    CA2406511A1|2001-11-01|
    AU5152201A|2001-11-07|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    2000-04-24|Priority to US55745300A
    2000-04-24|Priority to US09/557,453
    2001-04-11|Application filed by 킴벌리-클라크 월드와이드, 인크.
    2002-12-31|Publication of KR20020097226A
    2007-10-04|Application granted
    2007-10-04|Publication of KR100761639B1
    优先权:
    申请号 | 申请日 | 专利标题
    US55745300A| true| 2000-04-24|2000-04-24|
    US09/557,453|2000-04-24|
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