WO2004081570A1 - A chip matrix, a chip comprising the matrix and their preparation and application - Google Patents

Abstract

The present invention relates to an assay-chip matrix, a chip comprising the matrix by which an interest component in a sample, particularly in a biological sample can be analyzed qualitatively and/or quantitatively, and a scanning equipment which can be used to measure the chip. The chip matrix based on the present inventio consists of a base and an arbitrary isolation construction, and said base comprises a substrate and an arbitrary composite construction(s) selected from covers, coats, films and slices. And said substrate and/or at least one said composite construction can include an optical-signal relative substance(s) with regard to the absorption, emission and/or reflection of optical signals, such as luminescing substances, dyes, pigments, dulling agents and their direct or indirect capturing agents. The present invention also relates to a preparation method of the matrix and the chip, and the measurement of the chip.

Description

 Chip substrate, chip containing the substrate, and preparation method and application thereof

 The invention relates to a detection chip substrate for qualitative and / or quantitative analysis of a target object in a sample, especially a biological sample, and also relates to a detection chip including the substrate, a detection chip scanning device, and a detection chip. The preparation method of the substrate and chip is described. Background technique

The "detection chip" in the present invention, also referred to as "chip" for short, includes, but is not limited to, "Biochip", "Microarray", and "Bioarray" in English. It is a detection device in a specific and / or quantitative analysis. The specific reaction between the trace probe in the reactor and the target molecule in the sample can be identified in an addressable manner. The core of the chip is the reactor in which the core of the reactor is the chip base and the chip fixed on the chip In the chip of the present invention, the distribution density of the probes on the substrate is greater than 10 dots / cm ² , preferably greater than 20 dots / cm ² , and more preferably greater than 40 dots / cm ² , and each The area of the probe point is not greater than 1 mm

 Chips include biochips and non-biochips. Currently, the most commonly used are biochips. The most commonly used biochips are peptide chips and gene chips. The peptide chip is a biochip prepared by immobilizing a sequence structure of multiple amino acids (including proteins) on a substrate as a probe. A gene chip is a chip that hybridizes nucleic acids, nucleotides with complementary nucleic acids, and nucleotide probes in a sample to be tested to form a hybrid, or binds to a specific antibody, and then displays the detection result with a color reaction. Biochips have a wide range of applications, including gene expression detection, gene screening, drug screening, disease diagnosis and treatment, environmental monitoring and governance, and judicial identification. The core of the biochip is the probes fixed in it. Biochip probes include all biologically active substances that can be immobilized on a solid support, such as antigens, antibodies, single- and multi-stranded DNA, RNA, nucleotides, ligands, ligands, peptides, cells, Tissue components and other biological components.

The chip substrate in the present invention, referred to as a substrate for short, refers to a product based on the chip base and optionally combined with other structures (such as an isolation structure) to form a chip after the probe is fixed. There can be one or more substrate pools on the substrate. There is usually no isolation structure on a single-chip substrate. In this case, the substrate is a substrate, such as a commercially available amino glass slide. The multi-chip base substrate has an isolation structure. At this time, the substrate includes a base and an isolation structure. The chip-based cell forms a reactor after the probe is fixed. The wafer base forms a multi-reactor chip. The substrate is a solid-phase carrier used to fix probes and other auxiliary agents (if any). The surface chemical and optical properties are important factors affecting chip performance and cost.

 In addition, the chip signal detection instrument is an important condition for chip applications. The most commonly used chip signal detection instruments are scanners, including confocal scanners, CCD scanners, and general scanners. The chip compartment in the scanner is the place where the chip is placed during scanning, and its optical properties affect the scanning results.

 The following briefly describes the problems that need to be solved with existing substrates, chips, and chip signal detection instruments.

 The existing chip detection methods include two types of light detection methods and non-light detection methods. Examples of non-luminous detection methods are the SELDI-TOF-MS method (Surface-Enhanced Laser Dissociation and Laser Ionization Time-of-Flight Mass Spectrometry, Surface-Enhanced Laser Desorption / Ionization-Time of Flight-Mass Spectra), such as Ciphergen, USA It includes the MetalCM-based ProteinCMp Array system. However, the most widely used biochip detection method is still the luminescence detection method. Luminescence detection methods mainly include fluorescence detection method, chemical luminescence detection method and composite light irradiation detection method. The current chip detection methods are based on the minimization of background signals. In the existing chip detection methods, reducing background noise and improving the signal-to-noise ratio are an important aspect of improving detection sensitivity, and involve substrate preparation and detection conditions and signal detection instrument preparation and detection conditions. Even reducing background noise has become one of the reasons for the high cost of chip and signal detection instruments.

 At present, the substrates used in fluorescence detection methods are basically transparent glass slides (such as amination, aldolization, and polylysine film substrates), and the substrates used in chemiluminescence detection methods are basically transparent glass slides, Plastic plate or metal film (such as silver film), polymer film (such as PVDF film, nylon film, nitrocellulose film, acetate fiber film, etc.) that is basically diffusible in the substrate used in the composite light irradiation detection method. However, the current composite light irradiation detection method based on a diffusible polymer film base has low sensitivity; the current chemiluminescence detection method is not very sensitive; the current fluorescence detection method, although the sensitivity is higher than the previous two High, but the background noise of the slide substrate is not low, the cost of the activated substrate is high, the detection equipment is expensive and other deficiencies, which affect the large-scale application of the biochip method.

On the other hand, whether it is a chip or a signal detection instrument, the reduction of its background noise is always limited and sometimes even full of contradictions. For example, in order to increase the fixing amount of the probe, The detection sensitivity of the chip is necessary to increase the activity and / or concentration of the surface-active groups on the substrate, but at the same time increase the non-specific binding activity on the substrate except for the probe points, thereby increasing the background noise Increased risk. For another example, in order to increase the intensity of a detectable signal, a laser excitation light source with a larger power is required in the laser confocal scanner, but it increases the cost and increases the risk of photobleaching. For example, in order to increase the intensity of a detectable signal, a higher-power excitation light source is required in the CCD scanner, but it increases both the cost and the instrument noise (such as dark current).

 In short, the existing research and development of substrates has mainly done a lot of work on the surface chemistry of the substrate, and the research on the optical properties of the substrate is still insufficient, leading to the following problems: 1) The focus is completely on the background The signal minimization / signal-to-noise ratio is maximized, which greatly reduces the degree of freedom in film selection. As a result, the types of film substrates are very limited; 2) Even when the background signal is minimized / maximized Focusing on the development of colorless, especially transparent substrates, further reducing the degree of freedom in selecting substrates, as a result, the types of substrates invented are very, very limited; and 3) the degree of freedom is too small, making the increase in detection sensitivity affected Limits, and / or substrate cost reductions are limited. Chips and detection instruments have similar problems. Therefore, the invention of a substrate, a chip, and a detection instrument with lower cost and / or higher detection sensitivity is an urgent problem to be solved in the development of the chip. Summary of the Invention

 According to one aspect of the present invention, it provides a chip substrate, which includes a substrate and an optional isolation structure, the substrate includes a substrate and optionally one or more of the following composite structures: coating, Coatings, films, and flakes, the matrix and / or at least one of the composite structures contain a colorant and / or a capture agent that directly or indirectly captures the colorant.

 According to another aspect of the present invention, a chip is provided, which includes a reaction system and an optional labeling system. The reaction system includes a substrate as described above and a probe fixed in a substrate substrate pool. .

 According to yet another aspect of the present invention, it provides a chip signal detection instrument, wherein at least the surface of a portion directly below the chip during scanning is an ultra-black surface having an absorbance greater than 95%, preferably greater than 98%. The ultra-black surface contains an ultra-black metal salt and / or an ultra-black metal oxide.

According to yet another aspect of the present invention, it provides a method for preparing a chip substrate according to the present invention, which comprises introducing on the substrate a light-emitting substance and / or a dye and / or a coloring pigment and / Or matting agents in one or more of the following structures: coatings, coatings, films and flakes.

 According to another aspect of the present invention, a method for preparing a chip and a chip manufactured according to the method are provided. The preparation method includes mixing a probe with a dye, a color pigment, and / or a coating material containing the color pigment, and then spotting the sample into a film base pool.

 According to another aspect of the present invention, it provides a chip detection method, in which at least one substrate and / or at least one probe point of a reaction system of a used chip contains dyes and / or colored pigments that are not used as probes and / Or a paint containing a coloring pigment, the dye is selected from black, green, blue, purple, and red dyes including amino black, Coomassie brilliant blue, crystal violet, and Ponceau red; the coloring pigment is selected from Said one or more pigments: luminescent pigments including fluorescent substances, chemiluminescent pigments and electroluminescent pigments, black pigments including carbon black, metal salts, white pigments including titanium dioxide, and Yellow pigment, red pigment, blue pigment, green pigment, metallic pigment; the paint is selected from black paint, white paint, red paint, yellow paint, green paint, blue paint, indigo paint or purple paint Painted inside. detailed description

the term

 The term "analysis chip" or "chip" in the present invention includes but is not limited to

"Biocliip", "Microarray", and "Bioarray" are a kind of detection device in the specified and / or quantitative analysis. The specific reaction between the trace probe in the reactor and the target molecule in the sample can be addressed with an addressable address. Way to identify. The core of the chip is the reactor therein, and the core of the reactor is the chip substrate and the probe fixed on the chip substrate. The chip includes a microchannel chip (equivalent to in English) and a microarray chip (equivalent to "Biochip", "Microarray", "Bioarray" in English), but it is well known that it does not include existing rapid test reagent strips. The chip of the present invention contains a single reactor or multiple reactors with or without a labeling system. The distribution density of the probes on the substrate in the reactor is greater than 10 points W, and the preferred solution is greater than

20 points / cm ² , more preferably a solution of more than 40 points / cm ² , and the area of each probe point is not more than 1 mm

 The term "detection device" according to the present invention is a device used in a specified and / or quantitative analysis method, and includes a chip, a signal detection device, and the like.

The term "reactor" in the present invention refers to the place where the probe specifically reacts with the target and other related structures communicating with it, such as the reaction cell and Related isolation structures and inlet and outlet fluid structures.

 The term "substrate" in the present invention refers to a product based on a substrate, optionally combined with other structures (such as an isolation structure), and used to form a chip after the probe is fixed. There can be one or more substrate pools on the substrate. Monolithic substrates usually do not have an isolation structure on them. In this case, the substrate is a substrate (such as a commercially available amino slide). There is an isolation structure on the multi-chip base substrate. In this case, the substrate includes a base and an isolation structure. The chip-based cell forms a reactor after the probe is fixed, and the multi-chip base forms a multi-reactor chip. The substrate is a solid support used to hold probes and other additives (if any). The surface chemical and optical properties are important factors affecting chip performance and cost.

 The term “film base pool” in the present invention refers to a structure formed by a film base and its isolation structure.

 The term "probe" in the present invention refers to a substance, such as an antigen, an antibody, a nucleic acid, and the like, which is immobilized on a solid-phase carrier to identify a target substance in a sample.

式中 Eo为目标比辐 射率， Eb为背景比幅射率， Eo和 Eb分别与目标材料和背景材料的吸收 率有正比关系。 对红外隐身而言， C越大， 热像仪分辨率越高、 可探测 性越大。 反之， C趋于零时处于隐身最佳状态。 隐身技术致力于使目标 与背景有最小化的反差， 本发明的技术致力于使目标与背景有最大化的 反差。 在本发明中， 反差最大化结构是指使目标与背景有最大化反差 的结构。 The term "contrast maximization" in the present invention: The signal contrast between the target and the background is a sign of detectability. For example, for infrared stealth technology, Maclean et al. Used the contrast ratio radiation C to represent the detectability of a thermal imaging camera:

In the formula, Eo is the target specific emissivity, Eb is the background specific radiance, and Eo and Eb are directly proportional to the absorption of the target material and the background material, respectively. For infrared stealth, the larger C, the higher the resolution and the greater the detectability of the camera. Conversely, C is in the best state of stealth when it approaches zero. Stealth technology is dedicated to minimizing the contrast between the target and the background, and the technology of the present invention is dedicated to maximizing the contrast between the target and the background. In the present invention, the contrast-maximizing structure refers to a structure that maximizes the contrast between the target and the background.

 The term "background signal enhancement" in the present invention means that the background detection signal is made higher than the signal with the weakest target detection signal.

 The term "attenuation of the target signal" in the present invention means that the weakest target detection signal is made lower than the background detection signal.

 The term "achromatic color" in the present invention: when white light is irradiated on an object, if the reflectance of visible light of all wavelengths is greater than 80%, the object is white; if the reflectance of visible light of all wavelengths is less than 7%, the object is black; If the reflectance of visible light is uniformly greater than 7% and less than 80%, the object is gray; a type of color composed of white, gray, and black is called an achromatic color in the present invention.

The term "coating" in the present invention refers to immobilizing a substance in a solid state on a solid support, For example, coating points, coating surfaces, etc., on which the dye molecules are fixed on the glass slide by ion adsorption, affinity adsorption, and the like.

 The term “coating” in the present invention refers to a dry film having a thickness of less than 1 mm formed by coating a solid phase material with a coating material.

 The term "film" in the present invention refers to a non-porous or perforated planar material having a thickness of less than 0.3 mm. The term "sheet" in the present invention refers to a non-porous or perforated planar material having a thickness of 0.3 mm or more.

 The term “labeling substance” in the present invention refers to a substance used to form or participate in the formation of a detection signal, such as fluorescein, a labeling substance commonly used in chip detection.

 The term “ligand” in the present invention refers to a substance used to capture its ligand (Ligate) through affinity, such as one or more of the following: antigen, antibody, ligand, ligand, polypeptide And single-stranded or multi-stranded DNA, RNA, nucleotides.

 The term "coloring" in the present invention refers to making the preparation have certain properties of light absorption, reflection, refraction and the like so as to form optimized hue and / or lightness and / or saturation in visible light or under a selected wavelength of light.

 The term "colorant" as used herein refers to a substance that can color a preparation.

 The term "coloring pigment" in the present invention refers to a substance that has no affinity for the coloring object and is mainly colored by combining other film-forming materials such as resin and adhesive with the coloring object, such as carbon black, mica titanium pearlescent coloring pigment, and azo coloring pigment. , Phthalocyanine color pigment, colorless fluorescent color pigment. The coloring pigments in the present invention do not include extender pigments according to the custom of the coating industry. The extender pigments generally refer to bulk fillers or substances without coloring functions. Examples of extender pigment colors can be the metallic silver color of a silver foil base, and a film of a base film. Essence and so on.

 The term "dye" in the present invention refers to a substance that has an affinity for a colored object and can color the colored object.

 The term "paint" in the present invention refers to a substance applied to a substrate of a film to obtain a specific function of a dry film having a thickness of less than 1 mm. Many paints contain colored pigments, such as various paints and various inks.

The term "target" in the present invention refers to the entirety of all substances related to the detection signal at the probe point in the chip pool when the signal is detected, including the probe, the target (if any) captured by the probe, and the marker ( If so, in addition to the probe, a probe carrier (for example, a nanocarrier) and a dye, a coloring pigment, a marker, and the like of the present invention may be provided on the probe point. The term “film background” in the present invention refers to the entirety of all substances related to the detection signal in the detection area except the target in the film base pool when the signal is detected.

 The present invention aims to provide more alternative substrates / chips, and / or improve chip detection sensitivity. In fact, one of the prerequisites for providing more alternative substrates / chips is that they need to have a sufficiently high detection sensitivity. The objectives of the present invention are related to the objectives of current stealth technology. For example, for infrared stealth technology, Maclean et al. Used the contrast ratio radiation C to represent the detectability of the thermal imager: C2 Eo-Eb. Where Eo is the specific emissivity of the target, Eb is the specific emissivity of the background, and Eo and Eb are directly proportional to the absorption of the target and background materials, respectively. For infrared stealth, the larger the C, the higher the resolution and the greater the detectability of the camera. Conversely, C is in the best state of stealth when it approaches zero. Stealth technology is devoted to minimizing the contrast between the target and the background. The technology of the present invention is devoted to maximizing the contrast between the target and the background, thereby improving the chip detection sensitivity and / or providing more options with a sufficiently high detection sensitivity Substrate / chip. The technique of the present invention may be referred to as a manifestation technique, and the detection method of the present invention may be referred to as a manifestation technique detection method. In fact, in one embodiment of the invention, by increasing the background luminous ability of the chip substrate instead of reducing the current background luminous ability, we were surprised to find that not only did the detection sensitivity not decrease, but it was significantly improved.

Therefore, the research of the present invention selects "revealing body" as the technical basis, and seeks to minimize the background noise of the chip base and seek the signal of the weakest detection target (usually a negative detection target) of the chip base background signal. The maximization of the difference ' ⁵ and "the maximum optical contrast between the labeling substance and the substrate to ensure or improve the detection sensitivity" two technical routes to achieve this goal.

 The roughness Ra of the chip substrate surface of the present invention is between 0.02 and 5.0 μm, and preferably between 0.25 and 5.0 μm.

The chip substrate of the present invention includes a substrate and an optional isolation structure. The substrate includes a substrate and optionally one or more of the following composite structures: a coating, a coating, a film, and a sheet. The substrate And / or at least one of the composite structures contains a colorant and / or a capture agent that directly or indirectly captures the colorant. It should be noted here that the capture agent that captures the colorant directly or indirectly is not used to capture a probe. The current chip substrates are all transparent or uncolored substrates. The uncolored film base only retains its base color (such as the color of extender pigments) without adding dyes and coloring pigments. Examples thereof may be the metallic silver color of silver foil base, the film color of thin film base, and the like. In the chip substrate of the present invention, a coating, a coating, a film, and a wafer containing a light-signal-related substance In the front and / or back of the substrate and / or sandwich. In Example 1, a coating containing a fluorescent substance in the substrate (Table 1), a substrate containing a luminescent pigment film, a substrate surface containing a luminescent pigment capturing agent, a coating containing a substrate surface containing a luminescent pigment, The surface of the substrate contains a coating containing a luminescent pigment, the surface of the substrate contains a reflective film, and the surface of the substrate contains a flake containing a luminescent pigment, and the like.

 In the chip substrate of the present invention, the colorant and / or capture agent is formed or involved in forming a colored background and / or a signal weakening target at least under signal detection conditions when the substrate is applied, and the colored background includes One or more of the following backgrounds: a luminous background that emits signal light, a reflective background with a signal light reflectance greater than 50%, an achromatic color background, and a colored background, the signal weakens the target, and its detection signal is related to the optical signal The presence of matter weakens. In Example 1, an example of a substrate having a light-emitting background (for example, a substrate having a luminescent pigment film on the surface of the substrate) and a light-reflecting background (for example, a substrate having a reflective film on the surface of the substrate) is given. A substrate having a luminescent background and / or a reflective background is also referred to as a background signal enhancement substrate in the present invention. In Embodiment 4, an example of a substrate having an achromatic color background (black substrate, white substrate) and a color background (blue substrate) is given. A substrate having an achromatic color background and / or a colored background is also referred to as a color substrate in the present invention.

• In the chip substrate of the present invention, the signal attenuation target and / or colored background, preferably a luminescent background and / or a reflective background participate in forming a high noise signal ratio, and the high noise signal ratio refers to a background signal ratio to a negative target Or a blank control target or an extreme weak positive target has a high detection signal, preferably at least 50% higher, more preferably at least 300% higher. Increasing the noise-to-signal ratio as a method for improving sensitivity is an important point of the present invention. Different from the current technical route that everyone is working to reduce background signals (even recognized as noise), we surprisingly found through the embodiments of the present invention that by fixing rhodamine markers beyond the probe point on the substrate, The sensitivity of the chip to detect positive samples, especially weak positive samples, is not reduced, but increased. Glowing backgrounds and / or reflective backgrounds are also referred to as background signal backgrounds in the present invention. The enhanced background of the substrate can be enhanced by one or more of the following methods: adding a luminescent pigment (such as fluorescein) to the substrate, and adding a coating or coating containing a luminescent pigment or reflection on the front and / or back of the substrate (Such as a luminescent coating on the surface and / or back of the substrate on which the probe is fixed), a thin film (such as a luminescent film or reflective film on the surface and / or the back of the substrate), with or without detection targets Porous flakes (such as luminescent flakes covering the surface and / or back of the substrate), and active groups derived from the surface of the substrate that can directly or indirectly bind luminescent pigments (such as coating protein A on the substrate and then Binding of rhodamine-labeled IgG-example tablet before or after detection reaction). Signal weakening targets can be achieved by adding dyes, coloring pigments and / or coatings that reduce signal light emission and / or reflection or increase signal light absorption at the quasi-fixed probe points, and / or add Coatings, coatings (eg, matt coatings on the surface and / or back of the substrate) of these dyes, pigments and / or coatings, films (eg, covering the surface of the substrate on which the probe is fixed and / or The light-reducing film on the back surface), and / or the sheet (for example, a light-reducing sheet containing a test substance colorant covering the surface of the substrate and / or the back surface).

In the chip substrate of the present invention, the colored background, preferably an achromatic color background and / or a colored background participates in forming a high chromatic aberration ratio, and the high chromatic aberration ratio refers to the entire wavelength or a part of the wavelength between the background and the target. the absolute value of the signal light or the absorbance difference in reflectance of not less than 50%, preferably not less than 70% ₀ of the higher ratio means a significant color difference between background and object in hue, lightness and saturation, or The absolute value of the difference between the absorptance or reflectance of the signal light of all or part of the wavelength between the background and the target is not less than 50%, preferably not less than 70%. Through the embodiments of the present invention, we have invented to improve the color difference ratio as a method of improving sensitivity, which is another focus of the present invention. The difference between the selective absorption rate of the signal light between the background and the target (for example, the achromatic color difference between a black background with an absorption rate greater than 95% and a light-emitting target containing a fluorescent substance (the absorption rate is set to 0 in the present invention) The maximization of the value is conducive to the target's self-explanation and thus improves the sensitivity. The difference between the selective reflectivity of the signal light between the background and the target (for example, when the signal light is white, the reflectance of visible light of all wavelengths is greater than 80% on a white background Maximization of the color difference between a target with crystal violet and a visible light reflectance other than purple that is less than 10%) is also conducive to the target's appearance and thus improves sensitivity. In the present invention, the achromatic color background and / or color The background is also called a color background. Obtaining a color background includes adding dyes and / or coloring pigments and / or coatings containing coloring pigments to a substrate, or adding dyes and / or coloring pigments and / or coloring pigments to a base. One or more of the following structures of a coating: coating, coating, film, and sheet.

In the chip substrate of the present invention, the substrate is selected from the group consisting of modified or unmodified glass, plastic, and metal. For example, the derivative activation group of the modified glass slide may include one or a combination of any two or more of the derivative activation groups of the existing glass chip substrate: amino, epoxy, aldehyde, and hydrazide (-CO-NHN¾), aminoureido (¾N-NH-CONH-), diethylaminoethyl (DEAE), diethylmono (2-hydroxypropyl) aminoethyl (QAE), carboxymethyl ( CM), sulfopropyl (SP), mercaptoethylpyridine (MEP), siloxane group, thiol group. In the chip substrate of the present invention, the colorant includes one or more of the following substances: a light-emitting substance, a dye, a coloring pigment, and a matting agent. Luminescent substances, dyes, coloring pigments, and matting agents are a well-known concept in the coloring of materials such as plastics and coatings, and have certain contents. The luminescent substance is selected from one or more of the following substances: rhodamine, CY3, CY5, Alexa, seaweed protein, rare earth compound-based fluorescent substances, chemiluminescent substances, and electrochemical light-emitting substances. The dyes include amino black, Coomassie brilliant blue, crystal violet, Ponceau red, printed paint paddles (7701 FBRN BLACK FBRN, 6201 Scarlet FGG SCARLET FGG, 6101 F7G BRILLIANT YELLOW F7G), water-based vapor dyes (water-based blue, Water-based green, water-based white) including black, purple, green, blue, indigo water-based dyes, water and oil amphoteric dyes. The coloring pigment is selected from one or more of the following pigments: black pigments including carbon black, metal salts, white pigments including titanium dioxide, and yellow pigments, red pigments, blue pigments, green pigments, Color pigments including metallic pigments. The fluorescent coloring pigment includes one or more of the following substances: rhodamine, CY3, CY5. The dyes are black, purple, green, blue or indigo water-based dyes including amino black, Coomassie brilliant blue, crystal violet, water-oil amphoteric dyes, printing paint pastes, and the like.

 In the chip substrate of the present invention, the roughness Ra of the surface of the substrate is between 0.02 and 5.0 m, preferably between 0.25 and 5.0 m.

 In the chip substrate of the present invention, the paint of the coating layer is selected from black, white, and various colored paints and / or inks. The colors include red, yellow, green, blue, cyan, and purple colors. In the present invention, both the ink and the paint have the property of coloring the substrate.

 In the chip substrate of the present invention, the substrate containing the colorant in the substrate and / or the composite structure contains a substance capable of binding a probe. The probe-binding substance includes one or more of the following known probe-binding organic substances and derivatives thereof: nitrocellulose, polystyrene, polyvinyl chloride, amino resin, polysaccharide, polyamino acid, Polyacrylate, polysulfone, polyethersulfone, etc.

 In the chip substrate of the present invention, the color paint of the coating layer includes one or more of the following paints: pearl black, magic black, pearl white, pearl blue, matte black, claret, scarlet, medium blue, etc. .

In the chip substrate of the present invention, the capture agent includes one or more of the following ligands: protein A, protein G, biotin, avidin, antigen, antibody, anti-antibody, polypeptide, DNA, etc. . For example, an antibody may capture an anti- antibody that binds to rhodamine to indirectly capture rhodamine, and so on. In the chip substrate of the present invention, the substrate containing the optical signal related substance in the substrate and / or the composite structure contains a substance capable of binding a probe. The probe-binding substance includes one or more of the following organic substances and derivatives thereof: nitrocellulose, polystyrene, polyvinyl chloride, amino resin, polysaccharide, polyamino acid, polyacrylate, polysulfone, Polyethersulfone, etc.

 In another aspect, the present invention also provides a chip including a reaction system and an optional marking system, the reaction system including the substrate according to the present invention as described above and a probe fixed in a substrate substrate pool. needle. The chip of the present invention can detect a signal for detecting a reaction result by one of the following instruments: a confocal scanner, a CCD scanner, a visible light scanner, and the like.

 In the chip of the present invention, the substrate of the substrate and the marking system are one of the following combinations: The substrate is red, orange, yellow, green, blue, cyan or purple under white light, preferably black When the marking substance is a luminescent substance or a white, light red, light orange, light yellow, light green, light blue, light cyan, or light purple substance; the substrate is light red, light orange, light yellow, light green under white light When light blue, light cyan or light purple, preferably white, the marking substance is red, orange, yellow, green, blue, cyan or purple, and preferably, the black substance.

 In the chip of the present invention, the probe is fixed by being mixed with nano particles and fixed in the substrate, wherein the average particle diameter of the nano particles is 1 to 500 nm.

 In the chip of the present invention, the marking system contains a dye. The dye is a black, purple, green, blue or indigo dye including amino black, Coomassie brilliant blue, crystal violet, and the like.

 In yet another aspect, the present invention provides a chip signal detection instrument, in which at least the surface of the portion directly below the chip during scanning is an ultra-black surface with an absorbance greater than 95%, preferably greater than 98%. The ultra-black surface contains an ultra-black metal salt and / or an ultra-black metal oxide, and the like.

 In another aspect, the present invention provides a method for preparing a chip substrate, which includes introducing one or more of the following structures containing a luminescent substance and / or a dye and / or a coloring pigment and / or a matting agent on the substrate: Coatings, coatings, films and sheets.

In addition, the present invention also provides a method for preparing a chip, which comprises mixing a probe with a dye, a coloring pigment, and / or a coating material containing the coloring pigment, and then spotting the sample into the film base pool. The dyes are black or colored water-based dyes including amino black, Coomassie brilliant blue, crystal violet, etc., water and oil amphoteric dyes, or printing paint paste. The coloring pigment is selected from one or more of the following pigments: black pigments including carbon black, metal salts, etc., and white pigments including titanium dioxide And pigments, including yellow, red, blue, green, and metallic pigments. The paint is selected from black, white and various colored paints and / or inks.

 In the method for preparing a chip of the present invention, the coating contains a substance that can bind a probe. The probe-binding substance includes one or more of the following organic substances and derivatives thereof: nitrocellulose, polystyrene, polyacrylate, polysulfone, polyethersulfone, polyvinyl chloride, amino resin, polysaccharide , Polyamino acids, etc. Examples of the paint color paint include one or more of the following paints: pearl black, magic black, pearl white, pearl blue, matte black, scarlet, medium blue, and the like.

 -In another aspect, the present invention provides a chip in which at least one probe point in a reaction system contains the dye and / or the colored pigment and / or the coating Λ "that is not a probe.

 Finally, the present invention also provides a chip detection method, in which at least one substrate and / or at least one probe point of the reaction system of the used chip contains dyes and / or colored pigments and / or colored pigments that are not used as probes. The paint is selected from black, green, blue and indigo dyes including amino black, Coomassie brilliant blue, crystal violet; the coloring pigment is selected from one or more of the following pigments: including Light-emitting pigments including fluorescent substances, chemiluminescent pigments and electrochemical light-emitting pigments, black pigments including carbon black, metal salts, etc., white pigments including titanium dioxide, and yellow pigments, red pigments, A blue pigment, a green pigment, a metallic pigment, and the like; the paint is selected from a colored paint including black paint, white paint, red paint, yellow paint, green paint, blue paint, indigo paint, or purple paint.

 The advantages of the substrate according to the present invention are that it can impart a high detection sensitivity to the chip of the final product, or a high degree of freedom of selection, a low cost, etc., while it can have a sufficiently high detection sensitivity.

 The advantages of the chip of the present invention are high detection sensitivity, or a high degree of freedom of selection, low cost, etc. while a sufficiently high detection sensitivity can be achieved.

 The chip signal detection instrument of the invention has the advantage of high detection sensitivity.

 The advantages of the chip detection method of the present invention are high detection sensitivity, or a high degree of freedom of selection, low cost, etc., while a sufficiently high detection sensitivity can be achieved.

 The invention is now illustrated by the following non-limiting examples. Examples

The embodiments of the present invention give examples of implementation principles of the present invention, and cannot be understood that the present invention is limited to these embodiments. Embodiment of the present invention used in the slide dimensions 75 X 25 X 1.0 mm, the specific specifications shown in Table 1 below ₀

 *: For the method, please refer to the slide JIANG Zhonghua et al. “Immobilization Technology and Application of Biomolecules”, Chemical Industry Press, 1998).

 **: Method reference: Melnyk 0 et al., Peptide arrays for highly sensitive and special antibody-binding fluorescence arrays. Bioconjug. Chem. 13: 713-20, 2002. ***: The method refers to our other invention, "Composition and Application of Planar Matrix Coating Polymer", China Patent Application No. 03135618.4. In the embodiment of the present invention, the preparation of a plurality of substrate pool substrates, wherein the isolation structure of the substrate pool is coated on the above glass slide with a highly hydrophobic organic silicon coating (Chengdu Chenguang Chemical Design Institute), and dried to form a film (film thickness less than 0.05 mm) was formed on the substrate (refer to our other invention: "<a biochip with a minimum height of the reactor isolation structure and a preparation method", patent application number 03117397.7). Eight substrate cells are formed for each substrate. The size of each substrate cell is 4.5 mm X 4.5 mm, and the width of the isolation structure between the substrate cells is 4.5 mm.

The probes used in this example are HCV antigen (Institute of Liver Diseases, Beijing People's Hospital, China) and HIV _{1 + 2} antigen (Institute of Liver Diseases, Beijing People's Hospital, China). In a substrate pool, two antigens each have three points with a diameter of 200 μm, and the distance between them is 800 m, forming a 2 × 3 array. The probe density on the reactor substrate was greater than 96 points / cm ² .

In this example, sample 1 is HCV antibody positive serum, sample 2 is HIV _{1 + 2} antibody positive human serum, and sample 3 is a positive control (a mixture of HCV antibody and HIV _{1 + 2} antibody positive serum control ), Sample 4 is the negative control (HCV antibody and HIV _{1 + 2} antibody Are negative serum controls). All samples were pre-tested using the classic ELISA method under serum '20-fold dilution reaction conditions. Example 1

Preparation and application of background signal enhancement substrate and background signal enhancement chip

 1) Preparation of background signal enhancement substrate

 The background signal enhancement substrate of this embodiment includes a substrate having a luminescent background and a substrate having a reflective background. The reference substrate used in this embodiment is the amino slide in Table 1, and the prepared substrates are listed in Table 2. Background signal enhancement substrate

 Substrate background signal enhancement structure Substrate detection signal Noise ratio ***

 冃 negative

 0 * None Amino slide 18 20 0.1

1 Polystyrene containing luminescent substance base plus fluorescent substance 18267 -2699 7.76 mass olefinic base (base 1)

 2 Film containing luminescent substance Polyvinyl chloride film base 14149 -2870 4.9

 (Base 2)

 3 Can bind luminescent substance Protein A coated substrate

 Or (luminescent base 3) 28457 -9946 3.9 active group of substance

 4 Luminescent substance coating Rhodamine-Peptide coating 27561 -9717 3.8

5 Coating with luminescent substance Fluorescent substance on the front surface 15462 -9711 2.6 layer Coating base (base 5)

 6 (ibid.) Fluorescent substance on the back 13489 -8679 2.6 Coating base (base 6)

 7 Film with luminescent substance Hot hole with light emission on the front surface 15795 -7679 3.1 Film base (film base 7)

 8 '(same as above) Back side heat-emitting film 13756 -7684 2.8 substrate (film substrate 8) **

 9 Reflective film included Heat-reflective film on the back 12686 -6695 2.9 substrate (film substrate 9) **

 10 Luminescent substance-containing sheet Backside with fluorescent sheet base 16798 -6966 3.2

(Base 10) *: The control substrate is the amino slide in Table 1.

**: applied with target

 ***: noise signal ratio = [(background signal-negative signal) I negative signal] absolute value of the substrate 1 prepared in this embodiment, which is a polystyrene substrate with a fluorescent substance added thereto. The method for preparing a substrate includes adding a fluorescent pigment ZnS-Mn to a thermoplastic polystyrene used for preparing an enzyme-labeled plate, and then obtaining the size by molding, and the size is 75x25x1 mm. The method for preparing the multi-chip base is as described above.

 The film base 2 prepared in this embodiment is a polyvinyl chloride film-glass slide composite film base to which a fluorescent substance is added. The method for preparing the substrate includes thermally bonding a polyvinyl chloride film that reflects light at a wavelength of 532 nm to a glass slide described in Table 1. The method for preparing the multi-chip base is as described above.

 The base 3 prepared in this embodiment is a protein A-coated base. The preparation method includes coating protein A (Shanghai Biological Products Research Institute) on the amino slides described in Table 1 by using a general protein coating technology.

 The base 4 prepared in this example is a rhodamine-polypeptide coating substrate. The preparation method includes coating the formed rhodamine-polypeptide on a semicarbazide glass slide described in Table 1 by using a known rhodamine-polypeptide technology. The polypeptide used is Epstein-Barr virus, VCA antigen fragment.

 The film base 5 prepared in this embodiment is a film base with a fluorescent substance-containing coating on the front side, and the preparation method includes a rhodamine-white paint light-emitting film base on the front side. The thickness of the coating formed is around 30 μηι.

 The film base 6 prepared in this embodiment is a film base with a fluorescent substance-containing coating on the back surface, and the preparation method includes a rhodamine-white paint light-emitting film base on the back surface. The thickness of the coating formed is around 30 μm.

 The film base 7 prepared in this embodiment is a front hole light-emitting film base. The preparation method includes leaving holes in a place where a polyvinyl chloride film has a reaction cell, and bonding the top surface of the aldehyde-based glass slide by a thermal bonding process. . The light-emitting film is a polyvinyl chloride film.

 The film base 8 prepared in this embodiment is a light-emitting film base with a hole on the back. The preparation method includes leaving holes in the place where the polyvinyl chloride film has a reaction cell, and bonding the bottom surface of the PVP-coated glass through a thermal bonding process. . The light-emitting film is a polyvinyl chloride film.

In the film base 9 prepared in this embodiment, a preparation method includes combining a non-porous fluorescent film on the back of the amino dolphin slide with a thermal bonding process. Wherein, the slide glass is the amino slide glass described in Table 1. The substrate 10 prepared in this embodiment is a fluorescent substrate with holes on the back. The preparation method includes using an amino slide as described in Table 1 as the substrate, and spotting and coating by conventional methods. The perforated fluorescent sheet is a sheet of PVC compression molding containing a fluorescent substance, which is punched and cut into a sheet with a plane size of 75x25 mm (thickness of 80 μm, pore diameter of 200 μm, and the distribution of holes corresponds to spotting. Distribution of sample points).

 The prepared substrates were formed from the eight substrate pool substrates formed by the aforementioned multi-piece substrate formation method. The substrates 0 to 10 in Table 2 were prepared from the substrates 0 to 10. ·

The prepared substrate was determined to have an enhanced background signal as follows: A HCV antigen and HIV _{1 + 2} antigen mixture (2 mg / ml each) was spotted into a substrate pool on the substrate according to a known chip spotting method. The 4 points in the pool form a 2X2 square matrix with a diameter of 200 μm and a pitch of 800 m. Then take the No. 4 sample (serum control with negative HCV antibody and HIV _{1 + 2} antibody) as the target sample, rhodamine-labeled goat anti-human anti-antibody (Jackson ImmunoRresearch Laboratories, USA) as the label, and according to the known chip The detection method detected negative targets and substrate background signals (Table 2).

2) Preparation of background signal enhancement chip

The background signal enhancement chip of this embodiment is formed by fixing a probe in the substrate base pool of the above background signal enhancement substrate (chip 0-6 in Table 3), or after fixing the probe on an amino slide, the substrate is processed. Coating of capture agents or substances containing luminescent substances (chips 7 and 8 in Table 3). The method of fixing the probe is as follows: In each of the above-mentioned prepared substrate pools, add HCV antigen (1.5 mg / ml) and HIV1 + 2 antigen solution (1.5 mg / ml) to the nanoparticles (silica oxide nanoparticles (SP1) ), 15-25 legs, Zhejiang Zhoushan Mingri Nano Materials Co., Ltd.), and then spotted in the substrate pool to form a reactor. The coating method of the capture agent or the luminescent substance-containing substance is: The capture agent or the luminescent substance-containing substance at a concentration of about 0.1 mg / ml is performed according to a well-known chip blocking method. The prepared chips are listed in Table 3. Background signal enhancement chip

所制备芯片的背景信号增强结构的确定如下： 以人 HCV抗体阴性血 清和人 HIV抗体阴性血清的混合物 (1： 1) 为目标样品、 罗丹明标记的 羊抗人抗抗体 (美国 Jackson ImmunoRresearcli Laboratories公司) 为标记 物， 按公知的芯片检测方法检出阴性目标和片基背信号 (表 3) 背景信号增强检测方法

The background signal enhancement structure of the prepared chip was determined as follows: A mixture of human HCV antibody negative serum and human HIV antibody negative serum (1: 1) was used as a target sample, and rhodamine-labeled goat anti-human anti-antibody (Jackson ImmunoRresearcli Laboratories, USA) ) Is a marker, and a negative target and a substrate-back signal are detected according to a known chip detection method (Table 3). Background signal enhancement detection method

 The reference picture used is chip 0 in Table 3.

During the experiment, the aforementioned four samples were respectively added to the reactor of the chip described in Table 3. A 1: 500 dilution was added to each reactor. The sample volume was 15 μ1. After 30 minutes of reaction, washing was performed 5 times, and the washing solution was added in an amount of 25 μ1 each time. The amount of the labeled substance was 15 μ1, and the reaction solution was washed 5 times, and the washing solution was added each time at an amount of 25 μ1. After drying, scanning was performed. The scanner is a confocal laser scanner (ZoCSoftlmageBoost). The scanning light wavelength is 532 nm and the emission light wavelength is 570 nm. The laser intensity and gain are 35/50. The read signals are processed by processing software (ZoCSoftlmageBoost), and then averaged. The results are shown in Table 4. Table 4: Background signal enhancement chip detection results

表中阳性与阴性差的绝对值， 芯片 0仅为 2000左右， 而背景信号增 强芯片为 4000至 10000之间。 实施例 2

The absolute value of the difference between positive and negative in the table, chip 0 is only about 2000, while the background signal enhancement chip is between 4000 and 10,000. Example 2

 Preparation and application of substrate and chip containing target signal attenuation structure

 Preparation of target signal attenuation substrate

 The method for preparing a substrate in this embodiment is made by spotting a dye or coating with a spotter to multiple base cells, or forming a target signal weakening point on the back of a glass probe to which a probe is to be fixed. Obviously, a target signal weakening point can also be formed by spotting a dye or coating with a spotter to or unactivated the point where the probe is to be fixed on the slide or the back of the point where the probe is to be fixed on the slide. Then, according to the aforementioned method for forming a multi-chip base cell, a multi-chip base cell substrate is prepared.

 The substrates prepared in this example are listed in Table 5.

 The preparation of the substrate bl is as follows: Using a spotter, the crystal violet (company) solution with a concentration of 2 mg / ml is distributed according to the probe point to the 8-piece base cell glass prepared according to the aforementioned multi-chip base cell preparation method On a chip (2 X 3 array, spot diameter 200 μm), react at room temperature for 1 hour, then wash and dry.

The preparation of the substrate b2 is as follows: Using a spotter, a Coomassie brilliant blue (company) solution with a concentration of 2 mg / ml is spotted onto a pseudo-point probe on 8 base-cell amino slides prepared according to the aforementioned multi-chip base-cell preparation method The back of the spot (2 X 3 array, dot diameter 200 m) was washed at room temperature for 1 hour and then dried. The substrate b3 was prepared as follows: Using a spotter, pearl black paint (Shanghai Qifu Industrial Development Co., Ltd.) was distributed according to the probe point distribution points to the 8-piece base-loaded glass prepared by the above-mentioned multi-chip base-cell preparation methods. On-chip (2 × 3 array, spot diameter 200 μm), and then dried.

 The substrate b4 was prepared as follows: Using a spotter, pearl black paint (Shanghai Qifu Industrial Development Co., Ltd.) was spotted onto the quasi-point probe points on the 8 base cell amino slides prepared according to the aforementioned multi-chip base cell preparation method. Back side (2 X 3 array, spot diameter 200 μm), and then dried. Table 5: Target signal attenuation substrate

Noise signal ratio = [(background signal-negative signal) I negative signal] absolute value of the target signal of the substrate prepared in this example is determined as follows: HCV antigen and HIV _{1 + 2} antigen are determined according to the known chip spotting method The mixture (2 mg / ml each) was spotted into the substrate pool on the substrate, and 4 spots were formed in each substrate pool to form a 2X 2 square matrix with a diameter of 200 um and a pitch of 800 μm. Then take the No. 4 sample (serum control negative for both HCV antibody and HIV _{1 + 2} antibody) as the target sample, rhodamine-labeled goat anti-human anti-antibody (Jackson ImmunoRresearch Laboratories, USA) as the label, and press the well-known chip The detection method detected negative targets and substrate background signals (Table 5). Preparation of target signal attenuation chip

 In this embodiment, chip 0 is a comparative chip using 8 base pool amino slides as the base.

The chips bl, b2, b3, and b4 in this embodiment are prepared according to a known spotting method. HCV antigen (1.5 mg / ml) and HIV _{1 + 2} antigen (1.5 mg / ml) were spotted into the substrate pools of the corresponding substrates (substrates bl, b2, b3, b4) in Table 5, and HCV The antigen and HIV _{1 + 2} antigen dots were made by overlapping the target signal weakening dots (2 X 3 array, dot diameter 200 μm) (Table 6). Table 6: Target signal attenuation chip

本实施例制备的芯片其目标信号减弱结构的确定如下： 以人 HCV抗 体阴性血清和人 HIV₁₊₂抗体阴性血清的混合物 ( 1： 1 )为目标样品、 罗 丹明标记的羊抗人抗抗体 (美国 Jackson ImmunoRresearch Laboratories 公司） 为标记物， 按公知的芯片检测方法检出阴性目标和片基背景信号 (表 6) 。 目标信号减弱检测方法

The target signal attenuation structure of the chip prepared in this embodiment is determined as follows: A mixture of human HCV antibody negative serum and human HIV _{1 + 2} antibody negative serum (1: 1) is used as a target sample, and rhodamine-labeled goat anti-human anti-antibody (Jackson ImmunoRresearch Laboratories, USA) As a marker, a negative target and a substrate-based background signal were detected according to a known chip detection method (Table 6). Target signal attenuation detection method

During the experiment, the same four samples as in Example 1 were added to the reactor of the chip. Each of the 4 reactors of each biochip was filled with a 1: 500 diluted sample. The sample volume was 15 μ1. After 30 minutes of reaction, the product was washed five times, and the washing solution was added in an amount of 25 μ1 each time. The amount of the labeled substance was 15 ul, washed 5 times after the reaction, and the washing solution was added 25 μΐ each time. After drying, scanning was performed. The scanner is a confocal laser scanner (Afymetrix's GMS 418 chip scanner). The scanning light wavelength is 532 nm and the emission light wavelength is 570 nm. The read signal is processed by the processing software (ZoCSoft ImageBoost), and the average value is obtained. The results are shown in Table 7. Table 7: Target signal attenuation chip detection results

实施例 3

Example 3

 Preparation of chip containing substrate background signal enhancement structure and target signal attenuation structure

 Preparation of background signal enhancement / target signal attenuation substrate

 The background signal enhancement / target signal attenuation substrate prepared in this example is shown in Table 8. The method for preparing the substrate cl-c5 includes the background signal enhancement and the target signal attenuation: (1) The method for background signal enhancement is the same as the method for preparing the background signal enhancement substrate in Example 1 (the substrates cl, c2, c3, c4, c5 corresponds to the substrates 3, 4, 5, 6, 9 respectively; (2) The method for weakening the target signal is the same as the method for preparing the target signal attenuating substrate in Example 2. The pearl black paint (Shanghai Qifu) Industrial Development Co., Ltd.) Press the distribution points of the probe points to the front of the substrate base and dry. Clothing 0: Header Jj 'ί Signal enhancement / Target signal weakening substrate Substrate Background signal enhancement Target signal weakening Detection signal Noise to signal ratio Negative

 Substrate cl protein A coated film with target signal on the front side 28496 -9812 3.9 weakening point

 Substrate c2 front rhodamine-multi front with target signal 26557 -9655 3.8 peptide coating weakening point

 Substrate c3 contains fluorescent substance on the front side and contains target letter on the front side 12783 -7599 2.7 Base point of coated film

 Substrate c4 contains fluorescent substance on the back 11456 -6973 on the front 2.6 The weakening point of the base of the coated film

Substrate c5 has a reflective film on the front with target letter 10591 -7613 2.4 weakening point on the front The determination of the noise-to-signal ratio of the substrate prepared in this embodiment is as follows: A HCV antigen and an HIV antigen mixture (2 mg / ml each) are spotted into a substrate pool on the substrate according to a known chip spotting method. There are 4 points in the pool to form a 2 × 2 square matrix with a diameter of 200 μ η and a pitch of 800 μ π. Then take the No. 4 sample (serum control negative for both HCV antibody and HIV _{1 + 2} antibody) as the target sample, rhodamine-labeled goat anti-human anti-antibody (Jackson ImnmnoRresearcli Laboratories, USA) as the label, and according to the known chip The detection method detected negative targets and substrate background signals (Table 8).

 It can be seen that the difference between the background signal enhancement / target signal attenuation substrate background and the negative signal is greater than the difference between the background signal enhancement substrate or the target signal attenuation substrate background and the negative signal. Background signal enhancement / target signal attenuation chip

 The background signal enhancement / target signal attenuation chips prepared in this example are listed in Table 9.

Chips cl, c2, c3, c4, and c5 were prepared by spotting the HCV antigen (1.5 mg / ml) and HIV _{1 + 2} antigen (2 mg / ml) according to the well-known spotting method. In the film base pool (chips cl, c2, c3, c4, and c5), and the HCV antigen and HIV _{1 + 2} antigen spots are coincident with the target signal attenuation structure (2X 3 array, dot diameter 200 μm) and Made (Table 9). Background signal enhancement / target signal attenuation chip

本实施例制备的芯片， 其背景信号增强 /目标信号减弱的确定如上述背 景信号增强 /目标信号减弱片基。 背景信号增强 /目标信号减弱检测方法

For the chip prepared in this embodiment, the determination of the background signal enhancement / target signal attenuation is as described above. Scene signal increase / target signal decrease. Background signal enhancement / target signal attenuation detection method

 During the experiment, the same four kinds of samples as in Example 1 were respectively added to the reactor of the chip. Each of the 4 reactors of each biochip was filled with a 1:50 diluted sample. The sample volume was 15 μ1. After 30 minutes of reaction, it was washed 5 times, and the washing solution was added in an amount of 25 μ1 each time. The labeling amount was 15 μ1, and the reaction solution was washed twice. The washing solution was added 15 μ1 each time. After drying, scanning was performed. The scanner is a confocal laser scanner (Afymetrix's GMS 418 chip scanner). The scanning light wavelength is 532 nm and the emission light wavelength is 570 nm. The read signal is processed by the processing software (ZoCSoft lmageBoost), and the average value is obtained. The results are shown in Table 10. Table 10: Background signal enhancement / target signal attenuation chip detection results

实施例 4

Example 4

 Preparation of colored film base and colored chip

 Preparation of colored tablets

 The colored background substrates prepared in this example are listed in Table 11. First, a base is prepared, and the obtained base is then an 8-base base substrate formed by the aforementioned multi-base base formation method.

 The film base dl prepared in this embodiment is a black plastic film base, and the colorant is channel black, which is a thermoplastic polystyrene used for the preparation of microplates. After adding a filler such as channel black, it is molded. Made by molding, with dimensions of 75x25x1 mm.

Film bases d2, d3, and d4 are front-side colored coating film bases, and the coatings are pearl black spray paint (Shanghai Qifu Industrial Development Co., Ltd.), matte black (Shanghai Qifu Industrial Development Co., Ltd.) and In Chuanyang automatic white spray paint (Chengdu Hongguang Coating Factory), the colorants in the paint are the following pigments: trough carbon black, titanium oxide, pearl black and pearl green paints (pearlescent substances) are added as matting agents. The coatings are sprayed on the top surface of the glass slide and dried to form a colored coating (thickness of about 30 μm). Their roughness Ra is between 0.4 and 0.5 μm (as measured by Chengdu Metrological Supervision and Verification Test).

 The film base d5 is a back-colored coating film base, and the paint is pearl black (Shanghai Qifu Industrial Development Co., Ltd.). The coating is sprayed on the back of the semi-urea-based glass and dried to form a colored coating (thickness around 30 μm). Obviously, combined with the preparation of the substrates d2 and d5, a double-sided colored coating substrate can also be prepared.

 Film bases d6 and d7 are front-side colored film bases. The coloring films are black and white polystyrene films (about 45 m thick, self-made), and the coloring agents of the coloring films are channel black and titanium dioxide. The colored film was thermally bonded to the top surface of the glass slide.

 The film base d8 is a backing color film film base. The coloring film is a black polystyrene film (about 45 μm thick, self-made), and the coloring agent of the coloring film is channel black. The colored film was thermally bonded to the back of the semi-urea-based glass. Table 11: Colored background film base

 *: Reflectivity ( )

标记物的制备 The values of surface roughness and absorbance / reflectance in the table are multiple inspections on the substrate.

Preparation of markers

Rhodamine-labeled goat anti-human secondary antibody was purchased from Jackson ImmunoRresearch, USA Laboratories.

 Dye-labeled anti-antibodies, of which the anti-antibodies are goat anti-human secondary antibodies (Beijing Institute of Biological Products), the dyes are amino black (Chengdu Kelong Chemical Reagent Factory), Coomassie Blue (Shanghai Boao Biotechnology Co., Ltd.) and crystals Purple (Chengdu Kelong Chemical Reagent Factory). The dye was dissolved in PBS buffer to a concentration of 2 mg / ml, and then mixed with an equal volume of sheep anti-human secondary antibody at a concentration of 2 mg / ml, and reacted at room temperature for 1 hour. The free dye was then removed by purification to obtain a purified dye-labeled anti-antibody. . Optimize the preparation of contrast color chips

 The optimized contrast color chips (Table 12) in this embodiment are all 8-reaction cell chips. It is formed by fixing the aforementioned probe in a substrate pool by a known method. Table 12: Optimized Contrast Combination Chip

优化反差组合芯片应用

Optimized Contrast Combination Chip Application

 1) Optimized contrast combination detection using a black background chip

 The reference photo base used was a transparent amino-modified glass slide with a size of 75x25x 1 mm (Telece International, USA, hereinafter referred to as the base 0).

For chips 0, dl, d2, d3, d5, d6, and d8, the experiment is the same as in Example 1 Each sample was added to the reactor of the chip. Each of the 4 reactors of each biochip was filled with a 1: 500 diluted sample. The sample volume was 15 μ1. After 30 minutes of reaction, the product was washed five times, and the washing solution was added in an amount of 25 μ1 each time. The labeling amount was 15 μ1, and the reaction solution was washed 5 times. The washing solution was added each time at 15 μ1. After drying, scanning was performed. The scanner is a confocal laser scanner (Afymetrix's GMS 418 chip scanner). The scanning light wavelength is 532 nm and the emission light wavelength is 570 nm. The read signal is processed by the processing software (ZoCSoft ImageBoost), and the average value is obtained. Negative or positive results (Table 13).

 For chips d4, d7, d9, and dl0, the four samples same as those in Example 1 were added to the reactor of the chip respectively. Each of the 4 reactors of each biochip was charged with a 1:20 dilution. The sample volume was 15 μ1. After 30 minutes of reaction, it was washed 5 times. The washing solution was added in an amount of 25 μ1 each time. The labeling amount was 15 μ1, and the reaction solution was washed 5 times. The washing solution was added each time at 15 μ1, and the scanning was performed after drying. The scanner is an EPSON 1260 scanner (EPSON). The scanning light is white and the signal light is white. The read signal is processed by ZoCSoft ImageBoost, and then the average value is obtained to obtain a negative or positive reaction result (Table 13). Table 13: Test results of optimized contrast combi chip

Note: "+" in the table is a positive result, and "-" is a negative result. Example 5

 Method for preparing colored probe point chip and chip

 Chip preparation method

 After mixing the probe with the pigment or coating, the mixture is spotted on the substrate of Table 1, Table 2, Table 5, Table 8 or Table 11 by using a manual spotter to make a colored probe point chip. (Table 14).

The paint used in this embodiment is the same pearl black and pearl black as in Example 4, and the probes are the aforementioned HCV antigen and HIV _{1 + 2} antigen (final concentrations are 1.5 mg / ml each).

 Table 14 lists the chips prepared in this example. Table 14

检测方法

Detection method

During the experiment, the same four samples as in Example 1 were added to the reactor of the chip. In each of the four reactors of the biochip, a 1:50 diluted sample was added. The sample volume was 15 μ1. After 30 minutes of reaction, the product was washed five times, and the washing solution was added in an amount of 25 μ1 each time. The amount of the labeled substance was 15 μΐ. After the reaction, washing was performed 5 times. The washing solution was added each time at an amount of 15 μ 1, and the scanning was performed after drying. The scanner is a laser confocal scanner (Afymelxix GMS 418 chip scanner), which scans the excitation light wavelength of 532 nm and the emission light wavelength of 570 nm. The read signal is processed by the processing software (ZoCSoft lmageBoost), and then averaged to obtain The results are shown in Table 15. Table 15: Chip test results

 +: Positive result, one: negative result.

Claims

Claim 1. A chip substrate comprising a substrate and an optional isolation structure, the substrate comprising a substrate and optionally one or more of the following composite structures: a coating, a coating, a film and a sheet, The matrix and / or at least one of the composite structures contains a colorant and / or a capture agent that directly or indirectly captures the colorant.  2. The chip substrate according to claim 1, wherein the colorant and / or capture agent is formed or involved in forming a colored background and / or a signal attenuation target at least under signal detection conditions when the substrate is applied, and The colored background includes one or more of the following backgrounds: a luminous background that emits signal light, a reflective background with a signal light reflectance greater than 50%, a non-colored background, a colored background, and a colored background, where the signal is weakened and the target is detected The signal is attenuated by the presence of the optical signal-related substance.  3. The chip substrate according to claim 2, wherein the signal attenuation target and / or colored background, preferably a luminescent background and / or a reflective background participate in forming a high noise signal ratio, and the high noise signal ratio refers to a background The signal is higher than the negative target, blank target or extreme weak positive target, preferably at least 50% higher, more preferably at least 300% higher.  4. The chip substrate according to claim 2, wherein the colored background, preferably an achromatic color background and / or a colored background participates in forming a high color difference ratio, and the high color difference ratio refers to a contrast between the background and the target. The absolute value of the difference between the absorptance or reflectance of the signal light at all or a part of the wavelength is not less than 50%, preferably not less than 70%.  The chip substrate according to any one of claims 1 to 4, wherein the substrate is selected from the group consisting of modified or unmodified glass, plastic, and metal.  6. The chip substrate according to claim 1, wherein the colorant comprises one or more of the following: a light-emitting substance, a dye, a coloring pigment, and a matting agent.  7. The chip substrate according to claim 6, wherein: the light-emitting substance is selected from one or more of the following: including rhodamine, CY3, CY5, Alexa, seaweed protein, and rare earth compounds Fluorescent substances, chemiluminescent substances and electroluminescent substances.  8. The chip substrate according to any one of claims 1 to 7, wherein the dyes are black, purple, green, blue, indigo water-based dyes including amino black, Coomassie brilliant blue, crystal violet, Water and oil amphoteric dye or printing paint color paste. 9. The chip substrate according to any one of claims 1 to 8, wherein: The coloring pigment is selected from one or more of the following: black pigments including carbon black, metal salts, white pigments including titanium dioxide, and yellow pigments, red pigments, blue pigments, green pigments, metal pigments Color paints inside.  10. The chip substrate according to claim 1, wherein a roughness Ra of the surface of the substrate is between 0.02 and 5.0 μm, preferably between 0.25 and 5.0 μm.  11. The chip substrate according to any one of claims 1 to 10, wherein a coating material of the coating layer is selected from black, white, and various colored paints and / or inks.  12. The chip substrate according to any one of claims 1 to 11, wherein the substrate containing the colorant in the substrate and / or the composite structure contains a substance that can bind a probe.  13. The chip substrate according to claim 12, wherein the probe-binding substance includes one or more of the following organic substances and derivatives thereof: nitrocellulose, polystyrene, polyvinyl chloride, amino resin , Polysaccharide, polyamino acid, polyacrylate, polysulfone, polyethersulfone.  14. The chip substrate according to any one of claims 11 to 13, wherein the colored paint comprises one or more of the following paints: pearl black, magic black, pearl white, pearl blue, matte black, bayonet, Scarlet and medium blue.  15. The chip substrate according to any one of claims 1 to 14, wherein the capture agent comprises one or more of the following ligands: protein A, protein G, biotin, avidin, antigen, antibody, Antibodies, peptides, DNA  16. A chip comprising a reaction system and optionally a labeling system, said reaction system comprising a substrate according to any one of claims 1 to 15 and a probe fixed in a substrate substrate pool.  17. The chip according to claim 16, wherein the substrate of the substrate and the marking system are one of the following combinations: the substrate is red, orange, yellow, green, blue, cyan or When purple, preferably black, the marking substance is luminescent substance or white, light red, light orange, light yellow, light green, light blue, light cyan, or light purple substance; the base is light red, light orange, When light yellow, light green, light blue, light cyan, or light purple, preferably white, the marking substance is red, orange, yellow, green, blue, cyan, or purple, and preferably a black substance. 18. The chip according to claim 16 or 17, wherein the probe is immobilized in the base pool after being mixed with nanoparticles, and the average particle diameter of the nanoparticles is 1 to 500. 19. The chip according to any one of claims 16 to 18, wherein the marking system contains a dye.  20. The chip according to claim 19, wherein the dye is a black, purple, green, blue or indigo dye including amino black, Coomassie brilliant blue, crystal violet.  21. A method for preparing a chip substrate according to any one of claims 1 to 15, comprising introducing on the substrate a luminescent substance and / or a dye and / or a coloring pigment and / or a matting agent as described below. One or more structures: coating, coating, film and sheet.  22. A method for preparing a chip, comprising mixing a probe with a dye, a coloring pigment, and / or a coloring pigment-containing coating, and then spotting it into a film base pool.  23. The preparation method according to claim 22, wherein the coloring pigment is selected from one or more of the following: a black pigment including carbon black, a metal salt, a white pigment including titanium dioxide, and Color pigments including yellow, red, blue, green, and metallic pigments.  24. The preparation method according to claim 22 or 23, wherein the dye is a black or colored water-based dye including amino black, Coomassie brilliant blue, crystal violet, water and oil amphoteric dye or printing paint paste.  25. The preparation method according to any one of claims 22 to 24, wherein the paint is selected from black, white, various colored paints and / or inks.  26. The preparation method according to claim 25, wherein the coating contains a substance capable of binding a probe.  27. The preparation method according to claim 26, wherein the probe-binding substance comprises one or more of the following organic substances and derivatives thereof: nitrocellulose, polystyrene, polyacrylate, polysulfone, Polyethersulfone, polyvinyl chloride, amino resin, polysaccharide, polyamino acid.  28. A chip prepared according to the manufacturing method of any one of claims 22-27. 29. The chip according to claim 28, wherein at least one of the probe points contains a dye and / or a colored pigment and / or a paint that is not used as a probe. 30. A chip detection method, in which at least one substrate and / or at least one probe point of a reaction system of a chip used contains dyes and / or colored pigments and / or paints containing colored pigments that are not used as probes, The dye is selected from black, green, blue, and indigo dyes including amino black, Coomassie brilliant blue, and crystal violet; the coloring pigment is selected from one or more of the following pigments: including fluorescent substances, chemical Luminescence including luminescent pigments and electrochemical luminescent pigments Pigments, black pigments including carbon black, metal salts, white pigments including titanium dioxide, and color pigments including yellow pigments, red pigments, blue pigments, green pigments, and metal pigments; the coating is selected from Paints including black, white, red, yellow, green, blue, indigo or purple.