WO2005059553A1 - The biochip assay and its relative equipment - Google Patents

The biochip assay and its relative equipment Download PDF

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Publication number
WO2005059553A1
WO2005059553A1 PCT/CN2004/000713 CN2004000713W WO2005059553A1 WO 2005059553 A1 WO2005059553 A1 WO 2005059553A1 CN 2004000713 W CN2004000713 W CN 2004000713W WO 2005059553 A1 WO2005059553 A1 WO 2005059553A1
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WO
WIPO (PCT)
Prior art keywords
chip
colorant
probe
substrate
coloring
Prior art date
Application number
PCT/CN2004/000713
Other languages
French (fr)
Chinese (zh)
Inventor
Fanglin Zou
Chunsheng Chen
Jianxia Wang
Original Assignee
Chengdu Kuachang Medical Industrial Limited
Chengdu Kuachang Science & Technology Co., Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/CN2003/001091 external-priority patent/WO2004081570A1/en
Application filed by Chengdu Kuachang Medical Industrial Limited, Chengdu Kuachang Science & Technology Co., Ltd filed Critical Chengdu Kuachang Medical Industrial Limited
Publication of WO2005059553A1 publication Critical patent/WO2005059553A1/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic

Definitions

  • the invention relates to the field of detection chips, in particular to a method and a related device for performing qualitative and / or quantitative analysis of a target substance in a sample, especially a biological sample, by using a detection chip.
  • Relevant devices related to the present invention include a chip substrate, a chip substrate, a chip probe board, and a chip kit. Background technique
  • the term “detection chip”, also referred to as “chip” in the present invention includes, but is not limited to, a biochip (such as “Biochip”, “Microarray”, and “Bioarray” in English), which is one of designated and / or quantitative analysis.
  • a biochip such as "Biochip”, “Microarray”, and “Bioarray” in English
  • the chip of the present invention is a flat wafer chip, and the distribution density of the probes in the probe region on the wafer in the reactor is greater than 10 points / cm 2 , preferably more than 20 points / cm 2 , and more preferably more than 40 points. / cm 2 .
  • Chips include biochips and non-biochips.
  • 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 and a microarray chip, but it is well known that it does not include the existing rapid detection reagent strip.
  • the chip of the present invention contains one or more reactors, the reactors include a reaction surface and other structures optionally present, and the reaction surface includes a base surface and a probe point.
  • 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 has a wide range of applications, including gene expression detection, gene screening, drug screening, disease diagnosis and treatment, environmental monitoring and governance, forensic identification and other fields.
  • the existing chip detection includes two types of light detection and non-light detection.
  • non-luminous detection 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 the MetalChip-based ProteinChip Array System.
  • Luminescence detection mainly includes fluorescence detection, chemiluminescence detection and composite light irradiation detection.
  • the detection methods currently used are detection methods based on maximizing the signal-to-background ratio.
  • the substrate used in the fluorescence detection method is basically a transparent glass slide (for example, aminated, aldehyde-based, polylysine film substrate), and the substrate used in the chemiluminescence detection method is basically a transparent glass slide or a plastic plate. Or a metal film (such as a silver film), a polymer film (such as a PVDF film, a nylon film, a nitrocellulose film, an acetate fiber film, etc.) that is basically diffusible in the substrate used in the composite light irradiation detection method.
  • a transparent glass slide for example, aminated, aldehyde-based, polylysine film substrate
  • the substrate used in the chemiluminescence detection method is basically a transparent glass slide or a plastic plate.
  • a metal film such as a silver film
  • a polymer film such as a PVDF film, a nylon film, a nitrocellulose film, an acetate fiber film, etc.
  • 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 there are still some shortcomings such as the low background noise of the slide substrate, the high cost of the activated substrate, and the expensive detection equipment, which affect the large-scale application of the biochip method.
  • the existing chip detection methods mainly work on detection instruments and substrate surface chemistry, and there are still problems in the following areas: 1) The focus is completely on minimizing the background signal / maximizing the signal-to-noise ratio, greatly The degree of freedom of film substrate selection is reduced, as a result, the types of film substrates are very limited; 2) Even if the background signal is minimized / the signal-to-noise ratio is maximized, it is focused on the development of colorless, especially transparent film substrates , Further reducing the degree of freedom of the selection of the substrate, as a result, the type of the substrate of the invention is very limited; and 3) the degree of freedom is too small, so that the improvement of the detection sensitivity is limited, or the reduction of the substrate cost is limited. Chips and detection instruments have similar problems. Therefore, the invention of a detection method with higher detection sensitivity, and a substrate, chip and chip kit with lower cost or / and higher detection sensitivity are urgently needed in chip development. Problem to be Solved
  • a chip detection method for a sample which includes a chip kit preparation process and a sample detection signal formation process, and the method is characterized by including coloring the chip to make the background and The step of maximizing the color difference between the targets, the coloring includes adding a colorant or a colorant containing the colorant, and is aimed at the probe point in the chip reactor or / and the area surrounding the probe point, but does not include Labeling of probes and / or probe captures.
  • the present invention provides a chip detection method for samples, especially biological samples, which includes a chip kit preparation process and a sample detection signal formation process.
  • the method is characterized in that the detection signal is read in the following Under the conditions: the chip weak target signal-background ratio is less than 0.80, preferably less than 0.50, and more preferably less than 0.25.
  • a flat chip substrate which is characterized by containing a colorant or a colorant containing a colorant.
  • a chip substrate which includes a colored planar substrate and an optional isolation structure, wherein the colored planar substrate contains a colorant or a colorant containing a colorant.
  • a chip probe card which includes a flat substrate, a probe point fixed on the substrate, and an optional isolation structure.
  • the chip probe card is characterized by : A. the flat sheet base contains a colorant or a colorant containing a colorant; or / and B. the probe dot contains a colorant or a colorant containing a colorant, but does not contain a labeling substance.
  • a chip kit which includes a chip probe card and an optional labeling system.
  • the chip kit is characterized by: A. a planar sheet in the chip probe card
  • the base contains a colorant or a colorant-containing colorant; or / and B.
  • the probe points in the chip probe card contain a colorant or a colorant-containing colorant, but do not contain a labeling substance; or / and C. It also contains a coloring system containing a colorant or a colorant containing a colorant.
  • chip detection method in the present invention refers to a method for detecting by a chip, including preparation of a chip kit, formation of a sample pick-up signal, and a method of reading and analyzing a detection signal.
  • chip detection is considered as the whole process from chip preparation to reading and analysis of detection signals. In reality, this process can be broken down into several processes and can be performed in the same or more operating units.
  • the preparation of a chip kit includes: preparing a chip substrate at a chip substrate manufacturer, preparing a chip substrate at a chip substrate manufacturer, spotting a chip and a chip kit at a chip manufacturer, and the like;
  • the formation of the sample detection signal includes sample processing, labeling, background enhancement in the present invention, and the like.
  • the chip of the present invention can detect signals by one of the following instruments: a confocal scanner, a CCD scanner, a visible light scanner, and the like.
  • detection device is a device used in a specified and / or quantitative analysis method, and includes a chip, a signal detection device, and the like.
  • 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 in the open multi-reactor biochip and the related isolation structure and inlet and outlet liquid structure Wait.
  • chip flat substrate in the present invention, referred to as a substrate, refers to a planar solid-phase carrier used to fix probes and other auxiliary agents (if any) in a chip. Its surface chemical and optical properties affect the chip. Important factors in performance and cost.
  • the current substrate is selected from modified or unmodified glass, plastic, and metal.
  • an activated glass containing one or more of the following derivatized groups: amino, epoxy, aldehyde, acyl Hydrazine (-CO-NHN3 ⁇ 4), semicarbazide (3 ⁇ 4N-NH-CONH-), diethylaminoethyl (DEAE), diethylmono (2-hydroxypropyl) aminoethyl (QAE), carboxymethyl (CM), sulfopropyl (SP), mercaptoethylpyridine (MEP), siloxane group, thiol group.
  • chip substrate in the present invention, referred to as a substrate for short, refers to a product based on a substrate and optionally combined with other structures (such as an isolation structure) for forming a chip probe board after the probe is fixed.
  • the substrate is a substrate, such as a commercially available amino glass slide.
  • the multi-chip base substrate has an isolation structure.
  • the substrate includes a base and an isolation structure.
  • the chip pool is formed after the probe is fixed In the reactor, a plurality of substrates form a multi-reactor chip.
  • film base pool in the present invention refers to a structure formed by a film base and its isolation structure.
  • chip probe board in the present invention refers to an article formed by fixing a probe to a substrate of a chip substrate.
  • 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.
  • chip kit in the present invention refers to a kit containing a chip probe card, which includes a chip probe card and an optional labeling system, and an optional coloring system of the present invention.
  • chip kit coloring system in the present invention, referred to as a coloring system for short, refers to a system for coloring the chip background or / and probe points during the formation of a sample detection signal.
  • background signal enhancement in the present invention means that the background detection signal value is increased, for example, the color value of the background detection signal is increased by introducing a coloring substance into the substrate, the substrate surface, or / and the substrate back surface.
  • reduced weak target signal in the present invention means that the weak target detection signal is decreased.
  • coating in the present invention refers to a substance that is immobilized on a solid support in a molecular form, for example, a coating point, a coating surface, and the like that fix a dye molecule on a glass slide by ion adsorption, affinity adsorption, and the like.
  • coating in the present invention means that the thickness of the solid phase material formed by coating is less than
  • film in the present invention refers to a non-porous or perforated planar material having a thickness of less than 0.3 mm.
  • sheet in the present invention refers to a non-porous or perforated planar material having a thickness of 0.3 mm or more.
  • labeling substance in the present invention refers to a substance used to label a probe or a probe capture substance so as to obtain a positive result, for example, a fluorescent substance contained in a commonly used label in chip fluorescence detection.
  • 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.
  • coloring in the present invention is equivalent to English coloration, which means that the background of the chip (such as the substrate) or / and the probe point (but not including the probe capture object) has a defined light absorption and reflection, which is different from the mark. , Refraction, and other properties to form an optimized hue or / and brightness or / and saturation in visible light or under a selected wavelength of light, the chip is limited to For chips for detecting samples, especially biological samples, the coloring on the probe points does not use a labeling substance.
  • the coloring is completely different from the markers currently used in chip detection, which aims to obtain a positive result by using a labeling substance at the probe point as a means to increase the detection signal of the probe capture (For example, in the US patent application Al'20030032040 and the Chinese patent application CN A 1443854, the probe or the probe capture is marked to verify the effect of the probe fixing on the chip).
  • the purpose of coloring the probe points in the present invention is not to mark but to enlarge the difference between the negative target and the positive target, and the colorant used is not a labeling substance used for detection.
  • the purpose of coloring the chip background according to the present invention is not to mark but to enlarge the difference between the negative target and the positive target.
  • the colorant used is generally not a labeling substance (such as a luminescent agent) for detection, and when the colorant is the same as the labeling substance, it is only enhanced by the signal used as the background of the chip.
  • colored film base in the present invention refers to a film base that is colored.
  • colored substrate in the present invention refers to a substrate containing a colored substrate.
  • colored chip probe board in the present invention refers to a colored chip probe board, and includes a chip probe board containing a colored substrate or / and colored probe points.
  • colorant in the present invention refers to a colored substance that can color a preparation.
  • An example of a colorant is a pigment.
  • colored matter in the present invention means a substance containing a colorant that can color the preparation. Examples of coloring matter are: pigment-containing coatings, coatings, films, flakes, coatings, and complexes of pigments and substances with adsorption properties (such as rhodamine albumin).
  • colored pigment in the present invention refers to a substance that has no affinity for coloring objects and is mainly colored by combining other film-forming materials such as resins and adhesives with coloring objects, such as carbon black, mica titanium pearlescent colored pigments, and azo colored pigments. , Phthalocyanine colored pigments, fluorescent colored pigments (fluorescent substances).
  • the colored pigments in the present invention do not include extender pigments according to the custom of the coatings 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.
  • die in the present invention refers to a substance that has an affinity for a colored object and can color the colored object.
  • coating material refers to a material having a thickness of less than 1 mm, which can obtain a specific function after being applied to a substrate.
  • coloring coating material refers to a coating material containing colored pigments, such as various colored paints and Various inks.
  • target in the present invention refers to the entirety of all substances related to the detected signal at the probe point in the chip reactor when the signal is detected, for example, including the probe, the target captured by the probe (false If there is), a label (if any), in addition to the probe, a probe carrier (such as a nanocarrier) and a dye, a colored pigment, a colorant, and the like of the present invention may be provided on the probe point.
  • a probe carrier such as a nanocarrier
  • dye, a colored pigment, a colorant, and the like of the present invention may be provided on the probe point.
  • weak target in the present invention refers to the entirety of all substances related to the detected signal after the corresponding negative sample or blank control or extreme weak positive sample is added to the probe point in the chip reactor when the signal is detected; “Target samples” refers to these negative or blank controls or extreme weak positive samples.
  • background in the present invention refers to the entirety of all substances related to the detected signal in the detection area surrounding the target in the chip reactor when the signal is detected, for example, except for including the substrate, the sealed object fixed on the substrate, In addition to markers, etc., it may also include a base backing (especially in the case of a transparent base).
  • the backing of the substrate can exist on the back of the chip in one or more of the following forms when the chip is read signals: attached to the transparent substrate, as a separate component, attached to the chip holder of the signal reader.
  • the term "luminescent agent" in the present invention refers to a substance capable of emitting a detection signal.
  • the luminescent agent is selected from a luminescent pigment including a fluorescent substance, a chemiluminescent substance, and an electroluminescent substance.
  • the main purpose of the present invention is to provide a chip detection method with higher sensitivity, or / and to provide more alternative substrates / chips / chip kits. In fact, one of the prerequisites for providing more substrates / chips / chip kits is to make them have high enough detection sensitivity.
  • the method of the invention aims to maximize the signal contrast between the target and the background. In fact, 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 indicate the detectability of the thermal imager: C2 Eo-Eb.
  • Eo is the target specific emissivity
  • Eb is the background specific radiance
  • Eo and Eb are directly proportional to the absorption of the target material and the background material, respectively.
  • 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
  • the technology of the present invention is dedicated to maximizing the contrast between the target and the background. Therefore, the research of the present invention has selected "obviousness" as the technical basis.
  • the technology of the present invention can be referred to as the manifestation technology
  • the detection method of the present invention can be referred to as the manifestation technology detection method.
  • the first aspect of the present invention is achieved by coloring the chip background or / and probe points.
  • the signal contrast is maximized and thus achieves the objectives of the present invention.
  • a first aspect of the present invention is to provide a chip detection method for samples, especially biological samples, which includes a chip kit preparation process and a sample detection signal formation process, and the method is characterized by including coloring a chip.
  • the step of maximizing the color difference between the background and the target, the coloring includes adding a colorant or a colorant-containing colorant, and is directed to a probe point in a chip reactor or / and an area surrounding the probe point , But does not include labeling of probes and / or probe captures.
  • the "coloring" is equivalent to the English coloumtion, which means that the background of the chip (such as the substrate) or / and the probe point (but not including the probe capture object) have a certain light, which is different from the mark. Absorption, reflection, refraction and other properties to form an optimized hue or / and brightness or / and saturation in visible light or under a selected wavelength of light.
  • the chip is limited to the detection of samples, especially biological samples.
  • the chip is colored without using a labeling substance on the probe spot. It needs to be emphasized that the coloring is completely different from the current markers used in chip detection, which aims to obtain a positive result and uses a labeling substance as a means (the coloring of the probe points according to the present invention).
  • the colorant used is not a labeling substance used for detection), and is characterized by increasing the detection signal of the probe capture object (for example, in US patent application A1 20030032040, Chinese patent application CN A 1443854, Check the effect of the probe on the chip).
  • the purpose of the coloring is to form a high color difference ratio between the background and the target, preferably to maximize the color difference ratio.
  • the chromatic aberration ratio refers to the difference between the background and the target in the difference in hue, lightness, or saturation, including the absolute value of the difference between the background and the target with respect to the absorptance or reflectance of the signal light of all or part of the wavelength. Less than 50%, preferably not less than 70%.
  • the detection method of the present invention is also different from the so-called liquid chip or suspended particle chip detection methods (US Pat. Nos. 6,524,793, 6,649,414, and 6,632,526). Moreover, the so-called liquid chip or suspended particle chip is not a chip (flat chip chip) defined in the present invention.
  • the currently produced chips are all uncolored chips, which contain a transparent or uncolored substrate, and have a transparent or uncolored background when the chip is applied.
  • the uncolored film base only retains its base color (such as the color of extender pigments) without adding dyes and colored pigments. Examples thereof may be the metallic silver color of silver foil base, the film color of thin film base, and the like.
  • the colorant or colorant may be incorporated in one or more of the following portions of the chip: the substrate front, the substrate back, and the substrate.
  • the substrate front For example, one or more of the following structures of a dye or / and a colored pigment or / and a pigment-containing coating is added to a substrate, or a base containing a dye or / and a pigment or / and a pigment is added : Coatings, coatings, films and sheets.
  • maximizing the color difference between the background and the target includes one of the following options: A. When the labeling substance is a luminescent agent, make the chip background or / and the probe point Dark under white light, preferably black; B. When the labeling substance is a dark, preferably black non-luminous agent under white light, make the chip background or / and probe point light under white light, preferably The scheme is white; C. When the labeling substance is a light-colored, preferably white, non-luminous agent under white light, the chip background or / and the probe point is made dark, preferably black, under white light.
  • examples of the dark color include: red, orange, yellow, green, blue, cyan, purple, or black under white light; examples of the light color include: light red, light under white light Orange, light yellow, light green, light blue, light cyan, light purple, or white.
  • the coloring is to apply black paint and white paint to the front surface of the slide glass, respectively, and then use them for fluorescent label detection and gold-silver label detection, respectively. Surprisingly, with such a cheap and simple method, a chip with a sufficiently high sensitivity can also be obtained.
  • the colorant is selected from one or more of the following: a dye, a pigment, and a matting agent.
  • Dyes, pigments, and matting agents are a well-known concept in the coloring of materials such as plastics and coatings, and have certain contents.
  • the pigments are: black pigments including carbon black, metal salts, white pigments including titanium dioxide, and color pigments including yellow pigment, red pigment, blue pigment, green pigment, metal pigment, etc. Etc; the dye Examples are: Including amino black, Coomassie brilliant blue, crystal violet, Ponceau red, printing paint color paste
  • the matting agent-containing coating forms a coating having a surface roughness Ra between 0.02 and 3.0 ⁇ m, and preferably between 0.25 and 3.0 ⁇ m, on the surface of the glass slide.
  • the coloring matter is selected from a colored paint.
  • the colored paint is a paint containing the colorant.
  • the colored paint is a well-known concept with a certain content.
  • the colored paint includes black, white, various colored paints and / or inks.
  • the colors include red, yellow, green, blue, cyan, and purple colors.
  • both inks and paints have the property of coloring the substrate.
  • the colored paint can cover the front, back, or both sides of the substrate.
  • the front side of the substrate refers to the side on the substrate for fixing the probe.
  • the colored paint may include the colorant and a probe-binding substance, and examples of the probe-binding substance include one or more of the following organic substances and derivatives thereof: nitrocellulose, polystyrene, polystyrene Acrylate, polysulfone, polyethersulfone, polyvinyl chloride, amino resin, polysaccharide, polyamino acid.
  • the colored paint may further contain a matting agent.
  • the colored paint is preferably selected from colored paints, for example, one or more of the following colored paints: pearl black, magic black, pearl white, pearl blue, matte black, bayonet, scarlet, medium blue.
  • the coloring includes mixing a probe with the colored paint and then spotting the chip onto a substrate to make a chip.
  • the substrate is selected from modified or unmodified glass, plastic, metal, preferably glass.
  • the chip background or / and probe points are also colored, but at the same time, it is required to reduce the chip's weak target signal-background ratio to maximize the signal contrast and thereby achieve the objective of the present invention.
  • a second aspect of the present invention is to provide a chip detection method for a sample, particularly a biological sample, which includes a chip kit preparation process and a sample detection signal formation process.
  • the method is characterized in that the detection signal is read.
  • the chip weak target signal-background ratio is less than 0.80, preferably less than 0.50, and more preferably less than 0.25.
  • By raising the signal- Background ratio to improve detection sensitivity is the consensus of researchers in the chip field (for example, European patent EP A1 1279960, US patent US A1 20030032040, Chinese patent CN A 1443854). It is an important point of the present invention to improve the detection sensitivity by reducing the weak target signal-background ratio instead of increasing the signal-background ratio.
  • reducing the signal-to-background ratio eg, weak target signal-to-background ratio is less than 0.25 can even improve detection sensitivity.
  • the weak target signal-background ratio is obtained by coloring the chip.
  • the coloring includes increasing the background signal and / or reducing the weak target signal.
  • the signal enhancement can be achieved by one or more of the following methods: adding a luminescent pigment (such as fluorescein) to the substrate, and adding a coating or coating (such as coating) containing a luminescent pigment or light reflection on the front or / and back of the substrate Luminescent coating on the surface or / and back of the substrate on which the probe is fixed), film (such as luminescent film or reflective film covering the surface or / and the back of the substrate), with or without the detection target hole (Such as a luminous sheet covering the surface or / and the back of the substrate), and derivatization of active groups that can directly or indirectly bind luminescent colored pigments on the surface of the substrate (such as coating protein A on the substrate and then binding before or after the detection reaction) Rhodamine-labeled IgG-example tablet).
  • a luminescent pigment such as flu
  • the luminescent pigment or reflective coating may be formed by including one or more of the following intermediates as a medium: protein A, protein G, biotin, avidin, antigen, antibody, anti-antibody, polypeptide , DNA, etc. (for example, antibodies can capture rhodamine-bound antibodies to indirectly capture rhodamine, etc.).
  • the weak target signal can be reduced by adding dyes, colored pigments or / and coatings that can reduce the signal light emission or / and reflection, or can increase the signal light absorption at the quasi-fixed probe point, or / and on the front of the substrate or / and Add coatings, coatings (such as a matte coating on the surface of the substrate or / and the back surface) containing these dyes, colored pigments, and / or coatings, films (such as the surface of the substrate on which the probe is fixed) Or / and a light-reducing film on the back surface), or / and a sheet (for example, a light-reducing film containing a test substance colorant covering the surface of the substrate or / and the back surface).
  • coatings such as a matte coating on the surface of the substrate or / and the back surface
  • films such as the surface of the substrate on which the probe is fixed
  • a sheet for example, a light-reducing film containing a test substance colorant covering
  • the coloring is implemented by one or more methods selected from the group consisting of: A. coloring the chip background to enhance the signal during the preparation of the chip kit; B. coloring the sample during the formation of the sample detection signal The background of the chip is colored to enhance the signal; C. The probe point is colored to reduce the weak target signal during the preparation of the chip kit; D. The probe point is colored to reduce the weak target signal during the formation of the sample detection signal . This is very different from the current technical route that everyone is working on reducing background signals (even recognized as noise).
  • the illustrated examples surprisingly found that by increasing the background signal (eg, introducing a fluorescent substance) in the background of the chip reactor during the preparation of the chip kit or / and the formation of a sample detection signal, a confocal scanner was used.
  • the coloring of the enhanced signal includes introducing a luminescent agent or a luminescent agent-containing luminescent substance into the background.
  • the luminescent agent is selected from one or more of the following substances that can emit signal light: an excited luminescent substance including a fluorescent substance and an autonomous luminescent substance including a chemiluminescent substance and an electrochemical luminescent substance.
  • excited luminescent substances include rhodamine,
  • these luminescent agents are luminescent pigments, which are usually used as a labeling substance in a chip. It is only here that these labeling substances are used not only to label the detection reaction, but also to color the background to increase the background signal. When the detection signal is formed by the labeling substance actively or passively emitting light, the labeling substance can be used as a luminescent agent. An important aspect of current chip detection methods is to minimize the luminescent agent in the background.
  • the method of the present invention does the opposite, and in several embodiments, a labeling substance (such as rhodamine) is introduced in the background (such as the chip reactor reaction surface) to increase sensitivity.
  • the luminescent substance can also be selected from one or more of the following: luminescent agent-containing coatings, films, sheets, and complexes of luminescent agent and active molecules, which can be fixed on the area around the chip sample spot.
  • the active molecule includes one or more of the following polypeptides: albumin, protein A, protein G, biotin, avidin, antigen, antibody, anti-antibody, synthetic polypeptide, DNA, and the like.
  • antibodies can capture anti-rhodamine bound antibodies to capture rhodamine indirectly, and so on.
  • the reducing the coloring of the weak target signal includes applying a dark colorant or a dark colorant containing a dark colorant to the probe point, the dark color
  • the agent is selected from one or more of the following materials, which are dark, preferably black: dyes, colored pigments, matting agents. Dyes, pigments, and matting agents are a well-known concept in the coloring of materials such as plastics and coatings, and have certain contents. Examples of the pigments are black pigments including carbon black, metal salts, and the like; examples of the dyes are amino black and Coomassie brilliant blue, and the like. Matting agents in pigmented coatings generally refer to materials that can reduce the gloss of the coating surface.
  • the matting agent-containing coating forms a coating having a surface roughness Ra between 0.02 and 3.0 ⁇ m, preferably between 0.25 and 3.0 ⁇ m, on the surface of the glass slide.
  • the colorant is selected from one or more of the following: a coating, a film, a sheet containing the colorant, and a complex of the colorant and an active molecule that can be fixed during the formation of a sample detection signal On a chip sample spot other than the sample target captured by the probe.
  • the colored paint is a well-known concept and has a certain content.
  • 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.
  • the coloring includes mixing a probe with the colored paint and then spotting the chip onto a substrate to make a chip.
  • the coloring includes ft performed during the formation of the sample detection signal.
  • a luminous background is introduced to the chip during the formation of the sample detection signal.
  • the coloring includes adding a sample to the reactor after reacting the sample with the luminescent agent or luminescent substance, and enhancing the detection signal on the area surrounding the probe point by 200% or more, preferably 500% or more.
  • the detection method of the present invention is different from the so-called direct method of directly labeling a target substance in a detection sample with only a labeling substance.
  • a labeling substance is added to a detection sample, and its purpose is not only to label the target substance, but also to react with non-target substances; its effect, especially the latter effect is increased by non-specific adsorption outside the probe point.
  • the background signal to obtain the weak target signal-background ratio.
  • the serum is mixed with rhodamine and added to the peptide chip reactor without purification. After the reaction, not only the labeled target is captured by the corresponding immobilized probe, but the background signal is also increased and weak The target signal-background ratio is less than 0.1.
  • the detection method of the present invention has not only high sensitivity but also high specificity, unlike the direct method that usually reduces specificity.
  • the coloring further includes adding the sample to the reactor, and then adding the luminescent agent or luminescent substance to react, and enhancing the detection signal on the area surrounding the probe point therein by more than 200%, preferably 500% or more.
  • chip detection methods such as an antigen-antibody reaction-based chip detection method (including indirect method, double antigen sandwich method, double antibody sandwich method, etc.)
  • a high-purity label is usually required, and the higher the purity, the more it is good.
  • an impure label for example, a label-coloring mixture, wherein the label is rhodamine-labeled ligand, and the color is rhodamine-albumin
  • a polypeptide chip reactor for example, a label-coloring mixture, wherein the label is rhodamine-labeled ligand, and the color is rhodamine-albumin
  • the target captured by the probe is labeled, the background signal is also increased, and the weak target signal-background ratio is less than 0.1.
  • people's views on the existing chip detection methods including indirect method, double antigen sandwich method, double antibody sandwich method, etc.
  • the detection method of the present invention using impure labels not only Has high sensitivity and no decrease in specificity.
  • a planar chip substrate which is characterized by containing a colorant or a colorant containing a colorant.
  • the substrate includes a matrix and optionally one or more of the following composite structures: coatings, coatings, films, and sheets.
  • the term "colored film base" is a film base containing a colorant or a colorant.
  • the colorant may be contained in the matrix or / and the composite structure combined with the matrix (for example, colored coatings, colored coatings, colored films and colored sheets, etc.).
  • the substrate is selected from modified or unmodified glass, plastic, metal, preferably glass.
  • the front side of the substrate is the side on the substrate for fixing the probe.
  • the flat film base is the basis for forming a high color difference ratio between the background and the target, and preferably forming a maximum color difference ratio.
  • the current chip flat substrates are transparent or uncolored substrates, and have a transparent or uncolored background when the chip is applied. Examples of the transparent substrate include a transparent glass substrate, a transparent plastic substrate, and the like.
  • the uncolored film base only retains its base color (such as the color of extender pigments) without adding dyes and colored pigments. Examples include a silver foil base with a metallic silver color, a film base with a film quality or a film-glass slide composite. Film base, etc.
  • the chip base according to the present invention may be made by coloring according to one of the above aspects of the present invention.
  • the following chip in the embodiment of the present invention the colored base for enhancing the signal of the chip background (chip background signal enhanced base), the base for coloring the chip background by using a colorant or a substance containing a colorant (Colored film base), etc.
  • chip background signal enhancement substrates are: luminescent agent-containing substrate (in which a luminescent agent or a substance containing a luminescent agent is introduced), high-reflectivity substrate (signal light reflectance is greater than 5%, preferably greater than 10%, more preferred Greater than 30%), etc.
  • colored substrates are: dark, preferably black substrates (for example, black lacquer-slide base prepared by covering glass slides with black lacquer), and light, preferably white substrates (for example, glass slide over white lacquer). Preparation of white paint-glass slide base), and so on.
  • black substrates for example, black lacquer-slide base prepared by covering glass slides with black lacquer
  • white substrates for example, glass slide over white lacquer
  • the coloring is bulk coloring, surface coloring, or spot coloring.
  • the body coloring is the coloring of the substrate
  • the surface coloring includes front, back, front / back coloring
  • the spot coloring is the probe point front, back, front / back coloring.
  • the chip substrate according to the present invention comprises a glass substrate and the colored paint coating.
  • the colored paint is a paint containing the colorant.
  • the colored paint includes black and white Color, various colored paint or / and ink.
  • the colors include red, yellow, green, blue, cyan, and purple colors.
  • both the ink and the paint have the property of coloring the substrate.
  • the colored paint may cover the front side, the reverse side, or both sides of the substrate (or raw material base).
  • the front side of the substrate is the side on the substrate for fixing the probe.
  • the colored paint is preferably a black paint or a white paint.
  • the black coating is formed from a black paint, such as a black paint with or without a matting agent.
  • the white coating is formed from a white paint, such as a white paint with or without a matting agent.
  • the surface roughness Ra of the chip substrate of the present invention is between 0.02 and 3.0 ⁇ m, and preferably between 0.25 and 3.0 ⁇ m.
  • a chip substrate which includes a colored planar substrate and an optional isolation structure, wherein the colored planar substrate contains a colorant or a colorant containing a colorant.
  • the colored flat base is also referred to as a colored base
  • the flat base is also referred to as a base.
  • the colored substrate is a chip substrate according to the third aspect of the present invention.
  • a chip probe card including a flat substrate, a probe point fixed on the substrate, and an optional isolation structure.
  • the chip probe card is characterized by A.
  • the flat sheet base contains a colorant or a colorant containing a colorant; or / and B.
  • the probe dot contains a colorant or a colorant containing a colorant, but does not contain a labeling substance.
  • the substrate is a colored chip substrate according to the third aspect of the present invention.
  • the substrate may be the following chip probe board: A. chip probe board (background signal enhanced chip probe board) prepared by the coloring preparation process with signal enhancement on the chip background, ⁇ . Pair of chip probes
  • the chip probe board (weak target signal weakening chip probe board) prepared by the coloring preparation process for reducing the weak target signal is included at the point, C. Contains the signal strengthening function of the chip background and the signal weakening of the chip probe point.
  • the chip probe card (background signal enhancement-weak target signal attenuation chip probe card) prepared by the coloring preparation process, D. It is prepared by using the colorant or the substance containing the colorant to perform the coloring on the chip background.
  • Chip probe board (background colored chip probe board), ⁇ . Chip probe board (fixed-point colored chip probe board) prepared by the preparation process using the colorant or the substance containing the colorant to perform chip coloring on the chip probe points ), And F.
  • a chip probe board prepared by a process for preparing a chip background and a probe spot by using a colorant or a substance containing a colorant as described above ( View / probe Coloring probe card chip) and the like.
  • the probe point may contain the colorant or coloring Thing.
  • a chip kit which includes a chip probe card and an optional labeling system.
  • the chip kit is characterized by: A. a planar sheet in the chip probe card
  • the base contains a colorant or a colorant-containing colorant; or / and B.
  • the probe points in the chip probe card contain a colorant or a colorant-containing colorant, but do not contain a labeling substance; or / and C. It also contains a coloring system containing a colorant or a colorant containing colorant.
  • the coloring system refers to a system for coloring the background of the chip or / and the probe during the formation of a sample detection signal.
  • the chip kit according to the present invention may be a chip kit prepared according to the method of the first aspect of the present invention, for example, the following chip kit: A. A chip kit containing a background-colored chip, B. A probe point Chip kits for coloring chips, C. chip kits with background / probe spot coloring chips, and D. chip kits with colorants for coloring systems.
  • the weak target signal-background ratio is less than 0.80, preferably less than 0.50, and more preferably less than 0.25.
  • High-signal-background ratio chip kits are the basic development direction of current chip kits.
  • the weak target signal-background ratio is a ratio of detection signal values between the weak target and the background surface.
  • Such a low signal-to-background ratio is a feature of the chip kit of the present invention. We have surprisingly discovered through the examples of the present invention that the use of this low signal-background chip kit can even improve detection sensitivity.
  • the chip kit according to the present invention may also be a chip kit prepared according to the method of the second aspect of the present invention, for example, the following chip kit: A. A chip kit containing a background signal enhancement chip, B. A weak target Chip kit for signal attenuation chip, C. Chip kit with background signal enhancement-weak target signal attenuation chip, D. Chip kit for coloring system using luminescent agent as colorant, and E. Coloring system for luminescent material containing substance Chip kits for colorants, etc.
  • the chip kit of the present invention preferably contains a chip probe card according to the present invention.
  • the marking system and / or the coloring system contains the luminescent agent or / and the luminescent substance.
  • the chip base of the chip may be the above-mentioned coloring base, or may be a non-coloring base (eg, activated glass, plastic, metal).
  • the preferred non-colored film base in the embodiment of the present invention is an activated glass slide, for example, an activated glass slide containing one or more of the following derivatized groups: amino, epoxy, aldehyde, and hydrazide (-CO- NHN3 ⁇ 4), aminoureido (H 2 N-NH-CONH-), diethylaminoethyl (DEAE), Diethylmono (2-hydroxypropyl) aminoethyl (QAE), carboxymethyl (CM), sulfopropyl (SP), mercaptoethylpyridine (MEP), siloxenyl group, and thiol group.
  • the marking system and the coloring system may contain the same luminescent agent or different luminescent agents.
  • the luminescent agent may exist independently or coexist with other substances (for example, a substance capable of non-specific adsorption with the area around the chip probe point and a substance capable of specifically adsorbed with the area around the chip probe point).
  • the luminescent agent is rhodamine, CY3, etc., which is used to react with the sample, and not only marks a sample target molecule that can bind to the probe point, but also binds to the sample and can be adsorbed. Other substances on the substrate around the probe point. After adding the chip, it not only marks the probe-target reaction, but also enhances the background signal.
  • the luminescent substance contained in the coloring system is rhodamine, CY3, etc., which is a purified or unpurified product after reacting with the labeled ligand-albumin mixture, respectively. Not only is the probe-target response labeled, but the background signal is enhanced.
  • 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 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 size of the slide glass used in the embodiment of the present invention is 75 X 25 X 1.0 mm, and the specific specifications are shown in Table 1. Table 1
  • the method refers to the following articles: Melnyk 0, etc., "Peptide arrays for highly sensitive and special antibody-binding fluorescence arrays", Bioconjug C em. 13: 713-20. 2002, and Olivier C, etc., "oxo semicarbazone peptide oro; iggodeoxynucleotide microarrays ⁇ , Bioconjug Chem. 14: 430-9.2003.
  • the method refers to our other Chinese invention patent application CN03135618.4.
  • the probe used is HCV antigen (Beijing, China) Institute of Liver Diseases, People's Hospital) and HIV 1 + 2 Antigen (Institute of Liver Diseases, Beijing People's Hospital, China).
  • the probe has a spot diameter of 200 ⁇ m and a spot distance of 800 ⁇ m.
  • sample 1 is HCV antibody-positive serum
  • sample 2 is HIV 1 + 2 antibody-positive human serum
  • sample 3 is a positive control (HCV antibody and HIV 1 + 2 antibody A mixture of positive serum controls)
  • sample 4 was a negative control (a serum control where both HCV and HIV 1 + 2 antibodies were negative). All samples were pre-tested using the classic ELISA method under serum 20-fold dilution reaction conditions.
  • Example 1 Preparation of background signal enhancement substrate and substrate
  • the luminescent substance in the chip analysis is selected from one or more of the following substances that can emit signal light: excited luminescent substances including fluorescent substances and autonomous luminescent substances including chemiluminescent substances and electrochemical luminescent substances .
  • excited luminescent substances include rhodamine, CY3, CY5, Alexa, algae protein, rare earth compound fluorescence Matter.
  • these luminescent substances are luminescent pigments.
  • the substrate may be selected from modified or unmodified glass, plastic, and metal. In this embodiment, glass (glass slide) is preferred.
  • the preparation of the background signal-enhancing film base in this embodiment includes four methods: 1) preparing a film base containing a light-emitting substrate (such as the preparation of the film base 1); 2) preparing a film base containing a light-emitting film (such as the film bases 2, 7) And preparation of 8); 3) preparation of bases containing luminescent coatings (for example, preparation of bases 3 and 4); 4) preparation of bases containing luminescent coatings (for example, preparation of bases 5 and 6). Based on these methods, some other methods can be derived.
  • the substrate 1 prepared in this embodiment is a substrate containing a luminescent substrate (a polystyrene substrate with a fluorescent substance added).
  • the preparation method comprises adding a fluorescent pigment ZnS-Mii to a thermoplastic polystyrene used for preparing an enzyme-labeled plate, and then obtaining the size of 75 X 25 X 1 mm by molding.
  • the light-emitting film used in this embodiment is a polyvinyl chloride film to which a fluorescent substance is added.
  • the substrate 2 prepared in this embodiment is a substrate containing a light-emitting film (light-emitting film-glass slide composite substrate).
  • the preparation method includes thermally bonding a polyvinyl chloride film that reflects light with a wavelength of 532 nm to a glass slide described in Table 1.
  • the film bases 7 and 8 prepared in this embodiment are film bases containing light-emitting films (front and back light-emitting film bases, respectively).
  • the preparation method includes punching a 150 ⁇ m diameter hole in a polyvinyl chloride light-emitting film, The thermal bonding process is combined on the front or back of the aldehyde-based glass, and the position of the hole is the same as the position of the probe point manually spotted when preparing the chip.
  • the substrates 3 and 4 prepared in this embodiment are substrates (rhodamine-albumin-coated substrate and rhodamine-polypeptide-coated substrate) containing a light-emitting coating on the back.
  • the preparation method includes applying albumin (Shanghai Institute of Biological Products) and synthetic peptides (synthesized Epstein-Barr virus VCA antigen, self-made) are divided into 1 J and Rhodamine (5- (and-6) -carboxytetramethylrhodamine succinimidyl ester, referred to as 5 (6) -TAMRA SE, Anaspec, USA) Corporate company)
  • the known rhodamine-binding peptide technology is used to form rhodamine-albumin and rhodamine-polypeptide, and they are respectively coated on the back of the amino slide described in Table 1 using a general protein coating technique.
  • the luminescent paint used in this embodiment is a white paint containing a fluorescent substance.
  • the substrates 5 and 6 prepared in this embodiment are substrates containing a luminescent coating (the substrates are coated with a fluorescent substance on the front and the substrates are coated with a fluorescent substance on the back).
  • the preparation method includes: The back of the amino slide is coated with a luminescent paint. The thickness of the coating formed is around 30 m.
  • the substrates and substrates prepared in this embodiment are listed in Table 2.
  • the reference substrate is the amino slide in Table 1.
  • the detection signal is a relative signal value. Table 2
  • control substrate is the amino slide in Table 1.
  • the substrate according to the embodiment of the present invention is a multi-chip base substrate, including a base substrate and a base substrate isolation structure.
  • the isolation structure of the substrate pool is a highly hydrophobic organic silicon coating (Chengdu Chenguang Chemical Design Institute) formed by coating on the above substrate and drying to form a film (film thickness less than 0.25mm) (refer to our other invention patent application CN03117397.7).
  • Each substrate contains 8 substrate pools. The size of each substrate pool is 4.5mm ⁇ 4.5mm, and the width of the isolation structure between the substrate pools is 4.5mm.
  • an isolation structure may be formed on the substrate (or raw material substrate) first, and then a coloring process is performed to form a substrate.
  • 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 well-known chip spotting method. The 4 points in the pool form a 2 X 2 square matrix. Then take sample No. 4 (serum control with negative HCV antibody and HIV 1 + 2 antibody) as the weak target sample, rhodamine-labeled goat anti-human anti-antibody (Jackson ImmunoRresearch Laboratories, USA) as the marker, The chip detection method detected negative targets and substrate background signals (Table 2).
  • the weak target signal-to-background ratio of the film base and the substrate prepared in this embodiment are all less than 0.25, most are less than 0.10, and some are even less than 0.05.
  • the colored substrate, colored paint, and colored film each contain a colorant and a substance that can bind a probe.
  • the colorant is selected from one or more of the following: dyes, pigments, and matting agents. Dyes, pigments, and matting agents are a well-known concept in the coloring of materials such as plastics and coatings, and have certain contents. Examples of the pigment include: black pigments including carbon black and metal salts, white pigments including titanium dioxide, and color pigments including yellow pigment, red pigment, blue pigment, green pigment, metal pigment, and the like Wait.
  • matting agents in colored coatings refer to materials that can reduce the gloss of the surface of the coating.
  • a large class of colored paints are colored paints, for example, one or more of the following colored paints: pearl black, magic black, pearl white, pearl blue, matte black, bayonet, scarlet, and medium blue.
  • paints often contain substances that can bind probes, such as nitrocellulose, polystyrene, polyacrylate, polysulfone, polyethersulfone, polyvinyl chloride, amino resins, polysaccharides, and polyamino acids.
  • the substrate may be selected from modified or unmodified glass, plastic, and metal. In this embodiment, glass (glass slide) is preferred.
  • the preparation of the colored substrate in this embodiment includes three methods: 1) preparing a colored substrate containing a colored substrate; 2) preparing a colored substrate containing a colored paint; 3) preparing a colored substrate containing a colored film. Based on these methods, some other methods can also be derived (for example, methods combined from them).
  • the base 9 prepared in this embodiment is a colored base (black plastic base) containing a color base.
  • the preparation method is as follows: the colorant (carbon black) is added to the thermoplastic polystyrene used for the preparation of the microtiter plate, and then the size is 75 X 25 X limn.
  • the paints used in this example are pearl black spray paint (Shanghai Qifu Industrial Development Co., Ltd.), pearl green spray paint (Shanghai Qifu Industrial Development Co., Ltd.), matte black (Shanghai Qifu Industrial Development Co., Ltd.) and Chuanyang Auto White. Spray paint (Chengdu Hongguang Coating Factory), the colorants in the paint are the following pigments: channel black, titanium oxide, etc. Pearl black and precious pearl green paints are added with a (matte substance) matting agent.
  • the preparation method is that the coating is sprayed on the top surface (or the back surface) of the slide glass, and a colored coating layer (thickness of about 30 ⁇ ) is formed after drying, and their roughness Ra is between 0.4 and 0.5 m (measured in Chengdu) Supervised verification testing).
  • a double-sided colored coating substrate can also be prepared.
  • Part of the colored film base containing the color paint prepared in this embodiment is referred to as film base 10 (pearl black spray paint / slide), film base 11 (pearl green spray paint / slide), and film base 12 (white spray paint / slide).
  • Glass slides) and 13 epoxy glass slides / matte black spray paint).
  • the bases 10, 11, and 12 are front-side colored bases
  • the sheet 13 is a back-side colored base.
  • the colored film used in this embodiment is a black polystyrene film (thickness about 45 um, self-made), and the colorant of the coloring film is channel black. It was then thermally bonded to the front of the glass slide and the back of the semicarbazide-based glass, respectively.
  • Part of the colored substrate containing the colored film prepared in this embodiment is referred to as a base 14 (a front-side colored film) and a base 15 (a back-side colored film base).
  • the colored background substrates and substrates prepared in this example are listed in Table 3.
  • the substrate includes a colored substrate and an optional isolation structure. table 3
  • the values of surface roughness and absorbance in the table are the average value of the detection of multiple points on the substrate.
  • Example 3 Preparation of fixed-point colored film substrates and substrates
  • the method for preparing a fixed-point colored substrate is to spot the colored paint with a spotter onto a transparent substrate (the glass slide or activated glass shown in Table 1) to form colored spots on the front or / and the back (corresponding to the colored spot positions At the position of the probe point to be fixed).
  • a large category of colored paints are colored paints, for example, one or more of the following colored paints: pearl black, magic black, pearl blue, matte black, bayonet, scarlet, medium blue, pearl white.
  • matting agents in pigmented coatings are materials that reduce the gloss of the surface of the coating.
  • paints often contain probe-binding substances, such as nitrocellulose, polystyrene, polyacrylate, polysulfone, polyethersulfone, polyvinyl chloride, amino resins, polysaccharides, and polyamino acids.
  • Coloring can be performed in one of the following steps: direct spot coloring of the substrate, then spot coloring of the substrate, and spot coloring of the substrate in the substrate substrate pool.
  • the substrates prepared by spotting the black paint and the white paint on the front surface of the glass slide are respectively referred to as the substrates 16 and 17;
  • the substrates prepared to the back of the epoxy glass slide are referred to as substrates 18 and 19.
  • the diameter of the colored dots may be slightly larger than the diameter of the probe-like dots.
  • the black paint used is pearl black paint (containing matting agent, Shanghai Qifu Industrial Development Co., Ltd.), and the white paint used is pearl white paint (containing matting agent, Shanghai Qifu Industrial Development Co., Ltd.).
  • the method for preparing the substrate in this embodiment is the same as the method for preparing the substrate in Example 1.
  • the scanner is a confocal laser scanner (Afymetrix GMS 418), scanning the excitation light wavelength 532nm, the emission light wavelength 570mn, the laser intensity and gain are 60/69, the read signal is processed by processing software (JAGUARII), and then taken Results are obtained after averaging.
  • the signal value of the uncolored points is between 20 and 100, and the signal value of the colored points is less than 20.
  • Example 4 Preparation of a background signal enhancement chip probe card
  • the preparation of the background signal enhancement chip probe board of this embodiment includes three methods: 1) spot preparation of the background signal enhancement substrate prepared from Example 1; 2) spotting the unstained film base, and then chip probe Plate background coloring preparation; 3). Spot from the stained film base, and then prepare the chip probe plate background coloring. Based on these methods, some other methods can be derived.
  • the probes used in this example are: HCV antigen solution (1.5mg / ml), HIV1 + 2 antigen solution (1.5mg / ml), and HCV prepared from another of our inventions (PCT / CN2004 / 000077) Antigen-silica solution (1.5mg antigen / ml) and HIV1 + 2 antigen-silica solution (1.5mg antigen / ml).
  • the colored substrate used in this embodiment is the background signal enhancement substrate (substrates 1-8) prepared in Example 1. Then, the above-mentioned probe is spotted into the substrate pool according to a conventional spotting method. Each of the four probes has 3 samples, forming a 4x3 array. It is then sealed with or without sealing fluid (such as milk sealing fluid).
  • the chip probe boards prepared by this method are named chip probe boards 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 corresponding to the substrates used. 2) Method for preparing a background signal enhancement chip probe card by spotting an uncolored substrate and then coloring the background of the chip probe card
  • the activated glass slides (non-colored film bases) shown in Table 1 were prepared into 8 base pool uncolored substrates. Then, the above-mentioned probe is spotted into the substrate pool according to a conventional spotting method. Each of the four probes has 3 samples, forming a 4x3 array.
  • Rhodamine-albumin was used as a luminescent substance and coated on the front surface of the substrate (rhodamine-albumin was prepared according to the well-known rhodamine-binding peptide technology).
  • albumin In addition to albumin, other peptides (such as protein A, protein G, biotin, avidin, antigen, antibody, anti-antibody, peptide, DNA, etc.) that can be used to form a luminescent substance can also be used. This method is used.
  • peptides such as protein A, protein G, biotin, avidin, antigen, antibody, anti-antibody, peptide, DNA, etc.
  • an amino slide, an aldehyde-based slide, an epoxy-based slide, a semicarbazide slide, and a PVP-coated slide are used as non-colored slides.
  • the background signal enhancement chip probe boards prepared separately are named as chips. Probe boards 11, 12, 13, 14, and 15.
  • the colored substrates used in this embodiment are the background signal enhancement substrate 6 and the substrate 8 prepared in Example 1. Then, the above-mentioned probe is spotted into the substrate pool according to a conventional spotting method. Each of the four probes has 3 samples, forming a 4x3 array. Rhodamine-albumin was used as a luminescent substance and coated on the front side of the substrate.
  • the prepared background signal enhancement chip probe cards are named chip probe cards 16 and 17, respectively.
  • a chip probe board is referred to, and the uncolored substrate is spotted according to the above method, and then is sealed with a milk blocking solution.
  • the scanner is a confocal laser scanner (Afymetrix Corporation GMS 418), which scans the excitation light wavelength of 532nm, the emission light wavelength of 570nm, the laser intensity and gain are 60/69, and the read signal is processed by processing software (ZoCSoft lmageBoost), and then Take the average and get the result.
  • the background signal value of the reference chip probe card is between 20 and 100.
  • the background signal value of the background signal enhancement chip probe card prepared in this embodiment is much larger, both being greater than 5000, or even greater than 10,000.
  • Example 5 Preparation of a colored chip probe card
  • the preparation of the colored chip probe card in this embodiment is prepared by spotting the colored chip base.
  • the probe used in this example is the same as the probe used in Example 4.
  • the coloring substrate used in this embodiment is the coloring substrate (substrate 9-15) prepared in Example 2. Then, the above-mentioned probe is spotted into the substrate pool according to a conventional spotting method. Each of the four probes has 3 samples, forming a 4X 3 array. Seal with or without sealant (such as milk sealant).
  • the chip probe boards prepared by this method are named as chip probe boards 18 (black plastic chip-based chip probe boards) and 19 (pearl black spray paint / slide chip-based chip probes) according to the naming order of the used substrates.
  • the preparation of the spotted colored chip probe card in this embodiment includes two methods: 1) the spotted colored substrate is spotted and prepared; and 2) the non-stained sliced spotted substrate is prepared. Based on these methods, some other methods can be derived.
  • the probe used in this example is the same as the probe used in Example 4.
  • the fixed-point colored substrates used in this embodiment are the colored substrates 16-19 prepared in Example 3. Then, the above-mentioned probe is spotted on the spot of colored spots in the substrate pool according to a conventional spotting method. Each of the four probes has 3 samples, forming a 4x3 array. Then block it with or without a sealant (such as milk sealant).
  • the chip probe boards prepared by this method are named chip probe boards 25 (black paint fixed-point colored glass slide-based chip probe boards) and 26 (white paint fixed-point colored glass Chip-based chip probe board), 27 (fixed-point colored epoxy glass slide-based chip probe board with black paint) and 28 (fixed-point colored epoxy glass-based chip probe board with black paint).
  • the method is as follows: the probe is mixed with the colored paint, and then spotted on the substrate; Or seal without sealing fluid (such as milk sealing fluid).
  • the paints used in this embodiment are pearl black paint (containing matting agent, Shanghai Qifu Industrial Development Co., Ltd.) and pearl white paint (containing matting agent, Shanghai Qifu Industrial Development Co., Ltd.).
  • the substrate used in this example is the glass slide in Table 1.
  • the chip probe boards prepared in this embodiment are named chip probe boards 29 (black probe chip probe boards) and 30 (fixed-point white probe chip probe boards), respectively.
  • Example 7 Preparation of low-weak target signal-background ratio chip kit
  • the low-weak target signal-background ratio chip kit of this embodiment includes a chip probe board and an optional labeling system, and an optional coloring system of the present invention. Its preparation includes 2 methods: 1) Preparation from a background signal enhanced chip probe card or a fixed-point colored chip probe card; 2) Preparation from an uncolored chip probe card. Based on these methods, some other methods can be derived.
  • This preparation method of the chip kit of this embodiment is a combination of a background signal enhancement chip probe board or a fixed-point coloring chip probe board and a labeling system to meet a low-weak target signal-background ratio chip standard (less than 0.80, preferably less than 0.50 , More preferably a chip kit of less than 0.25).
  • the labeling systems used in this embodiment are the rhodamine labeling system and the CY3 labeling system, respectively.
  • the labels used are rhodamine-labeled goat anti-human secondary antibody and CY3-labeled goat anti-human secondary antibody. Marking method).
  • the chip probe card used in this embodiment includes the background signal enhancement chip probe card (chip probe card 1-17) prepared in Example 4 and the fixed-point colored chip probe card prepared in Example 6. (Fixed-point black chip probe boards 25, 27, 28).
  • the prepared chip kits were named chip kits 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 respectively in this order. , 19, and 20.
  • This method of preparing the chip kit of this embodiment is a combination of an uncolored chip probe card with a labeling system and a coloring system
  • a chip kit with a weak target signal-background ratio chip standard (less than 0.80, preferably less than 0.50, more preferably less than 0.25) in accordance with the detection method of the present invention.
  • the uncolored chip probe card used in this embodiment uses the epoxy glass slide and the semicarbazide glass in Table 1 as non-colored substrates, and is made into a non-colored substrate according to the isolation structure forming method of Embodiment 1. Spotting method and closed method.
  • the probe solution used was an anti-human hepatitis B surface antibody (HBsAb) coating solution (2 mg / ml, Institute of Liver Diseases, Beijing People's Hospital).
  • HBsAb anti-human hepatitis B surface antibody
  • a luminescent agent or a luminescent substance is used as a labeling substance of a labeling system and a coloring substance of a coloring system, and then a kit is formed with an uncolored chip probe card.
  • the luminescent agents used in this embodiment are rhodamine and CY3, respectively, and their amounts are optimized according to the type of sample to be detected.
  • the prepared chip kits are: chip kit 21 (unstained chip probe board based on epoxy glass slide, rhodamine luminescent agent), chip kit 22 (unstained chip probe based on semicarbazide slide) Plate, rhodamine luminescent agent) and chip kit 23 (unstained chip probe board based on semicarbazide glass, CY3 luminescent agent).
  • the labeled ligand and active molecule are mixed into a mixture at a preferred ratio, and then a preferred amount of luminescent agent is reacted with this mixture and bound to the labeled ligand and active molecule, respectively.
  • the purified or unpurified product is The marker-colorant mixture is then combined with the uncolored chip probe plate to form a kit.
  • the luminescent agents used in this example are rhodamine and CY3
  • the labels used are rhodamine and CY3 labeled anti-human hepatitis B surface antibody (HBsAb) (anti-human hepatitis B surface antibody for labeling, Institute of Liver Diseases, Beijing People's Hospital).
  • the compounds were rhodamine and CY3 labeled human albumin, respectively.
  • the prepared chip kits are: Chip kit 24 (unstained chip probe board based on epoxy slides, rhodamine anti-human hepatitis B surface antibody and rhodamine chemical albumin) and chip kit 25 (based on Unstained chip probe plate of epoxy glass slide, CY3 anti-human hepatitis B surface antibody and CY3 albumin).
  • the marker and the color can be prepared separately and mixed, or not even mixed.
  • the weak target samples used in this example are the above-mentioned negative samples 3 (which are also HBs Ag negative serum).
  • Identification test of chip kit 20 Dilute negative sample 3 to 1/20 and add them to the reactor of chip kit, respectively, and the sample volume is 15 ⁇ 1. After 30 minutes of reaction, it was washed 5 times, and the washing solution was added in an amount of 25 ⁇ 1 each time. The labeling amount was 15 ⁇ 1, and the reaction was washed 5 times after the reaction, and scanned after drying.
  • Identification experiments of chip kits 21, 22, and 23 Dilute negative sample 3 to 1/10, and then mix with the luminescent agent solution (preferred concentration) in the chip kit in equal volumes and react at room temperature for 30 minutes, then prepare this The samples were respectively added to the reactor of the chip kit, and the loading amount was 15 ⁇ 1. After 30 minutes of reaction, it was washed 5 times. Scan after drying.
  • Identification experiments of chip kits 24 and 25 Dilute negative sample 3 to 1/20 and add them to the reactor of the chip kit, respectively, and the sample volume is 15 ⁇ 1. After 30 minutes of reaction, it was washed 5 times, and the washing solution was added in an amount of 25 ⁇ 1 each time. Then add the above-mentioned marker-luminescence solution (preferred concentration) in an amount of 15 ⁇ l, wash 5 times after the reaction, and scan after drying.
  • the scanner is a confocal laser scanner (Afymetrix GMS 418).
  • the scanning light wavelength is 532nm
  • the emission light wavelength is 570nm
  • the laser intensity and gain are 60/69
  • the read signals are processed by the software ZoCSoft ImageBoost and averaged Get the result after value.
  • the background signal values of the chips are all greater than 10,000
  • the target signal values of the negative samples are less than 2000.
  • their weak target signal-background ratios are all less than 0.2, some are less than 0.08, and even some reagents are less than 0.03.
  • Example 8 Preparation of a colored chip kit
  • the colored chip kit of this embodiment includes a chip probe card and an optional labeling system, and a preparation method thereof:
  • the color difference chip probe card and the labeling system are combined by color difference.
  • the typical method of the preparation method is the following two methods. Based on these methods, some other colored chip kits can also be prepared.
  • the selection criterion is to maximize the signal contrast between the marking result and the background.
  • Marking used in this example The system is a fluorescent labeling system.
  • the label used is rhodamine-labeled goat anti-human secondary antibody.
  • the labeling method used is a well-known labeling method.
  • the dark chip probe board used in this embodiment is selected from the dark chip probe board prepared in Example 5 [chip probe board 18 (black plastic chip-based chip probe board), 19 (pearl black spray paint / glass Chip-based chip probe board), 20 (Pearl green spray paint / slide chip-based chip probe board), 22 (Epoxy glass slide / matte black spray-painted chip-based chip probe board), 23 (front side sticker Stained film-based chip probe board), and 24 (Stained film-based chip probe board on the back)], and the chip kits prepared from these chip probe boards are named chip kits 26 in this order.
  • Black Plastic Chip Kit 27 (Pearl Black Spray Paint / Slide Chip Kit), 28 (Pearl Green Spray Paint / Slide Chip Kit), 29 (Epoxy Slide / Matte black spray chip chip kit), 30 (black film chip chip kit on the front), and 31 (black film chip chip kit on the back).
  • the labeling system used in this embodiment is a gold-silver labeling system (refer to the "Method for Identifying and / or Quantifying Target Compounds" of Chinese Patent Application No. 00807744.4).
  • the labeling method used is gold-labeled goat anti-human secondary antibody. It is a well-known marking method.
  • the light-colored chip probe board used in this embodiment is selected from the light-colored chip probe board [chip probe board 21 (white spray paint / slide-based chip probe board) prepared in Example 5].
  • the chip kit was named Chip Kit 32 (white spray paint / slide-based chip kit).
  • Example 9 Preparation of spot dye chip kit
  • the fixed-point colored chip kit of this embodiment includes a chip probe board and a labeling system.
  • the preparation method is as follows: It is a combination of selecting a fixed-point colored chip probe card and a labeling system, and the selection criterion is to maximize the signal contrast between the labeling result and the background.
  • the labeling system used in this embodiment is a fluorescent labeling system
  • the labeling substance is rhodamine-labeled goat anti-human secondary antibody
  • the labeling method used is a well-known labeling method.
  • the fixed-point colored chip probe board used in this embodiment is a fixed-point dark chip probe board, which is selected from the fixed-point colored chip probe board prepared in Example 6 [chip probe board 25 (black paint fixed-point colored slide glass base Chip probe board), 27 (black paint fixed-point colored epoxy glass slide-based chip probe board), 28 (Black paint fixed-point colored epoxy based glass slide chip probe board) and 29 (black probe chip probe board)].
  • chip kits 32 black paint fixed-point stained glass slide chip chip kit
  • 33 black paint fixed-point stained epoxy glass slide chip kits
  • Chip Kits 34 (Black Paint Spotted Epoxy Stained Slides Chip Kit) and 35 (Black Probe Chip Kit).
  • the fixed-point light-colored chip probe board prepared in Example 6 [Chip Probe Board 26 (White paint fixed-point colored slide glass-based chip probe board) and 30 (Fixed-point white probe chip probe board)] can also be pressed The same method as the above kit preparation method is used together with a dark labeling system (such as a gold-silver labeling system) to make a kit.
  • a dark labeling system such as a gold-silver labeling system
  • the low-weak target signal-background ratio chip kit used in this example is part of the low-weak target signal-background ratio chip kit prepared in Example 7. [Chip kits 1, 2, 3, 4, 5, 6, 7, 8, , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21], the samples used are as described above (samples 1-4), and the weak target sample used is the negative sample among them.
  • the reference chip kit used in this embodiment is a fluorescently labeled chip kit (referred to as chip kit 0) containing the same probe and prepared in the conventional manner with the epoxy glass slide in Table 1 as a base.
  • the aforementioned four samples were respectively added to the reactor of the chip kit, and the amount of each sample was 15 ⁇ 1. After 30 minutes of reaction, it was washed 5 times. The labeling amount was 15 ⁇ 1, and the reaction was washed 5 times after the reaction. Scan after drying.
  • the scanner is a confocal laser scanner (Afymetrix GMS 418).
  • the scanning light wavelength is 532nm and the emission light wavelength is 570nm.
  • the laser intensity and gain are 60/69 respectively.
  • the read signals are processed by software ZoCSoft ImageBoost ( Target signal-background ratio chip kit, where the target readings are the difference between their absolute readings and background values) and JAGUARII (for the reference chip kit), and then average the results to get the results.
  • the detection results (negative, positive) obtained by using the above-mentioned low-weak target signal-background ratio chip kit are consistent with the sample negative.
  • sample 1 was negative when diluted to 100 times.
  • the sample 1 was still diluted to 500 times. Is a positive result.
  • Table 4 gives some test results.
  • the detection results of other low-weak target signal-background ratio chip kits are similar to these detection results. Table 4
  • the low-weak target signal-background ratio chip kit used in this example is part of the low-weak target signal-background ratio chip kit prepared in Example 7.
  • Chip kit chip kit 21 unstained chip based on epoxy glass slides
  • Chip Kit 22 Unstained Chip Probe Board Based on Semicarbazide, Rhodamine
  • Chip Kit 23 Unstained Chip Probe Board Based on Semicarbazide, CY3
  • the sample used was the negative control and positive control in the ELISA kit (Beijing Tiantan Biological Products Co., Ltd.) for the detection of hepatitis B surface antigen, and the weak target sample was used as the negative control.
  • the reference chip kit used in this example is the fluorescently labeled chip kit (Chip Kit 0) in Example 10.
  • the scanner is a confocal laser scanner (Afymetrix GMS 418 or Chengdu Optoelectronics Institute Scan-2).
  • the scanning light wavelength is 532nm and the emission light wavelength is 570nm.
  • the laser intensity and gain are 60/69, and the read signals are processed separately.
  • Software ZoCSoft lmageBoos t (For low-weak target signal-background ratio chip kit) and JAGUARII (for reference chip kit), and then average the results to get the results. Detection using low-weak target signal-background ratio chip kit, the weak target signal-background ratio is less than 0.1, and the negative and positive results are consistent with the sample used.
  • the low-weak target signal-background ratio chip kit used in this example is part of the low-weak target signal-background ratio chip kit prepared in Example 7.
  • Chip Kit 24 Unstained chip probe board based on epoxy glass slides
  • Rhodamine anti-hepatitis B surface antibody and rhodamine albumin Chip kit 24
  • chip kit 25 unstained chip probe board based on epoxy slides, CY3 anti-human hepatitis B surface antibody and CY3 albumin
  • the samples used and the weak target samples are the same as in Example 11.
  • the reference chip kit used in this example is the fluorescently labeled chip kit (Chip Kit 0) in Example 10.
  • Operation method When using the low-weak target signal-background ratio chip kit for detection, dilute the two samples to between 1/20 and 1/10000, and add them to the reactor of the chip kit. 1. After the reaction was completed, washing was performed 5 times. The optimal concentration of the labeling substance-the amount of luminescent substance was 15 ⁇ 1, and the solution was washed 5 times after the reaction, and the washing solution was added 25 ⁇ 1 each time. After drying, scanning was performed.
  • the operation method is the same as that of the reference chip kit in Example 11.
  • the scanner is a confocal laser scanner (Afymetrix GMS 418).
  • the scanning light wavelength is 532nm
  • the emission light wavelength is 570nm
  • the laser intensity and gain are 60/69
  • the read signals are processed by software ZoCSoft ImageBoost (for low and strong The target signal-background ratio chip kit) and JAGUARII (for the reference chip kit) are processed, and then the average value is obtained to obtain the result.
  • Detection using low-weak target signal-background ratio chip kit the weak target signal-background ratio is less than 0.1, and the negative and positive results are consistent with the sample used.
  • the positive control was negative when diluted to 50 times.
  • the positive control was still positive when diluted to 500 times.
  • Example 13 Color chip detection
  • samples used in this example are as described above (samples 1-4), and the weak target sample used is the negative sample among them.
  • the detection in this embodiment is respectively a dark chip detection and a light chip detection.
  • the chip kits used were the dark chip kits 26 (black plastic chip-based chip kits), 27 (pearl black spray paint / slide chip-based chip kits), 28 (pearl green spray paint / loaded) prepared in Example 8, respectively.
  • the reference chip kit used was a fluorescently labeled chip kit prepared in accordance with conventional methods using the amino slides in Table 1 as a base.
  • the aforementioned four samples were respectively added to the reactor of the chip reagent box, and the sample volume was 15 ul. After 30 minutes of reaction, it was washed 5 times. The labeling amount was 15 ul, washed 5 times after the reaction, and scanned after drying.
  • the scanner is a confocal laser scanner (Afymetrix GMS 418). The scanning light wavelength is 532nm and the emission light wavelength is 570im. The laser intensity and gain are 60/69.
  • the read signals are processed by the processing software JAGUARII and then averaged. Get the result after value.
  • the detection using dark chip kits showed negative and positive results consistent with the samples used. For the test using the reference chip kit, the positive control 1 was negative when diluted to 100 times; for the test using the dark chip kit, the positive control was still positive when diluted to 200 times.
  • the chip kit used was the light-colored chip kit prepared in Example 8 [chip kit 32 (white spray paint / slide-based chip kit)].
  • the sample and weak target sample used in this example are the same as in Example 13.
  • the chip kits used in this example are the fixed-point colored chip kits prepared in Example 9 [chip kit 32 (black paint fixed-point colored slide glass-based chip kit), 33 (black paint fixed-point colored epoxy glass slides) Chip-based chip kits), 34 (black paint fixed-point stained epoxy-based glass slide chip-based kits) and 35 (black probe chip kits)].
  • the aforementioned four samples were respectively added to the reactor of the chip reagent cartridge, and the sample loading amounts were all 15 ⁇ 1. After 30 minutes of reaction, it was washed 5 times. The labeling amount was 15 ⁇ 1. After the reaction, it was washed 5 times and dried and then scanned.
  • the scanner is a confocal laser scanner (Afymetrix Corporation GMS 418). The scanning light wavelength is 532nm, the emission light wavelength is 570nm, the laser intensity and gain are 60/69, and the read signal is processed by the processing software JAGUARII, and then averaged. After getting the results. The detection using the fixed-point staining chip kit, the negative and positive results are consistent with the samples used.

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Abstract

The invention relates to a qualitative and /or quantitative analysis method of targets in sample, especially in biologic sample by a chip, which includes coloring the chip other than labels during the preparation of the chip kit or/and the forming of the detection signal, in order to the maximization of the chromatism ratio of the background and the target signal. Especially the ratio of the weak signal to the background is less than 0.80, preferred less than 0.50, more preferred less than 0.25, to improve the detection sensitivity. The invention also relates to a film base of a chip, a substrate of a chip, a probe plate of a chip and a chip kit which contain stain and pigment prepared during the preparation of the chip kit.

Description

芯片检测方法及相关装置  Chip detection method and related device
技术领域 Technical field
本发明涉及检测芯片领域， 特别是利用检测芯片对样品中、尤其是 生物样品中的目标物进行定性和 /或定量分析的方法及相关装置。本发明 涉及的相关装置包括芯片片基、 芯片基片、 芯片探针板和芯片试剂盒。 背景技术  The invention relates to the field of detection chips, in particular to a method and a related device for performing qualitative and / or quantitative analysis of a target substance in a sample, especially a biological sample, by using a detection chip. Relevant devices related to the present invention include a chip substrate, a chip substrate, a chip probe board, and a chip kit. Background technique
本发明术语"检测芯片"，又简称 "芯片"，包括但不限于生物芯片（例 如英语中的 "Biochip"、 "Microarray"、 "Bioarray") ， 是指定性和 /或 定量分析中的一种检测装置， 其反应器中微量探针同样品中的目标分子 发生特异反应的结果可以以可寻址的方式进行识别。 本发明的芯片， 为 平面片基芯片， 其反应器中探针在片基上探针区内的分布密度大于 10 点 /cm2、优选方案大于 20点 /cm2、更优选方案大于 40点 /cm2。 芯片包括 生物芯片和非生物芯片， 目前最常用的是生物芯片， 而最常用的生物芯 片是多肽芯片和基因芯片。 芯片的核心是其中的反应器， 反应器的核心 是其中的芯片片基和固定在芯片片基上的探针。 芯片包括微通道芯片和 微阵列芯片， 但众所周知不包括现有的快检试剂条。 本发明的芯片含一 个或多个反应器， 所述反应器包括反应面和任选存在的其它结构， 所述 反应面包括片基面及探针点。 生物芯片的探针， 包括所有可以固定在固 相载体上的具有生物活性的物质， 例如抗原、 抗体、 单链和多链 DNA、 RNA、 核苷酸、 配体、 配基、 多肽、 细胞、 组织成分等生物成分。 芯片 有着广泛的应用范围， 包括基因表达检测、 基因筛选、 药物筛选、 疾病 诊断治疗、 环境监测和治理、 司法鉴定等领域。 The term "detection chip", also referred to as "chip" in the present invention, includes, but is not limited to, a biochip (such as "Biochip", "Microarray", and "Bioarray" in English), which is one of designated and / or quantitative analysis. In the detection device, the result of 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 chip of the present invention is a flat wafer chip, and the distribution density of the probes in the probe region on the wafer in the reactor is greater than 10 points / cm 2 , preferably more than 20 points / cm 2 , and more preferably more than 40 points. / cm 2 . Chips include biochips and non-biochips. Currently, the most commonly used are biochips, and the most commonly used biochips are peptide chips and gene chips. 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 and a microarray chip, but it is well known that it does not include the existing rapid detection reagent strip. The chip of the present invention contains one or more reactors, the reactors include a reaction surface and other structures optionally present, and the reaction surface includes a base surface and a probe point. 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 has a wide range of applications, including gene expression detection, gene screening, drug screening, disease diagnosis and treatment, environmental monitoring and governance, forensic identification and other fields.
现有的芯片检测， 包括发光检测和非发光检测两类。非发光检测的例 子有 SELDI-TOF-MS方法（表面增强的激光解离和激光离子化的飞时质 谱， Surface-Enhanced Laser Desorption/ Ionization-Time of Flight-Mass Spectra) ， 例如美国 Ciphergen公司之包含金属片基的 ProteinChip Array 系统。 发光检测主要包括荧光检测、 化学发光检测和复合光照射检测。 目前使用的检测方法均以信号-背景比最大化为基础的检测方法， 在芯片 的制备及利用所制备的芯片进行检测时， 降低背景噪音、 提高信噪比是 提高检测灵敏度的一个重要方面， 涉及基片制备与检测条件和信号检出 仪器制备与检测条件的专利很多，例如美国专利第 6,573，048、 6,496,307、 5,827,66 K 5,279,93和 6，664,060号。 甚至于降低背景噪音成为目前芯片 和信号检出仪器成本居高不下的原因之一。 The existing chip detection includes two types of light detection and non-light detection. Examples of non-luminous detection 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 the MetalChip-based ProteinChip Array System. Luminescence detection mainly includes fluorescence detection, chemiluminescence detection and composite light irradiation detection. The detection methods currently used are detection methods based on maximizing the signal-to-background ratio. When preparing the chip and using the prepared chip for detection, reducing the background noise and improving the signal-to-noise ratio is An important aspect of improving detection sensitivity is that there are many patents related to substrate preparation and detection conditions and signal detection equipment preparation and detection conditions, such as U.S. Patent Nos. 6,573,048, 6,496,307, 5,827,66 K 5,279,93 and 6,664,060 . Even reducing background noise has become one of the reasons why the cost of chip and signal detection instruments is still high.
荧光检测法中所用的基片基本上是透明玻片（例如氨基化、醛基化、 多聚赖氨酸片基） ， 化学发光检测法中所用片基基本上是透明的玻片、 塑料板或金属膜（例如银薄膜） ， 复合光照射检测法中所用片基基本上 可扩散的聚合物膜（例如 PVDF膜、 尼龙膜、 硝酸纤维膜、 醋酸纤维膜 等）。然而， 目前以可扩散的聚合物膜片基为基础的复合光照射检测法， 其灵敏度不高； 目前的化学发光检测法灵敏度也不很高； 目前的荧光检 测法， 虽然灵敏度比前两者高， 但仍存在玻片片基的背景噪声不低、 活 化片基成本高、 检测仪器昂贵等不足之处， 影响了生物芯片法的大规模 应用。  The substrate used in the fluorescence detection method is basically a transparent glass slide (for example, aminated, aldehyde-based, polylysine film substrate), and the substrate used in the chemiluminescence detection method is basically a transparent glass slide or a plastic plate. Or a metal film (such as a silver film), a polymer film (such as a PVDF film, a nylon film, a nitrocellulose film, an 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 there are still some shortcomings such as the low background noise of the slide substrate, the high cost of the activated substrate, and the expensive detection equipment, which affect the large-scale application of the biochip method.
另一方面， 无论是芯片还是信号检出仪器， 其背景噪音的降低总是 有限的， 有时甚至是充满矛盾的。 例如， 为提高探针的固着量从而提高 芯片的检测灵敏度，有必要提高片基表面活化基团的活性或 /和浓度，然 而同时又增大了片基上除探针点以外的区域上的非特异结合活性， 因而 增大了背景噪音提高的风险。 又例如， 为提高可检出信号的强度， 要求 激光共聚焦扫描仪中有较大功率的激发光源，然而其既增加成本又增大 了光漂白风险。 例如， 为提高可检出信号的强度， 要求 CCD扫描仪中 有较大功率的激发光源， 然而其既增加成本又增大了仪器噪音（例如暗 电流） 。  On the other hand, whether it is a chip or a signal detection instrument, the reduction of background noise is always limited, and sometimes it is even full of contradictions. For example, in order to increase the fixing amount of the probe and thereby increase the detection sensitivity of the chip, it is necessary to increase the activity or / and concentration of the surface-active groups on the substrate, but at the same time increase the area on the substrate other than the probe point. Non-specific binding activity, thus increasing the risk of increased background noise. As another example, in order to increase the intensity of a detectable signal, a laser confocal scanner is required to have a relatively powerful excitation light source. However, it increases both the cost and 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 a CCD scanner, but it increases both the cost and the instrument noise (such as dark current).
总之， 现有的芯片检测方法， 主要在检测仪器和片基表面化学方面 下功夫， 尚存在以下几方面的问题： 1 ) 重点完全放在背景信号最低化 / 信噪比最大化上， 大大地减小了片基选择的自由度， 结果是发明的片基 种类非常有限； 2) 即便是达到背景信号最低化 /信噪比最大化， 也集中 在无色、 特别是透明片基的开发上， 进一步减小了片基选择的自由度， 结果是发明的片基种类非常非常有限； 以及 3 ) 自由度太小， 使得检测 灵敏度的提高受限、或 /和片基成本的降低受限。芯片和检出仪器， 也有 类似的问题。 因而， 发明具有更高检测灵敏度的检测方法， 及更低成本 或 /和更高检测灵敏度的片基、芯片和芯片试剂盒，是芯片发展中迫切需 要解决的问题 发明内容 In short, the existing chip detection methods mainly work on detection instruments and substrate surface chemistry, and there are still problems in the following areas: 1) The focus is completely on minimizing the background signal / maximizing the signal-to-noise ratio, greatly The degree of freedom of film substrate selection is reduced, as a result, the types of film substrates are very limited; 2) Even if the background signal is minimized / the signal-to-noise ratio is maximized, it is focused on the development of colorless, especially transparent film substrates , Further reducing the degree of freedom of the selection of the substrate, as a result, the type of the substrate of the invention is very limited; and 3) the degree of freedom is too small, so that the improvement of the detection sensitivity is limited, or the reduction of the substrate cost is limited. Chips and detection instruments have similar problems. Therefore, the invention of a detection method with higher detection sensitivity, and a substrate, chip and chip kit with lower cost or / and higher detection sensitivity are urgently needed in chip development. Problem to be Solved
根据本发明的一个方面， 其提供一种样品、 特别是生物样品的芯 片检测方法， 其包括芯片试剂盒制备过程和样品检测信号形成过程， 该方法的特征在于包含对芯片进行着色以使背景与目标之间的色差最 大化的步骤， 所述着色包含加入着色剂或含着色剂的着色物， 并且针 对的是芯片反应器中的探针点或 /和包围探针点的区域， 但不包括对探 针或 /和探针捕获物的标记。  According to an aspect of the present invention, a chip detection method for a sample, particularly a biological sample, is provided, which includes a chip kit preparation process and a sample detection signal formation process, and the method is characterized by including coloring the chip to make the background and The step of maximizing the color difference between the targets, the coloring includes adding a colorant or a colorant containing the colorant, and is aimed at the probe point in the chip reactor or / and the area surrounding the probe point, but does not include Labeling of probes and / or probe captures.
根据本发明的再一个方面， 其提供一种样品、 特别是生物样品的 芯片检测方法， 其包括芯片试剂盒制备过程和样品检测信号形成过程， 该方法的特征在于使得检测信号的读取在下述条件下进行： 芯片弱目 标信号 -背景比小于 0.80、 优选小于 0.50、 更优选小于 0.25。  According to another aspect of the present invention, it provides a chip detection method for samples, especially biological samples, which includes a chip kit preparation process and a sample detection signal formation process. The method is characterized in that the detection signal is read in the following Under the conditions: the chip weak target signal-background ratio is less than 0.80, preferably less than 0.50, and more preferably less than 0.25.
根据本发明的另一个方面， 其提供一种平面芯片片基， 其特征在 于包含着色剂或含着色剂的着色物。  According to another aspect of the present invention, it provides a flat chip substrate, which is characterized by containing a colorant or a colorant containing a colorant.
根据本发明的又一个方面， 其提供一种芯片基片， 其包含着色平 面片基及任选存在的隔离结构， 所述着色平面片基含着色剂或含着色 剂的着色物。  According to yet another aspect of the present invention, a chip substrate is provided, which includes a colored planar substrate and an optional isolation structure, wherein the colored planar substrate contains a colorant or a colorant containing a colorant.
根据本发明的再一个方面，其提供一种芯片探针板， 其包含平面片 基、 固定在所述片基上的探针点及任选存在的隔离结构， 该芯片探针板 的特征在于： A.所述平面片基含着色剂或含着色剂的着色物；或 /和 B. 所 述探针点含着色剂或含着色剂的着色物，但不含标记物质。  According to still another aspect of the present invention, a chip probe card is provided, which includes a flat substrate, a probe point fixed on the substrate, and an optional isolation structure. The chip probe card is characterized by : A. the flat sheet base contains a colorant or a colorant containing a colorant; or / and B. the probe dot contains a colorant or a colorant containing a colorant, but does not contain a labeling substance.
根据本发明的再一个方面， 其提供一种芯片试剂盒， 其包括芯片 探针板和任选存在的标记系统， 该芯片试剂盒的特征在于： A. 所述芯 片探针板中的平面片基含着色剂或含着色剂的着色物;或 /和 B. 所述芯 片探针板中的探针点含着色剂或含着色剂的着色物， 但不含标记物质； 或 /和 C. 其还含着色系统， 所述着色系统含着色剂或含着色剂的着色 物。 具体实施方式 According to yet another aspect of the present invention, it provides a chip kit, which includes a chip probe card and an optional labeling system. The chip kit is characterized by: A. a planar sheet in the chip probe card The base contains a colorant or a colorant-containing colorant; or / and B. The probe points in the chip probe card contain a colorant or a colorant-containing colorant, but do not contain a labeling substance; or / and C. It also contains a coloring system containing a colorant or a colorant containing a colorant. detailed description
术语 the term
本发明术语"芯片检测方法"， 是指利用芯片进行检测的方法,包括 芯片试剂盒的制备、 样品捡测信号的形成和检测信号的读取与分析的方 法。 在本发明中， 芯片检测被视作从芯片制备直到检测信号的读取与分 析的全过程。 在现实中， 此一过程可以分解为若干过程并可以在同一个 或多个操作单位中进行。 例如， 芯片试剂盒的制备包括： 在芯片片基制 造者那里制备芯片片基、 在芯片基片制造者那里制备芯片基片、 在芯片 制造者那里点样制备芯片和芯片试剂盒、 等等； 样品检测信号的形成包 括样品处理、 标记、 本发明中的背景增强、 等等。 本发明的芯片可通过 下述之一种仪器检出信号： 共聚焦扫描仪、 CCD扫描仪、 可见光扫描仪 等等。  The term "chip detection method" in the present invention refers to a method for detecting by a chip, including preparation of a chip kit, formation of a sample pick-up signal, and a method of reading and analyzing a detection signal. In the present invention, chip detection is considered as the whole process from chip preparation to reading and analysis of detection signals. In reality, this process can be broken down into several processes and can be performed in the same or more operating units. For example, the preparation of a chip kit includes: preparing a chip substrate at a chip substrate manufacturer, preparing a chip substrate at a chip substrate manufacturer, spotting a chip and a chip kit at a chip manufacturer, and the like; The formation of the sample detection signal includes sample processing, labeling, background enhancement in the present invention, and the like. The chip of the present invention can detect signals by one of the following instruments: a confocal scanner, a CCD scanner, a visible light scanner, and the like.
本发明术语 "检测装置"是指定性和 /或定量分析方法中所使用的装 置， 包括芯片和信号检出装置等等。 · 本发明术语 "反应器"是指探针与目标物发生特异性反应的场所及 与其连通的其它相关结构， 例如开放式多反应器生物芯片中的反应池和 相关的隔离结构和进出液结构等。  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 in the open multi-reactor biochip and the related isolation structure and inlet and outlet liquid structure Wait.
本发明术语 "芯片平面片基" ， 简称片基， 是指芯片中用以固定探 针及其它助剂 （假如有的话） 的平面状固相载体， 其表面化学性质和光 学性质是影响芯片性能及成本的重要因素。 目前的片基选自于改性或未 改性的玻璃、 塑料、 金属。 其可用作本发明的片基、 基片或芯片探针板 的原料片基， 例如， 含下述一种或多种衍生基团的活化玻片： 氨基、 环 氧基、 醛基、 酰肼基 （-CO-NHN¾) 、 氨基脲基 （¾N-NH-CONH-) 、 二乙氨乙基 （DEAE) 、 二乙基一 （2—羟丙基） 氨乙基 （QAE) 、 羧甲 基 （CM) 、 磺酸丙基 （SP) 、 巯乙基吡啶 （MEP) 、 硅氧烷基、 硫醇 基。  The term "chip flat substrate" in the present invention, referred to as a substrate, refers to a planar solid-phase carrier used to fix probes and other auxiliary agents (if any) in a chip. Its surface chemical and optical properties affect the chip. Important factors in performance and cost. The current substrate is selected from modified or unmodified glass, plastic, and metal. It can be used as the base of the substrate, substrate or chip probe board of the present invention, for example, an activated glass containing one or more of the following derivatized groups: amino, epoxy, aldehyde, acyl Hydrazine (-CO-NHN¾), semicarbazide (¾N-NH-CONH-), diethylaminoethyl (DEAE), diethylmono (2-hydroxypropyl) aminoethyl (QAE), carboxymethyl (CM), sulfopropyl (SP), mercaptoethylpyridine (MEP), siloxane group, thiol group.
本发明术语 "芯片基片" ， 简称基片， 是指以片基为基础并任选地 结合其它结构 （例如隔离结构） 的、 用以在固定探针后形成芯片探针板 的产品。 基片上可以有一个或多个片基池。 单片基池基片上通常没有隔 离结构， 此时基片即是片基， 例如市售的氨基玻片。 多片基池基片上有 隔离结构， 此时基片包括片基和隔离结构。 片基池在固定上探针后形成 反应器， 多片基池片基形成多反应器芯片。 本发明术语 "片基池"是指 片基与其隔离结构形成的结构。 The term "chip substrate" in the present invention, referred to as a substrate for short, refers to a product based on a substrate and optionally combined with other structures (such as an isolation structure) for forming a chip probe board 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 pool is formed after the probe is fixed In the reactor, a plurality of substrates form a multi-reactor chip. The term “film base pool” in the present invention refers to a structure formed by a film base and its isolation structure.
本发明术语 "芯片探针板"是指通过将探针固定在芯片基片的片基 上形成的制品。 本发明术语 "探针"是指固定于固相载体上用以识别样 品中的目标物的物质， 如抗原、 抗体、 核酸等。  The term "chip probe board" in the present invention refers to an article formed by fixing a probe to a substrate of a chip substrate. 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.
本发明术语 "芯片试剂盒"是指含有芯片探针板的试剂盒， 其包括 芯片探针板和任选存在的标记系统，以及任选存在的本发明的着色系统。 本发明术语 "芯片试剂盒着色系统"， 简称着色系统，是指用以在样品检 测信号形成过程时对所述芯片背景或 /和探针点着色的系统。  The term "chip kit" in the present invention refers to a kit containing a chip probe card, which includes a chip probe card and an optional labeling system, and an optional coloring system of the present invention. The term "chip kit coloring system" in the present invention, referred to as a coloring system for short, refers to a system for coloring the chip background or / and probe points during the formation of a sample detection signal.
本发明术语 "背景信号增强"是指使所述背景检出信号值提高,例如 通过在片基内、片基面或 /和片基背面引入发光物的着色后背景检出信号 值的提高。  The term "background signal enhancement" in the present invention means that the background detection signal value is increased, for example, the color value of the background detection signal is increased by introducing a coloring substance into the substrate, the substrate surface, or / and the substrate back surface.
本发明术语 "弱目标信号降低"是指使所述弱目标检出信号降低。 本发明术语 "包被"是指将某种物质以分子形态固定在固相载体， 例如通过离子吸附、 亲和吸附等作用将染料分子固定在玻片上的包被 点、 包被面等等。  The term "reduced weak target signal" in the present invention means that the weak target detection signal is decreased. The term "coating" in the present invention refers to a substance that is immobilized on a solid support in a molecular form, for example, a coating point, a coating surface, and the like that fix a dye molecule on a glass slide by ion adsorption, affinity adsorption, and the like.
本发明术语 "涂层"是指由涂料涂覆在固相材料上形成的厚度小于 The term "coating" in the present invention means that the thickness of the solid phase material formed by coating is less than
1 mm的干膜。 1 mm dry film.
本发明术语 "薄膜"是指厚度小于 0.3 mm的无孔或有孔平面材料。 本发明术语 "薄片"是指厚度大于或等于 0.3 mm的无孔或有孔平 面材料。  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.
本发明术语 "标记物质"是指用以标记探针或探针捕获物从而获得 阳性结果的物质， 例如芯片荧光检测中的常用标记物中含的标记物质荧 光素。  The term "labeling substance" in the present invention refers to a substance used to label a probe or a probe capture substance so as to obtain a positive result, for example, a fluorescent substance contained in a commonly used label in chip fluorescence detection.
本发明术语 "配基" （Ligand) 是指用以通过亲和作用捕获其配体 (Ligate) 的物质， 例如下述之一种或多种物质： 抗原、 抗体、 配体、 配基、 多肽和单链或多链 DNA、 RNA、 核苷酸。  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.
本发明术语 "着色"，相当于英语的 colouration,是指区别于标记 的、 使芯片背景 （例如片基） 或 /和探针点 (但不包括探针捕获物)具有确 定的光线吸收、 反射、 折射等性质从而在可见光下或在选定波长的光线 下形成优化的色调或 /和明度或 /和饱和度的过程,所述芯片仅限于用以进 行样品、 特别是生物样品检测的芯片， 所述探针点上的着色不使用标记 物质。需要强调的是，所述着色完全区别于目前芯片检测中使用的标记, 后者以获得阳性结果为目的、 以在探针点上引入标记物质为手段、 以增 大探针捕获物的检测信号为特征 （例如美国专利申请 Al'20030032040， 中国专利申请 CN A 1443854中通过对探针或探针捕获物进行标记以检 验芯片上探针固定效果） 。 本发明所述对探针点的着色， 目的不是标记 而是扩大阴性目标与阳性目标的差异， 所用着色剂不是检测所用标记物 质。 本发明所述对芯片背景的着色， 目的不是标记而是扩大阴性目标与 阳性目标的差异，。所用着色剂一般不是检测所用标记物质 （例如发光 剂） ， 而当所用着色剂与标记物质相同时其仅被用作芯片背景的信号加 强。 The term "coloring" in the present invention is equivalent to English coloration, which means that the background of the chip (such as the substrate) or / and the probe point (but not including the probe capture object) has a defined light absorption and reflection, which is different from the mark. , Refraction, and other properties to form an optimized hue or / and brightness or / and saturation in visible light or under a selected wavelength of light, the chip is limited to For chips for detecting samples, especially biological samples, the coloring on the probe points does not use a labeling substance. It needs to be emphasized that the coloring is completely different from the markers currently used in chip detection, which aims to obtain a positive result by using a labeling substance at the probe point as a means to increase the detection signal of the probe capture (For example, in the US patent application Al'20030032040 and the Chinese patent application CN A 1443854, the probe or the probe capture is marked to verify the effect of the probe fixing on the chip). The purpose of coloring the probe points in the present invention is not to mark but to enlarge the difference between the negative target and the positive target, and the colorant used is not a labeling substance used for detection. The purpose of coloring the chip background according to the present invention is not to mark but to enlarge the difference between the negative target and the positive target. The colorant used is generally not a labeling substance (such as a luminescent agent) for detection, and when the colorant is the same as the labeling substance, it is only enhanced by the signal used as the background of the chip.
本发明术语 "着色片基"是指被着色的片基。 本发明术语 "着色基 片"是指含着色片基的基片。 本发明术语 "着色芯片探针板"是指着色 的芯片探针板，包括含着色片基或 /和着色探针点的芯片探针板。  The term "colored film base" in the present invention refers to a film base that is colored. The term "colored substrate" in the present invention refers to a substrate containing a colored substrate. The term "colored chip probe board" in the present invention refers to a colored chip probe board, and includes a chip probe board containing a colored substrate or / and colored probe points.
本发明术语 "着色剂"是指可对制备物进行着色的有色物质。 着色 剂的一个例子是颜料。 本发明术语 "着色物"是指含着色剂可对制备物 进行着色的物质。着色物的例子有： 含颜料的涂料、涂层、 薄膜、 薄片、 包被、 及颜料与有吸咐活性的物质的复合物 （例如罗丹明化白蛋白） 。  The term "colorant" in the present invention refers to a colored substance that can color a preparation. An example of a colorant is a pigment. The term "colored matter" in the present invention means a substance containing a colorant that can color the preparation. Examples of coloring matter are: pigment-containing coatings, coatings, films, flakes, coatings, and complexes of pigments and substances with adsorption properties (such as rhodamine albumin).
本发明术语 "有色颜料"是指对着色对象无亲和力、 主要靠树脂、 胶粘剂等其它成膜材料与着色对象相结合而进行着色的物质， 例如炭 黑、 云母钛珠光有色颜料、 偶氮有色颜料、 酞菁有色颜料、 荧光有色颜 料（荧光物质）。本发明中的有色颜料按涂料行业习惯不包括体质颜料， 体质颜料通常指体积填充剂或不具有着色功能的物质， 体质颜料颜色例 子可以是银箔片基的金属银本色、 薄膜片基的膜本色等等。  The term "colored pigment" in the present invention refers to a substance that has no affinity for coloring objects and is mainly colored by combining other film-forming materials such as resins and adhesives with coloring objects, such as carbon black, mica titanium pearlescent colored pigments, and azo colored pigments. , Phthalocyanine colored pigments, fluorescent colored pigments (fluorescent substances). The colored pigments in the present invention do not include extender pigments according to the custom of the coatings 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.
本发明术语 "染料"是指对着色对象有亲和力、 可对着色对象进行 着色的物质。  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.
本发明术语 "涂料"是指施于片基基质上后可获得特定功能的、 厚 度小于 1 mm的干膜的材料；而 "有色涂料" 是指含有有色颜料的涂料， 例如各种色漆和各种油墨。  In the present invention, the term "coating material" refers to a material having a thickness of less than 1 mm, which can obtain a specific function after being applied to a substrate. The "coloring coating material" refers to a coating material containing colored pigments, such as various colored paints and Various inks.
本发明术语 "目标"是指信号检出时芯片反应器中探针点处与检出 信号有关的全部物质的整体， 例如包括探针、 被探针捕获的目标物（假 如有） 、 标记物 （假如有） ， 探针点上除探针外还可以有探针载体 （例 如纳米载体）和本发明的染料、 有色颜料及着色物、 等等。 本发明术语 "弱目标"是指信号检出时芯片反应器中探针点处加入相应阴性样品或 空白对照物或极限弱阳性样品反应后与检出信号有关的全部物质的整 体;而 "弱目标样品" 是指这些阴性样品或空白对照物或极限弱阳性样 品。 The term "target" in the present invention refers to the entirety of all substances related to the detected signal at the probe point in the chip reactor when the signal is detected, for example, including the probe, the target captured by the probe (false If there is), a label (if any), in addition to the probe, a probe carrier (such as a nanocarrier) and a dye, a colored pigment, a colorant, and the like of the present invention may be provided on the probe point. The term “weak target” in the present invention refers to the entirety of all substances related to the detected signal after the corresponding negative sample or blank control or extreme weak positive sample is added to the probe point in the chip reactor when the signal is detected; "Target samples" refers to these negative or blank controls or extreme weak positive samples.
本发明术语 "背景"是指信号检出时芯片反应器中包围目标的检出 区域内与检出信号有关的全部物质的整体， 例如除包括片基、 及片基上 固定的封密物、 标记物、 等等外， 还可能包括片基后衬（特别在透明片 基的情况下） 。 片基后衬在芯片被读取信号时可以下述之一种或多种形 态存在于芯片背面： 依附于透明片基、 作为独立部件、 依附于读取信号 仪的芯片托板上。  The term "background" in the present invention refers to the entirety of all substances related to the detected signal in the detection area surrounding the target in the chip reactor when the signal is detected, for example, except for including the substrate, the sealed object fixed on the substrate, In addition to markers, etc., it may also include a base backing (especially in the case of a transparent base). The backing of the substrate can exist on the back of the chip in one or more of the following forms when the chip is read signals: attached to the transparent substrate, as a separate component, attached to the chip holder of the signal reader.
本发明术语 "信号-背景比"是指目标的信号强度 a与背景的信号强 度 b之比 c=a/b。本发明术语 "弱目标信号-背景比"是指弱目标的信号 强度 al与背景的信号强度 b之比 c=al/b。  The term "signal-background ratio" in the present invention refers to the ratio c = a / b of the target signal strength a to the background signal strength b. The term "weak target signal-background ratio" in the present invention refers to the ratio c = al / b of the signal strength al of the weak target to the signal strength b of the background.
本发明术语 "发光剂"是指能够发射检测信号的物质。所述发光剂 选自于包括荧光物质、化学发光物质和电化学发光物质在内的发光颜料。  The term "luminescent agent" in the present invention refers to a substance capable of emitting a detection signal. The luminescent agent is selected from a luminescent pigment including a fluorescent substance, a chemiluminescent substance, and an electroluminescent substance.
本发明以提供一种更高灵敏度的芯片检测方法、或 /和提供更多供选 择的基片 /芯片 /芯片试剂盒为主要目的。 实际上， 提供更多供选择的基 片 /芯片 /芯片试剂盒的前提之一也是需使它们有足够高的检测灵敏度。 本发明的方法， 致力于使目标与背景有最大化的信号反差。 实际上， 目 标与背景之间的信号反差是可探测性的一个表征。 例如， 对红外隐身技 术而言， Maclean等人用反差比幅射 C的大小来表示热像仪的可探测性： C二 Eo-Eb。 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。 因而， 本发明的研究选择了 "显 身"作为技术基础， 本发明的技术可称为显身技术， 本发明的检测方法 可称为显身技术检测方法。  The main purpose of the present invention is to provide a chip detection method with higher sensitivity, or / and to provide more alternative substrates / chips / chip kits. In fact, one of the prerequisites for providing more substrates / chips / chip kits is to make them have high enough detection sensitivity. The method of the invention aims to maximize the signal contrast between the target and the background. In fact, 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 indicate the detectability of the thermal imager: C2 Eo-Eb. 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 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 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. Therefore, the research of the present invention has selected "obviousness" as the technical basis. The technology of the present invention can be referred to as the manifestation technology, and the detection method of the present invention can be referred to as the manifestation technology detection method.
本发明的第一个方面， 是通过对芯片背景或 /和探针点着色来实现 信号反差最大化， 并从而实现本发明的目标。 The first aspect of the present invention is achieved by coloring the chip background or / and probe points. The signal contrast is maximized and thus achieves the objectives of the present invention.
具体而言， 本发明的第一个方面是提供一种样品、特别是生物样品 的芯片检测方法，其包括芯片试剂盒制备过程和样品检测信号形成过程， 该方法的特征在于包含对芯片进行着色以使背景与目标之间的色差最大 化的步骤， 所述着色包含加入着色剂或含着色剂的着色物， 并且针对的 是芯片反应器中的探针点或 /和包围探针点的区域， 但不包括对探针或 / 和探针捕获物的标记。本发明中,所述"着色" ,相当于英语的 coloumtion, 是指区别于标记的、 使芯片背景 （例如片基） 或 /和探针点 (但不包括探 针捕获物)具有确定的光线吸收、 反射、折射等性质从而在可见光下或在 选定波长的光线下形成优化的色调或 /和明度或 /和饱和度的过程,所述芯 片仅限于用以进行样品、 特别是生物样品检测的芯片， 所述探针点上的 着色不使用标记物质。 需要强调的是， 所述着色完全区别于目前芯片检 测中使用的标记,后者以获得阳性结果为目的、 以在探针点上引入标记物 质为手段 (本发明所述对探针点的着色，所用着色剂不是检测所用标记物 质)、 以增大探针捕获物的检测信号为特征 （例如美国专利申请 A1 20030032040, 中国专利申请 CN A 1443854中通过对探针或探针捕获物 进行标记以检验芯片上探针固定效果） 。 在本发明中， 所述着色的目的 是在背景与目标之间形成高色差比、 优选形成最大化色差比。 所述色差 比是指背景与目标之间在色调、 明度或和饱和度的差别、 包括背景与目 标之间的对全部波长或部分波长的信号光线的吸收率或反射率之差的绝 对值不低于 50%、优选不低于 70%。于是， 本发明的检测方法也区别于 所谓的液体芯片或悬浮微粒芯片检测方法 （美国专利第 6,524,793、 6,649,414和 6,632,526号） 。 而且， 所谓的液体芯片或悬浮微粒芯片也 不是本发明中定义的芯片 （平面片基芯片） 。  Specifically, a first aspect of the present invention is to provide a chip detection method for samples, especially biological samples, which includes a chip kit preparation process and a sample detection signal formation process, and the method is characterized by including coloring a chip. The step of maximizing the color difference between the background and the target, the coloring includes adding a colorant or a colorant-containing colorant, and is directed to a probe point in a chip reactor or / and an area surrounding the probe point , But does not include labeling of probes and / or probe captures. In the present invention, the "coloring" is equivalent to the English coloumtion, which means that the background of the chip (such as the substrate) or / and the probe point (but not including the probe capture object) have a certain light, which is different from the mark. Absorption, reflection, refraction and other properties to form an optimized hue or / and brightness or / and saturation in visible light or under a selected wavelength of light.The chip is limited to the detection of samples, especially biological samples. The chip is colored without using a labeling substance on the probe spot. It needs to be emphasized that the coloring is completely different from the current markers used in chip detection, which aims to obtain a positive result and uses a labeling substance as a means (the coloring of the probe points according to the present invention). , The colorant used is not a labeling substance used for detection), and is characterized by increasing the detection signal of the probe capture object (for example, in US patent application A1 20030032040, Chinese patent application CN A 1443854, Check the effect of the probe on the chip). In the present invention, the purpose of the coloring is to form a high color difference ratio between the background and the target, preferably to maximize the color difference ratio. The chromatic aberration ratio refers to the difference between the background and the target in the difference in hue, lightness, or saturation, including the absolute value of the difference between the background and the target with respect to the absorptance or reflectance of the signal light of all or part of the wavelength. Less than 50%, preferably not less than 70%. Therefore, the detection method of the present invention is also different from the so-called liquid chip or suspended particle chip detection methods (US Pat. Nos. 6,524,793, 6,649,414, and 6,632,526). Moreover, the so-called liquid chip or suspended particle chip is not a chip (flat chip chip) defined in the present invention.
目前的芯片检测方法中， 均未有对芯片背景进行着色。 目前制作的 芯片均为未着色芯片， 其含透明或未着色的片基， 在芯片应用时有透明 或未着色的背景。未着色片基仅保留其基质本色（例如体质颜料的颜色） 而未加入染料、 有色颜料， 其例子可以是银箔片基的金属银本色、 薄膜 片基的膜本色等等。通过本发明的实施例我们发明了以背景着色或 /和探 针点着色提高目标-背景色差比的方法， 这是本发明的一个重点。 背景与 目标之间对信号光线的选择性吸收率之差（例如吸收率大于 95 %的黑色 背景与含有荧光物质的发光目标 （本发明中设定为吸收率为 0) 之间的 非彩色色差） 的最大化、 或背景与目标之间对信号光线的选择性反射率 之差（例如信号光线为白光时， 对所有波长的可见光反射率大于 80%的 白色背景与含有结晶紫的对紫色以外的可见光反射率小于 10%的目标 之间的彩色色差） 的最大化是有利于目标显身从而提高灵敏度的。 本发 明的实施例将说明这种处理及其对实现本发明目标的意义。 None of the current chip detection methods has colored the chip background. The currently produced chips are all uncolored chips, which contain a transparent or uncolored substrate, and have a transparent or uncolored background when the chip is applied. The uncolored film base only retains its base color (such as the color of extender pigments) without adding dyes and colored pigments. Examples thereof may be the metallic silver color of silver foil base, the film color of thin film base, and the like. Through the embodiments of the present invention, we have invented a method for improving the target-background color difference ratio by using background coloring and / or probe point coloring, which is an important point of the present invention. The difference between the selective absorption of signal light between the background and the target (e.g. black with an absorption rate greater than 95% Maximize the achromatic color difference between the background and the luminescent target containing the fluorescent substance (the absorption rate is set to 0 in the present invention), or the difference between the selective reflectance of the signal light between the background and the target (for example, the signal When the light is white, the maximum color difference between a white background with a visible light reflectance of greater than 80% for all wavelengths and a target with a visible light reflectance other than violet that contains crystal violet is less than 10%. Thus improving sensitivity. Embodiments of the present invention will illustrate this process and its significance for achieving the objectives of the present invention.
所述着色剂或着色物可引入于芯片的下述之一个或多个部位：片基 正面、 片基背面、 和片基内。 例如，将染料或 /和有色颜料或 /和含有色颜 料的涂料加入基质，或在片基中加入含染料或 /和有色颜料或 /和含有色颜 料的涂料的下述一种或多种结构： 包被、 涂层、 薄膜和薄片。  The colorant or colorant may be incorporated in one or more of the following portions of the chip: the substrate front, the substrate back, and the substrate. For example, one or more of the following structures of a dye or / and a colored pigment or / and a pigment-containing coating is added to a substrate, or a base containing a dye or / and a pigment or / and a pigment is added : Coatings, coatings, films and sheets.
在根据本发明的检测方法中， 所述背景与目标之间的色差最大化包 括下述之一种选择： A. 当标记物质为发光剂时， 使所述芯片背景或 /和 探针点在白光下为深色、优选方案为黑色； B. 当标记物质为在白光下深 色、优选黑色的非发光剂时， 使所述芯片背景或 /和探针点在白光下为浅 色、优选方案为白色； C. 当标记物质为在白光下浅色、优选白色的非发 光剂时， 使所述芯片背景或 /和探针点在白光下为深色、 优选为黑色。 本 发明中， 所述深色的例子有： 在白光下呈红色、 橙色、 黄色、 绿色、 蓝 色、 青色、 紫色、 或黑色； 所述浅色的例子有： 在白光下呈浅红色、 浅 橙色、 浅黄色、 浅绿色、 浅蓝色、 浅青色、 浅紫色、 或白色。 在本发明 的一个实施方案中， 着色是在载玻片正面分别涂黑色涂料和白色涂料， 然后分别用于荧光标记检测和金-银标记检测。令人'惊奇的是， 应用如此 低廉、 简单的方法制备的芯片， 竞也可获得灵敏度足够高的检测结果。 我们在研究中发现， 这些色漆涂层中用以固定探针的组分对于固定探针 从而提高灵敏度是重要， 但色漆涂层中用以着色的组分对提高灵敏度也 是重要的。 例如含相同固定探针组分和不同着色剂的不同色漆， 其灵敏 度悬殊很大。  In the detection method according to the present invention, maximizing the color difference between the background and the target includes one of the following options: A. When the labeling substance is a luminescent agent, make the chip background or / and the probe point Dark under white light, preferably black; B. When the labeling substance is a dark, preferably black non-luminous agent under white light, make the chip background or / and probe point light under white light, preferably The scheme is white; C. When the labeling substance is a light-colored, preferably white, non-luminous agent under white light, the chip background or / and the probe point is made dark, preferably black, under white light. In the present invention, examples of the dark color include: red, orange, yellow, green, blue, cyan, purple, or black under white light; examples of the light color include: light red, light under white light Orange, light yellow, light green, light blue, light cyan, light purple, or white. In one embodiment of the present invention, the coloring is to apply black paint and white paint to the front surface of the slide glass, respectively, and then use them for fluorescent label detection and gold-silver label detection, respectively. Surprisingly, with such a cheap and simple method, a chip with a sufficiently high sensitivity can also be obtained. We found in research that the components used to fix the probes in these paints are important for fixing the probes to improve the sensitivity, but the components used to paint in the paints are also important to improve the sensitivity. For example, different lacquers containing the same fixed probe components and different colorants have very different sensitivities.
在根据本发明的检测方法中， 所述着色剂选自下述一种或多种物 质： 染料、 颜料、 消光剂。 染料、 颜料、 消光剂在塑料、 涂料等材料着 色中是一个公知的概念， 具有确定的内容。 所述颜料的例子有： 包括炭 黑、金属盐在内的黑颜料， 包括二氧化钛在内的白颜料， 和包括黄颜料、 红颜料、 蓝颜料、 绿颜料、 金属颜料在内的彩色颜料， 等等； 所述染料 的例子有： 包括氨基黑、 考马斯亮蓝、 结晶紫、 丽春红、 印花涂料色浆In the detection method according to the present invention, the colorant is selected from one or more of the following: a dye, a pigment, and a matting agent. Dyes, pigments, and matting agents are a well-known concept in the coloring of materials such as plastics and coatings, and have certain contents. Examples of the pigments are: black pigments including carbon black, metal salts, white pigments including titanium dioxide, and color pigments including yellow pigment, red pigment, blue pigment, green pigment, metal pigment, etc. Etc; the dye Examples are: Including amino black, Coomassie brilliant blue, crystal violet, Ponceau red, printing paint color paste
(7701 FBRN BLACK FBR > 6201大红 FGG SCARLET FGG、 6101 F7G BRILLIANT YELLOW F7G) 、 水性汽巴染料 （水性蓝、 水性绿、 水性 白） 在内的黑色、 紫色、 绿色、 蓝色、 靛色水性染料、 水油两性染料， 等等。 通常有色涂料中的消光剂是指能降低涂层表面光泽度的材料。 降 低涂层表面光泽度可以通过增加微观粗糙度来实现。 在本发明的一个实 施例中， 含消光剂的涂料在载玻片表面形成表面粗糙度 Ra在 0.02— 3.0 μ m之间、 优选方案在 0.25— 3.0 μ ιη之间的涂层。 (7701 FBRN BLACK FBR> 6201 Scarlet FGG SCARLET FGG, 6101 F7G BRILLIANT YELLOW F7G), water-based Ciba dyes (water-based blue, water-based green, water-based white) including black, purple, green, blue, indigo water-based dyes, Water and oil amphoteric dyes, etc. Matting agents in pigmented coatings generally refer to materials that can reduce the gloss of the coating surface. Reducing the surface gloss of the coating can be achieved by increasing the micro-roughness. In one embodiment of the present invention, the matting agent-containing coating forms a coating having a surface roughness Ra between 0.02 and 3.0 μm, and preferably between 0.25 and 3.0 μm, on the surface of the glass slide.
在根据本发明的检测方法中， 所述着色物选自有色涂料。所述有色 涂料为含所述着色剂的涂料。 所述有色涂料是一个公知的概念， 具有确 定的内容。 所述有色涂料包括黑色、 白色、 各种彩色的油漆或 /和油墨。 所述彩色包括红、 黄、 绿、 蓝、 青、 紫等色。 在本发明中， 油墨与油漆 都具有可对片基着色的性质。所述有色涂料可以覆盖在片基正面、反面、 或正反两面。 在本发明中， 片基正面是指片基上用以固定探针的一面。  In the detection method according to the present invention, the coloring matter is selected from a colored paint. The colored paint is a paint containing the colorant. The colored paint is a well-known concept with a certain content. The colored paint includes black, white, various colored paints and / or inks. The colors include red, yellow, green, blue, cyan, and purple colors. In the present invention, both inks and paints have the property of coloring the substrate. The colored paint can cover the front, back, or both sides of the substrate. In the present invention, the front side of the substrate refers to the side on the substrate for fixing the probe.
所述有色涂料可包含所述着色剂和可结合探针的物质，该可结合探 针的物质的例子包括下述一种或多种有机物及其衍生物： 硝化纤维素、 聚苯乙烯、 聚丙烯酸脂、 聚砜、 聚醚砜、 聚氯乙烯、 氨基树脂、 聚多糖、 聚氨基酸。  The colored paint may include the colorant and a probe-binding substance, and examples of the probe-binding substance include one or more of the following organic substances and derivatives thereof: nitrocellulose, polystyrene, polystyrene Acrylate, polysulfone, polyethersulfone, polyvinyl chloride, amino resin, polysaccharide, polyamino acid.
所述有色涂料还可含消光剂。  The colored paint may further contain a matting agent.
所述有色涂料优选选自于色漆，例如下述一种或多种色漆：珍珠黑、 魔力黑、 珍珠白、 珍珠蓝、 亚光黑、 枣红、 猩红、 中蓝。  The colored paint is preferably selected from colored paints, for example, one or more of the following colored paints: pearl black, magic black, pearl white, pearl blue, matte black, bayonet, scarlet, medium blue.
在根据本发明的检测方法中，所述着色包括将探针与所述有色涂料 混合后再点样至片基上制作芯片。所述片基选自于改性或未改性的玻璃、 塑料、 金属、 优选为玻璃。  In the detection method according to the present invention, the coloring includes mixing a probe with the colored paint and then spotting the chip onto a substrate to make a chip. The substrate is selected from modified or unmodified glass, plastic, metal, preferably glass.
本发明的第二个方面， 也是通过对芯片背景或 /和探针点着色， 但 同时要求降低芯片弱目标信号 -背景比来实现信号反差最大化， 并从而 实现本发明的目标。  In the second aspect of the present invention, the chip background or / and probe points are also colored, but at the same time, it is required to reduce the chip's weak target signal-background ratio to maximize the signal contrast and thereby achieve the objective of the present invention.
具体而言， 本发明的第二个方面是提供一种样品、特别是生物样品 的芯片检测方法，其包括芯片试剂盒制备过程和样品检测信号形成过程， 该方法的特征在于使得检测信号的读取在下述条件下进行： 芯片弱目标 信号 -背景比小于 0.80、 优选小于 0.50、 更优选小于 0.25。 通过提高信号- 背景比来提高检测灵敏度， 是目前芯片领域中的研究者的共识 （例如， 欧洲专利 EP A1 1279960， 美国专利 US A1 20030032040， 中国专利 CN A 1443854) 。 通过降低弱目标信号-背景比、 而不是提高信号 -背景比来提 高检测灵敏度， 是本发明的一个重点。 我们通过本发明的实施例惊奇地 发现， 降低信号-背景比（例如弱目标信号 -背景比小于 0.25)甚至可以提 高检测灵敏度。 Specifically, a second aspect of the present invention is to provide a chip detection method for a sample, particularly a biological sample, which includes a chip kit preparation process and a sample detection signal formation process. The method is characterized in that the detection signal is read. The following conditions are adopted: The chip weak target signal-background ratio is less than 0.80, preferably less than 0.50, and more preferably less than 0.25. By raising the signal- Background ratio to improve detection sensitivity is the consensus of researchers in the chip field (for example, European patent EP A1 1279960, US patent US A1 20030032040, Chinese patent CN A 1443854). It is an important point of the present invention to improve the detection sensitivity by reducing the weak target signal-background ratio instead of increasing the signal-background ratio. We have surprisingly discovered through embodiments of the present invention that reducing the signal-to-background ratio (eg, weak target signal-to-background ratio is less than 0.25) can even improve detection sensitivity.
所述弱目标信号 -背景比是通过对芯片进行着色获得的。 所述着色 包括使背景信号增强或 /和弱目标信号降低。所述信号增强可通过下述一 种或多种方法实现： 在基质中加入发光颜料 （例如荧光素） ， 在基质正 面或 /和背面加入含发光颜料或反光的包被、涂层（例如覆涂在固定探针 的片基表面或 /和背面上的发光涂层） 、 薄膜 （例如覆盖在片基表面或 / 和背面上的发光薄膜、 反光薄膜） 、 含或不含检测目标孔的薄片 （例如 覆盖在片基表面或 /和背面的发光薄片） ， 以及在基质表面衍生可直接或 间接结合发光有色颜料的活性基团（例如在基质上包被蛋白质 A然后在 检测反应前或后结合罗丹明标记的 IgG-实施例片基） 。 所述含发光颜料 或反光的包被的形成可通过包括下述一种或多种中间体作介质而形成： 蛋白质 A、 蛋白质 G、 生物素、 亲和素、 抗原、 抗体、 抗抗体、 多肽、 DNA、 等等， （例如， 抗体可捕获结合有罗丹明的抗抗体从而间接地捕 获罗丹明， 等等） 。 所述弱目标信号降低可通过在拟固定探针点加入可 降低信号光线发射或 /和反射、 或可提高信号光线吸收的染料、 有色颜料 或 /和涂料， 或 /和在基质正面或 /和背面加入含这些染料、 有色颜料或 /和 涂料的包被、 涂层 （例如覆涂在片基表面或 /和背面上的减光涂层） 、 薄 膜（例如覆盖在固定探针的片基表面或 /和背面上的减光薄膜）、或 /和薄 片（例如覆盖在片基表面或 /和背面的含有检测物着色剂的减光薄片）来 实现。  The weak target signal-background ratio is obtained by coloring the chip. The coloring includes increasing the background signal and / or reducing the weak target signal. The signal enhancement can be achieved by one or more of the following methods: adding a luminescent pigment (such as fluorescein) to the substrate, and adding a coating or coating (such as coating) containing a luminescent pigment or light reflection on the front or / and back of the substrate Luminescent coating on the surface or / and back of the substrate on which the probe is fixed), film (such as luminescent film or reflective film covering the surface or / and the back of the substrate), with or without the detection target hole (Such as a luminous sheet covering the surface or / and the back of the substrate), and derivatization of active groups that can directly or indirectly bind luminescent colored pigments on the surface of the substrate (such as coating protein A on the substrate and then binding before or after the detection reaction) Rhodamine-labeled IgG-example tablet). The luminescent pigment or reflective coating may be formed by including one or more of the following intermediates as a medium: protein A, protein G, biotin, avidin, antigen, antibody, anti-antibody, polypeptide , DNA, etc. (for example, antibodies can capture rhodamine-bound antibodies to indirectly capture rhodamine, etc.). The weak target signal can be reduced by adding dyes, colored pigments or / and coatings that can reduce the signal light emission or / and reflection, or can increase the signal light absorption at the quasi-fixed probe point, or / and on the front of the substrate or / and Add coatings, coatings (such as a matte coating on the surface of the substrate or / and the back surface) containing these dyes, colored pigments, and / or coatings, films (such as the surface of the substrate on which the probe is fixed) Or / and a light-reducing film on the back surface), or / and a sheet (for example, a light-reducing film containing a test substance colorant covering the surface of the substrate or / and the back surface).
所述着色是通过选自以下组中的一种或多种方法而实施的： A. 在 芯片试剂盒的制备过程中对芯片背景进行加强信号的着色； B. 在样品检 测信号形成过程中对芯片背景进行加强信号的着色； C. 在芯片试剂盒的 制备过程中对探针点进行降低弱目标信号的着色； D. 在样品检测信号形 成过程中对探针点进行降低弱目标信号的着色。 与目前大家致力于降低 背景信号 （甚至于被公认为噪音） 的技术路线大相庭径， 我们通过本发 明的实施例惊奇地发现， 通过在芯片试剂盒的制备、或 /和样品检测信号 的形成时在芯片反应器背景中信号增大所述背景信号 （例如引入荧光物 质） ， 用共聚焦扫描仪进行荧光物质信号扫描时， 所读取的信号经分析 计算后，检测灵敏度不是降低了、而是提高了。 目前除了经典标记以外， 对探针点没有本发明的着色处理。 本发明的实施例将说明这种处理及其 对实现本发明目标的意义。 The coloring is implemented by one or more methods selected from the group consisting of: A. coloring the chip background to enhance the signal during the preparation of the chip kit; B. coloring the sample during the formation of the sample detection signal The background of the chip is colored to enhance the signal; C. The probe point is colored to reduce the weak target signal during the preparation of the chip kit; D. The probe point is colored to reduce the weak target signal during the formation of the sample detection signal . This is very different from the current technical route that everyone is working on reducing background signals (even recognized as noise). The illustrated examples surprisingly found that by increasing the background signal (eg, introducing a fluorescent substance) in the background of the chip reactor during the preparation of the chip kit or / and the formation of a sample detection signal, a confocal scanner was used. When the fluorescent substance signal is scanned, after the read signal is analyzed and calculated, the detection sensitivity is not reduced but increased. Except for the classical labeling, currently, there is no coloring treatment of the present invention for the probe points. Embodiments of the present invention will illustrate this process and its significance for achieving the objectives of the present invention.
具体而言，所述加强信号的着色包括在所述背景中引入发光剂或含 发光剂的发光物。 所述发光剂选自于下述一种或多种可发射信号光线的 物质： 包括荧光物质在内的激发发光物质和包括化学发光物质和电化学 发光物质在内的自主发光物质。其中，激发发光物质的例子包括罗丹明、 Specifically, the coloring of the enhanced signal includes introducing a luminescent agent or a luminescent agent-containing luminescent substance into the background. The luminescent agent is selected from one or more of the following substances that can emit signal light: an excited luminescent substance including a fluorescent substance and an autonomous luminescent substance including a chemiluminescent substance and an electrochemical luminescent substance. Among them, examples of excited luminescent substances include rhodamine,
CY3、 CY5、 Alexa、 海藻蛋白、 稀土化合物类荧光物质。 实际上， 这些 发光剂均为发光颜料， 在芯片中通常是作为标记物质使用的。 只是在这 里， 这些标记物质不仅用来标记检测反应， 而且被用来对背景着色增大 所述背景信号。 当检测信号是标记物质主动或被动发射光线形成的时， 可以用标记物质作为发光剂。 目前的芯片检测方法中， 一个重要方面是 尽量降低所述背景中的发光剂。 本发明的方法反其道而行之， 在若干实 施例中在所述背景中 （例如芯片反应器反应面） 引入标记物质 （例如罗 丹明）而提高了灵敏度。 CY3, CY5, Alexa, seaweed protein, rare earth compound fluorescent substance. In fact, these luminescent agents are luminescent pigments, which are usually used as a labeling substance in a chip. It is only here that these labeling substances are used not only to label the detection reaction, but also to color the background to increase the background signal. When the detection signal is formed by the labeling substance actively or passively emitting light, the labeling substance can be used as a luminescent agent. An important aspect of current chip detection methods is to minimize the luminescent agent in the background. The method of the present invention does the opposite, and in several embodiments, a labeling substance (such as rhodamine) is introduced in the background (such as the chip reactor reaction surface) to increase sensitivity.
所述发光物还可选自于下述一种或多种： 含发光剂的涂料、 薄膜、 片及发光剂与活性分子的复合物， 所述活性分子可固定在芯片样品点周 围区域上。 在本发明中， 所述活性分子包括下述一种或多种多肽： 白蛋 白、 蛋白质 A、 蛋白质 G、 生物素、 亲和素、 抗原、 抗体、 抗抗体、 合 成多肽、 DNA、 等等。 例如， 抗体可捕获结合有罗丹明的抗抗体从而间 接地捕获罗丹明， 等等。  The luminescent substance can also be selected from one or more of the following: luminescent agent-containing coatings, films, sheets, and complexes of luminescent agent and active molecules, which can be fixed on the area around the chip sample spot. In the present invention, the active molecule includes one or more of the following polypeptides: albumin, protein A, protein G, biotin, avidin, antigen, antibody, anti-antibody, synthetic polypeptide, DNA, and the like. For example, antibodies can capture anti-rhodamine bound antibodies to capture rhodamine indirectly, and so on.
在根据第一和第二方面的检测方法中，所述降低弱目标信号的着色 包括对探针点弓 I入深色着色剂或含深色着色剂的深色着色物， 所述深色 着色剂选自下述一种或多种颜色为深色、 优选为黑色的物质： 染料、 有 色颜料、 消光剂。 染料、 颜料、 消光剂在塑料、 涂料等材料着色中是一 个公知的概念， 具有确定的内容。 所述颜料的例子为包括炭黑、 金属盐 在内的黑颜料， 等等； 所述染料的例子有氨基黑和考马斯亮蓝， 等等。 通常有色涂料中的消光剂是指能降低涂层表面光泽度的材料。 降低涂层 表面光泽度可以通过增加微观粗糙度来实现。 在本发明的一个实施方案 中， 含消光剂的涂料在载玻片表面形成表面粗糙度 Ra在 0.02— 3.0 μ ιη之 间、 优选方案在 0.25— 3.0 μ ιη之间的涂层。 In the detection method according to the first and second aspects, the reducing the coloring of the weak target signal includes applying a dark colorant or a dark colorant containing a dark colorant to the probe point, the dark color The agent is selected from one or more of the following materials, which are dark, preferably black: dyes, colored pigments, matting agents. Dyes, pigments, and matting agents are a well-known concept in the coloring of materials such as plastics and coatings, and have certain contents. Examples of the pigments are black pigments including carbon black, metal salts, and the like; examples of the dyes are amino black and Coomassie brilliant blue, and the like. Matting agents in pigmented coatings generally refer to materials that can reduce the gloss of the coating surface. Reduce coating Surface gloss can be achieved by increasing micro-roughness. In one embodiment of the present invention, the matting agent-containing coating forms a coating having a surface roughness Ra between 0.02 and 3.0 μm, preferably between 0.25 and 3.0 μm, on the surface of the glass slide.
所述着色物选自下述一种或多种： 含所述着色剂的涂料、 薄膜、 片 及所述着色剂与活性分子的复合物， 所述活性分子在样品检测信号形成 过程中可固定在芯片样品点中除探针捕获的样品目标物以外的物质上。 所述有色涂料是一个公知的概念， 具有确定的内容。 所述可结合探针的 物质包括下述一种或多种有机物及其衍生物： 硝化纤维素、 聚苯乙烯、 聚丙烯酸脂、 聚砜、 聚醚砜、 聚氯乙烯、 氨基树脂、 聚多糖、 聚氨基酸。  The colorant is selected from one or more of the following: a coating, a film, a sheet containing the colorant, and a complex of the colorant and an active molecule that can be fixed during the formation of a sample detection signal On a chip sample spot other than the sample target captured by the probe. The colored paint is a well-known concept and has a certain content. 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.
所述着色包括将探针与所述有色涂料混合后再点样至片基上制作 芯片。  The coloring includes mixing a probe with the colored paint and then spotting the chip onto a substrate to make a chip.
另外， 所述着色包括在所述样品检测信号的形成过程中进行的着 ft。 例如，在所述样品检测信号的形成过程中对芯片引入发光背景。  In addition, the coloring includes ft performed during the formation of the sample detection signal. For example, a luminous background is introduced to the chip during the formation of the sample detection signal.
所述着色包含将样品与所述发光剂或发光物反应后再加入反应器、 并使其中包围探针点的区域上的检测信号增强 200%以上、 优选 500%以 上。 本发明的检测方法不同于所谓仅仅用标记物质标记来直接标记检测 样品中的目标物的直接法。本发明的检测方法标记物质加入检测样品中， 其目的不仅是标记目标物、 而且也与非目标物反应； 其效果、 特别是后 者的效果是通过探针点外的非特异性吸附增大了所述背景信号、 从而获 得所述弱目标信号-背景比。 在本发明的一个实施例中， 血清与罗丹明混 合反应后不经纯化即加入多肽芯片反应器中， 反应后不仅标记目标物被 相应固定化探针捕获、背景信号还被增大、且弱目标信号 -背景比小于 0.1。 令人'惊奇的是， 与直接法通常降低特异性大为不同， 本发明的检测方法 不仅具有高的灵敏度且具有高的特异性。  The coloring includes adding a sample to the reactor after reacting the sample with the luminescent agent or luminescent substance, and enhancing the detection signal on the area surrounding the probe point by 200% or more, preferably 500% or more. The detection method of the present invention is different from the so-called direct method of directly labeling a target substance in a detection sample with only a labeling substance. In the detection method of the present invention, a labeling substance is added to a detection sample, and its purpose is not only to label the target substance, but also to react with non-target substances; its effect, especially the latter effect is increased by non-specific adsorption outside the probe point. The background signal to obtain the weak target signal-background ratio. In one embodiment of the present invention, the serum is mixed with rhodamine and added to the peptide chip reactor without purification. After the reaction, not only the labeled target is captured by the corresponding immobilized probe, but the background signal is also increased and weak The target signal-background ratio is less than 0.1. Surprisingly, the detection method of the present invention has not only high sensitivity but also high specificity, unlike the direct method that usually reduces specificity.
所述着色还包含将样品加入反应器后再加入所述发光剂或发光物 进行反应、并使其中包围探针点的区域上的检测信号增强 200%以上、优 选 500%以上。 在现有的芯片检测方法、 例如基于抗原-抗体反应的芯片 检测方法 （包括间接法、 双抗原夹心法、 双抗体夹心法、 等等） 中， 通 常要求高纯度标记物， 且纯度越高越好。 在本发明的一个实施例中， 不 纯标记物 （例如标记物-着色物混合物,其中标记物为罗丹明化标记配基, 着色物为罗丹明化白蛋白） 加入多肽芯片反应器中， 反应后不仅固定化 探针捕获的目标物被标记、 背景信号还被增大、 且弱目标信号-背景比小 于 0.1。 令人惊奇的是， 与人们对现有的芯片检测方法 （包括间接法、 双 抗原夹心法、 双抗体夹心法、 等等） 的看法相异， 本发明的使用不纯标 记物的检测方法不仅具有高的灵敏度且未见降低特异性。 The coloring further includes adding the sample to the reactor, and then adding the luminescent agent or luminescent substance to react, and enhancing the detection signal on the area surrounding the probe point therein by more than 200%, preferably 500% or more. In the existing chip detection methods, such as an antigen-antibody reaction-based chip detection method (including indirect method, double antigen sandwich method, double antibody sandwich method, etc.), a high-purity label is usually required, and the higher the purity, the more it is good. In one embodiment of the present invention, an impure label (for example, a label-coloring mixture, wherein the label is rhodamine-labeled ligand, and the color is rhodamine-albumin) is added to a polypeptide chip reactor, and reacts Not only fixed The target captured by the probe is labeled, the background signal is also increased, and the weak target signal-background ratio is less than 0.1. Surprisingly, people's views on the existing chip detection methods (including indirect method, double antigen sandwich method, double antibody sandwich method, etc.) are different. The detection method of the present invention using impure labels not only Has high sensitivity and no decrease in specificity.
根据本发明的第三个方面， 其提供一种平面芯片片基， 其特征在于 含着色剂或含着色剂的着色物。 所述片基包括基质以及任选存在下述一 种或多种复合结构： 包被、 涂层、 薄膜和薄片。 本发明中， 术语 "着色 片基"是含着色剂或着色物的片基。 着色剂可以含于其基质内或 /和与基 质结合的复合结构内 （例如： 有色包被、 有色涂层、 有色薄膜和有色薄 片、 等等） 。 所述基质选自于改性或未改性的玻璃、 塑料、 金属、 优选 为玻璃。 在本发明中， 片基正面为片基上用以固定探针的一面。 其中， 平面片基是使背景与目标之间形成高色差比、 优选形成最大化色差比的 基础。 目前的芯片平面片基， 均为透明或未着色的片基， 在芯片应用时 有透明或未着色的背景。 透明片基的例子有透明玻璃片基、 透明塑料片 基、 等等。 未着色片基仅保留其基质本色 （例如体质颜料的颜色） 而未 加入染料、 有色颜料， 其例子有具金属银本色的银箔片基、 具膜本色的 薄膜片基或膜-玻片复合片基、 等等。  According to a third aspect of the present invention, it provides a planar chip substrate, which is characterized by containing a colorant or a colorant containing a colorant. The substrate includes a matrix and optionally one or more of the following composite structures: coatings, coatings, films, and sheets. In the present invention, the term "colored film base" is a film base containing a colorant or a colorant. The colorant may be contained in the matrix or / and the composite structure combined with the matrix (for example, colored coatings, colored coatings, colored films and colored sheets, etc.). The substrate is selected from modified or unmodified glass, plastic, metal, preferably glass. In the present invention, the front side of the substrate is the side on the substrate for fixing the probe. Among them, the flat film base is the basis for forming a high color difference ratio between the background and the target, and preferably forming a maximum color difference ratio. The current chip flat substrates are transparent or uncolored substrates, and have a transparent or uncolored background when the chip is applied. Examples of the transparent substrate include a transparent glass substrate, a transparent plastic substrate, and the like. The uncolored film base only retains its base color (such as the color of extender pigments) without adding dyes and colored pigments. Examples include a silver foil base with a metallic silver color, a film base with a film quality or a film-glass slide composite. Film base, etc.
根据本发明的芯片片基可以是通过根据本发明上述方面之一进行 着色而制成的。 例如本发明实施例中的下述芯片： 对芯片背景进行加强 信号的所述着色的片基 （芯片背景信号加强片基） 、 利用着色剂或含着 色剂的物质对芯片背景进行着色的片基 （着色片基） 、 等等。 芯片背景 信号加强片基的例子有： 含发光剂片基 （其中引入有发光剂或含发光剂 的物质） 、 高反射率片基（信号光线反射率大于 5 %、 优选大于 10%、 更 优选大于 30% ) 、 等等。 着色片基的例子有： 深色、 优选方案黑色片基 (例如载玻片覆黑漆制备的黑漆 -玻片片基） ， 浅色、 优选方案白色片基 (例如载玻片覆白漆制备的白漆 -玻片片基） ， 等等。  The chip base according to the present invention may be made by coloring according to one of the above aspects of the present invention. For example, the following chip in the embodiment of the present invention: the colored base for enhancing the signal of the chip background (chip background signal enhanced base), the base for coloring the chip background by using a colorant or a substance containing a colorant (Colored film base), etc. Examples of chip background signal enhancement substrates are: luminescent agent-containing substrate (in which a luminescent agent or a substance containing a luminescent agent is introduced), high-reflectivity substrate (signal light reflectance is greater than 5%, preferably greater than 10%, more preferred Greater than 30%), etc. Examples of colored substrates are: dark, preferably black substrates (for example, black lacquer-slide base prepared by covering glass slides with black lacquer), and light, preferably white substrates (for example, glass slide over white lacquer). Preparation of white paint-glass slide base), and so on.
在根据本发明的芯片片基中，所述着色为体着色、面着色或定点着 色。 在本发明中， 体着色为基质作色， 面着色包括正面、 背面、 正 /背面 着色， 定点着色为探针点正面、 背面、 正 /背面着色。  In the chip base according to the present invention, the coloring is bulk coloring, surface coloring, or spot coloring. In the present invention, the body coloring is the coloring of the substrate, the surface coloring includes front, back, front / back coloring, and the spot coloring is the probe point front, back, front / back coloring.
优选地， 根据本发明的芯片片基包含玻璃基质和所述有色涂料涂 层。 所述有色涂料为含所述着色剂的涂料。 所述有色涂料包括黑色、 白 色、 各种彩色的油漆或 /和油墨。 所述彩色包括红、 黄、 绿、 蓝、 青、 紫 等色。 在本发明中， 油墨与油漆都具有可对片基着色的性质。 所述有色 涂料可以覆盖在基质 （或原料片基） 正面、 反面、 或正反两面。 在本发 明中， 片基正面为片基上用以固定探针的一面。 Preferably, the chip substrate according to the present invention comprises a glass substrate and the colored paint coating. The colored paint is a paint containing the colorant. The colored paint includes black and white Color, various colored paint or / and ink. 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. The colored paint may cover the front side, the reverse side, or both sides of the substrate (or raw material base). In the present invention, the front side of the substrate is the side on the substrate for fixing the probe.
所述有色涂料优选为黑色的漆或白色的漆。 黑色涂层由黑色涂料 (例如含或不含消光剂的黑漆） 形成。 白色涂层由白色涂料 （例如含或 不含消光剂的白漆） 形成。  The colored paint is preferably a black paint or a white paint. The black coating is formed from a black paint, such as a black paint with or without a matting agent. The white coating is formed from a white paint, such as a white paint with or without a matting agent.
本发明的芯片片基的表面粗糙度 Ra在 0.02— 3.0 μ m之间、 优选在 0.25—3.0 μ ιη之间。  The surface roughness Ra of the chip substrate of the present invention is between 0.02 and 3.0 μm, and preferably between 0.25 and 3.0 μm.
根据本发明的第四个方面， 其提供一种芯片基片， 其包含着色平面 片基及任选存在的隔离结构， 所述着色平面片基含着色剂或含着色剂的 着色物。 本发明中， 着色平面片基又简称着色片基， 平面片基又简称片 基。 优选地， 所述着色片基为根据本发明第三方面的芯片片基。  According to a fourth aspect of the present invention, a chip substrate is provided, which includes a colored planar substrate and an optional isolation structure, wherein the colored planar substrate contains a colorant or a colorant containing a colorant. In the present invention, the colored flat base is also referred to as a colored base, and the flat base is also referred to as a base. Preferably, the colored substrate is a chip substrate according to the third aspect of the present invention.
根据本发明的第五方面，其提供一种芯片探针板，其包含平面片基、 固定在所述片基上的探针点及任选存在的隔离结构， 该芯片探针板的特 征在于 .·Α.所述平面片基含着色剂或含着色剂的着色物;或 /和 Β.所述探针 点含着色剂或含着色剂的着色物，但不含标记物质。  According to a fifth aspect of the present invention, there is provided a chip probe card including a flat substrate, a probe point fixed on the substrate, and an optional isolation structure. The chip probe card is characterized by A. The flat sheet base contains a colorant or a colorant containing a colorant; or / and B. The probe dot contains a colorant or a colorant containing a colorant, but does not contain a labeling substance.
在本发明的探针板中，所述片基为根据本发明第三方面的着色芯片 片基。例如， 可以是下述芯片探针板： Α. 含对芯片背景进行信号加强的 所述着色的制备过程制备的芯片探针板 （背景信号加强芯片探针板） 、 Β. 含对芯片探针点进行降低弱目标信号的所述着色的制备过程制备的 芯片探针板（弱目标信号减弱芯片探针板） 、 C. 含对芯片背景进行信号 加强和对芯片探针点进行信号减弱的所述着色的制备过程制备的芯片探 针板 （背景信号加强-弱目标信号减弱芯片探针板） 、 D. 含利用着色剂 或含着色剂的物质对芯片背景进行所述着色的制备过程制备的芯片探针 板（背景着色芯片探针板） 、 Ε. 含利用着色剂或含着色剂的物质对芯片 探针点进行所述着色的制备过程制备的芯片探针板 （定点着色芯片探针 板） 、 以及 F. 含利用着色剂或含着色剂的物质对芯片背景和探针点进行 所述着色的制备过程制备的芯片探针板 （背景 /探针点着色芯片探针板） 等。  In the probe card of the present invention, the substrate is a colored chip substrate according to the third aspect of the present invention. For example, it may be the following chip probe board: A. chip probe board (background signal enhanced chip probe board) prepared by the coloring preparation process with signal enhancement on the chip background, Β. Pair of chip probes The chip probe board (weak target signal weakening chip probe board) prepared by the coloring preparation process for reducing the weak target signal is included at the point, C. Contains the signal strengthening function of the chip background and the signal weakening of the chip probe point. The chip probe card (background signal enhancement-weak target signal attenuation chip probe card) prepared by the coloring preparation process, D. It is prepared by using the colorant or the substance containing the colorant to perform the coloring on the chip background. Chip probe board (background colored chip probe board), Ε. Chip probe board (fixed-point colored chip probe board) prepared by the preparation process using the colorant or the substance containing the colorant to perform chip coloring on the chip probe points ), And F. A chip probe board prepared by a process for preparing a chip background and a probe spot by using a colorant or a substance containing a colorant as described above ( View / probe Coloring probe card chip) and the like.
在根据权利要求的芯片探针板中，探针点可包含所述着色剂或着色 物。 In the chip probe card according to claim, the probe point may contain the colorant or coloring Thing.
根据本发明的最后一个方面， 其提供一种芯片试剂盒， 其包括芯片 探针板和任选存在的标记系统， 该芯片试剂盒的特征在于： A. 所述芯 片探针板中的平面片基含着色剂或含着色剂的着色物;或 /和 B. 所述芯 片探针板中的探针点含着色剂或含着色剂的着色物， 但不含标记物质； 或 /和 C. 其还含着色系统，所述着色系统含着色剂或含着色剂的着色物。 本发明中， 所述着色系统是指用以在样品检测信号形成过程时对所述芯 片背景或 /和探针卓作色的系统。  According to the last aspect of the present invention, a chip kit is provided, which includes a chip probe card and an optional labeling system. The chip kit is characterized by: A. a planar sheet in the chip probe card The base contains a colorant or a colorant-containing colorant; or / and B. The probe points in the chip probe card contain a colorant or a colorant-containing colorant, but do not contain a labeling substance; or / and C. It also contains a coloring system containing a colorant or a colorant containing colorant. In the present invention, the coloring system refers to a system for coloring the background of the chip or / and the probe during the formation of a sample detection signal.
根据本发明的芯片试剂盒可以是根据本发明的第一个方面的方法 制备的芯片试剂盒， 例如是下述芯片试剂盒： A. 含背景着色芯片的芯片 试剂盒、 B. 含探针点着色芯片的芯片试剂盒、 C. 含背景 /探针点着色芯 片的芯片试剂盒、 以及 D. 着色系统含着色剂的芯片试剂盒等。  The chip kit according to the present invention may be a chip kit prepared according to the method of the first aspect of the present invention, for example, the following chip kit: A. A chip kit containing a background-colored chip, B. A probe point Chip kits for coloring chips, C. chip kits with background / probe spot coloring chips, and D. chip kits with colorants for coloring systems.
在此情况下， 弱目标信号 -背景比小于 0.80、 优选小于 0.50、 更优选 小于 0.25。 高信号-背景比芯片试剂盒， 是目前芯片试剂盒的基本开发方 向。 其中， 所述弱目标信号-背景比为所述弱目标与所述背景面之间的检 测信号值的比。如此低的信号-背景比，是本发明芯片试剂盒的一个特征。 我们通过本发明的实施例惊奇地发现， 使用此低信号 -背景芯片试剂盒甚 至可以提高检测灵敏度。  In this case, the weak target signal-background ratio is less than 0.80, preferably less than 0.50, and more preferably less than 0.25. High-signal-background ratio chip kits are the basic development direction of current chip kits. The weak target signal-background ratio is a ratio of detection signal values between the weak target and the background surface. Such a low signal-to-background ratio is a feature of the chip kit of the present invention. We have surprisingly discovered through the examples of the present invention that the use of this low signal-background chip kit can even improve detection sensitivity.
另外，根据本发明的芯片试剂盒还可以是根据本发明第二方面的方 法制备的芯片试剂盒， 例如是下述芯片试剂盒： A. 含背景信号加强芯片 的芯片试剂盒、 B. 含弱目标信号减弱芯片的芯片试剂盒、 C. 含背景信 号加强-弱目标信号减弱芯片的芯片试剂盒、 D. 着色系统以发光剂为着 色剂的芯片试剂盒、 以及 E. 着色系统以含发光剂物质为着色剂的芯片试 剂盒等。  In addition, the chip kit according to the present invention may also be a chip kit prepared according to the method of the second aspect of the present invention, for example, the following chip kit: A. A chip kit containing a background signal enhancement chip, B. A weak target Chip kit for signal attenuation chip, C. Chip kit with background signal enhancement-weak target signal attenuation chip, D. Chip kit for coloring system using luminescent agent as colorant, and E. Coloring system for luminescent material containing substance Chip kits for colorants, etc.
本发明的芯片试剂盒优选包含根据本发明的芯片探针板。  The chip kit of the present invention preferably contains a chip probe card according to the present invention.
在根据本发明的芯片试剂盒中， 所述标记系统和 /或着色系统含所 述发光剂或 /和所述发光物。 在本发明芯片试剂盒包含所述着色系统时， 所述芯片的片基可是上述着色片基， 也可是非着色片基 （例如活化的玻 璃、 塑料、 金属） 。 本发明实施例中优选的非着色片基为活化玻片， 例 如， 含下述一种或多种衍生基团的活化玻片： 氨基、 环氧基、 醛基、 酰 肼基（-CO-NHN¾)、氨基脲基（H2N-NH-CONH-)、二乙氨乙基（DEAE)、 二乙基一（2—羟丙基）氨乙基（QAE)、羧甲基（CM)、磺酸丙基（SP)、 巯乙基吡啶 （MEP) 、 硅氧垸基、 硫醇基。 所述标记系统和着色系统， 可含相同所述发光剂， 也可含不相同所述发光剂。 发光剂可独立存在， 也可以与其它物质 （例如可与芯片探针点周围区域发生非特异性吸附的 物质、 可与芯片探针点周围区域发生特异性吸附的物质） 共存的方式存 在。 例如， 本发明实施例中， 所述发光剂为罗丹明、 CY3、 等等， 其被 用于与样品反应， 不仅标记了可与探针点结合的样品目标分子， 而且结 合在样品中可吸附在探针点周围的片基上的其它物质。 加入芯片后， 其 不仅标记了探针-目标物反应， 而且增强了背景信号。 又例如， 本发明实 施例中， 所述着色系统所含含发光剂物质为罗丹明、 CY3、 等等分别与 标记配基-白蛋白混合物反应后纯化或未纯化的产物， 加入芯片后， 其不 仅标记了探针-目标物反应， 而且增强了背景信号。 In the chip kit according to the present invention, the marking system and / or the coloring system contains the luminescent agent or / and the luminescent substance. When the chip kit of the present invention includes the coloring system, the chip base of the chip may be the above-mentioned coloring base, or may be a non-coloring base (eg, activated glass, plastic, metal). The preferred non-colored film base in the embodiment of the present invention is an activated glass slide, for example, an activated glass slide containing one or more of the following derivatized groups: amino, epoxy, aldehyde, and hydrazide (-CO- NHN¾), aminoureido (H 2 N-NH-CONH-), diethylaminoethyl (DEAE), Diethylmono (2-hydroxypropyl) aminoethyl (QAE), carboxymethyl (CM), sulfopropyl (SP), mercaptoethylpyridine (MEP), siloxenyl group, and thiol group. The marking system and the coloring system may contain the same luminescent agent or different luminescent agents. The luminescent agent may exist independently or coexist with other substances (for example, a substance capable of non-specific adsorption with the area around the chip probe point and a substance capable of specifically adsorbed with the area around the chip probe point). For example, in the embodiment of the present invention, the luminescent agent is rhodamine, CY3, etc., which is used to react with the sample, and not only marks a sample target molecule that can bind to the probe point, but also binds to the sample and can be adsorbed. Other substances on the substrate around the probe point. After adding the chip, it not only marks the probe-target reaction, but also enhances the background signal. As another example, in the embodiment of the present invention, the luminescent substance contained in the coloring system is rhodamine, CY3, etc., which is a purified or unpurified product after reacting with the labeled ligand-albumin mixture, respectively. Not only is the probe-target response labeled, but the background signal is enhanced.
本发明的芯片检测方法的优点是高的检测灵敏度，或在可以有足够 高的检测灵敏度的同时有高的选择自由度、 低的成本、 等等。  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.
根据本发明的基片的优点是可以赋于最终产品芯片高的检测灵敏 度， 或在可以有足够高的检测灵敏度的同时有高的选择自由度、 低的成 本等。  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.
本发明的芯片的优点是高的检测灵敏度，或在可以有足够高的检测 灵敏度的同时有高的选择自由度、 低的成本等。  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.
本发明的芯片试剂盒的优点是高的检测灵敏度，或在可以有足够高 的检测灵敏度的同时有高的选择自由度、 低的成本等。 现在通过以下非限制性的实施例对本发明进行说明。 实施例  The advantages of the chip kit 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
本发明实施例给出本发明实施原理的例子， 不能被理解为本发明仅 限于这些实施例。  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.
本发明实施例中所用玻片尺寸为 75 X25 X 1.0mm，具体的规格见表 1 所示。 表 1 The size of the slide glass used in the embodiment of the present invention is 75 X 25 X 1.0 mm, and the specific specifications are shown in Table 1. Table 1
Figure imgf000019_0001
Figure imgf000019_0001
*: 方法参考玻片蒋中华等《生物分子固定化技术及应用》， 化学工业出 版社， 1998)。  *: For the method, please refer to the slide "Jiang Zhonghua" and "Biomolecular Immobilization Technology and Application", Chemical Industry Press, 1998).
* *：方法参考以下文章： Melnyk 0等， " Peptide arrays for highly sensitive and special antibody-binding fluorescence arrays" ， Bioconjug C em. 13： 713-20. 2002, 以及 Olivier C等， " oxo semicarbazone peptide oro; iggodeoxynucleotide microarrays^ ， Bioconjug Chem. 14： 430-9.2003。 ***： 方法参考我们另一中国发明专利申请 CN03135618.4。 在实施例中， 除特别说明的地方， 所用探针为 HCV抗原 （中国北 京人民医院肝病研究所） 和 HIV1+2抗原 （中国北京人民医院肝病研究 所） 。 * *: The method refers to the following articles: Melnyk 0, etc., "Peptide arrays for highly sensitive and special antibody-binding fluorescence arrays", Bioconjug C em. 13: 713-20. 2002, and Olivier C, etc., "oxo semicarbazone peptide oro; iggodeoxynucleotide microarrays ^, Bioconjug Chem. 14: 430-9.2003. ***: The method refers to our other Chinese invention patent application CN03135618.4. In the examples, unless otherwise specified, the probe used is HCV antigen (Beijing, China) Institute of Liver Diseases, People's Hospital) and HIV 1 + 2 Antigen (Institute of Liver Diseases, Beijing People's Hospital, China).
在本实施例中， 探针点点径 200 μ ηι， 点距 800 y m。  In this embodiment, the probe has a spot diameter of 200 μm and a spot distance of 800 μm.
在本实施例中， 除特别说明的地方， 1号样为 HCV抗体阳性血清， 2号样为 HIV1+2抗体阳性人血清， 3号样为阳性对照物 （HCV抗体和 HIV1+2抗体阳性血清对照物的混合物） ， 4号样品为阴性对照物 （HCV 抗体和 HIV1+2抗体都为阴性的血清对照物） 。所有的样品， 均经使用经 典的 ELISA方法在血清 20倍稀释反应条件下预先检测。 实施例 1 : 背景信号增强片基和基片的制备 In this example, unless otherwise specified, sample 1 is HCV antibody-positive serum, sample 2 is HIV 1 + 2 antibody-positive human serum, and sample 3 is a positive control (HCV antibody and HIV 1 + 2 antibody A mixture of positive serum controls), and sample 4 was a negative control (a serum control where both HCV and HIV 1 + 2 antibodies were negative). All samples were pre-tested using the classic ELISA method under serum 20-fold dilution reaction conditions. Example 1: Preparation of background signal enhancement substrate and substrate
众所周知， 芯片分析中的发光物选自于下述一种或多种可发射 信号光线的物质： 包括荧光物质在内的激发发光物质和包括化学发光 物质和电化学发光物质在内的自主发光物质。 其中， 激发发光物质的 例子包括罗丹明、 CY3、 CY5、 Alexa, 海藻蛋白、 稀土化合物类荧光 物质。 实际上， 这些发光物均为发光颜料。 所述基质可选自于改性或 未改性的玻璃、 塑料、 金属。 本实施例中优选为玻璃 （载玻片） 。 It is well known that the luminescent substance in the chip analysis is selected from one or more of the following substances that can emit signal light: excited luminescent substances including fluorescent substances and autonomous luminescent substances including chemiluminescent substances and electrochemical luminescent substances . Among them, examples of excited luminescent substances include rhodamine, CY3, CY5, Alexa, algae protein, rare earth compound fluorescence Matter. In fact, these luminescent substances are luminescent pigments. The substrate may be selected from modified or unmodified glass, plastic, and metal. In this embodiment, glass (glass slide) is preferred.
1 )背景信号增强片基的制备 1) Preparation of background signal enhancement film base
本实施例背景信号增强片基的制备， 包括四种方法： 1 ) 制备含发 光基质的片基（例如片基 1的制备）； 2) 制备含发光薄膜的片基（例 如片基 2、 7和 8的制备） ； 3 ) 制备含发光包被物的片基（例如片基 3和 4的制备）； 4)制备含发光涂料的片基（例如片基 5和 6的制备）。 基于这些方法， 还可衍生出一些其它方法。  The preparation of the background signal-enhancing film base in this embodiment includes four methods: 1) preparing a film base containing a light-emitting substrate (such as the preparation of the film base 1); 2) preparing a film base containing a light-emitting film (such as the film bases 2, 7) And preparation of 8); 3) preparation of bases containing luminescent coatings (for example, preparation of bases 3 and 4); 4) preparation of bases containing luminescent coatings (for example, preparation of bases 5 and 6). Based on these methods, some other methods can be derived.
( 1 )含发光基质的片基的制备方法 (1) Preparation method of film base containing luminescent matrix
本实施例所制备的片基 1， 其为含发光基质的片基 （加有荧光物质 的聚苯乙烯片基）。其制备方法包括将荧光颜料 ZnS-Mii加入用以制备 酶标板的热塑性聚苯乙烯，再经模压成型制得，尺寸为 75 X25 X 1 mm。  The substrate 1 prepared in this embodiment is a substrate containing a luminescent substrate (a polystyrene substrate with a fluorescent substance added). The preparation method comprises adding a fluorescent pigment ZnS-Mii to a thermoplastic polystyrene used for preparing an enzyme-labeled plate, and then obtaining the size of 75 X 25 X 1 mm by molding.
(2) 含发光薄膜的片基的制备方法 (2) Preparation method of film base containing luminescent film
本实施例所用发光薄膜为加有荧光物质的聚氯乙烯薄膜。  The light-emitting film used in this embodiment is a polyvinyl chloride film to which a fluorescent substance is added.
本实施例制备的片基 2，其为含发光薄膜的片基（发光薄膜-玻片复 合片基） 。 其制备方法包括将对 532nm波长光线反光的聚氯乙烯薄膜 热贴合在表 1所述载玻片上。  The substrate 2 prepared in this embodiment is a substrate containing a light-emitting film (light-emitting film-glass slide composite substrate). The preparation method includes thermally bonding a polyvinyl chloride film that reflects light with a wavelength of 532 nm to a glass slide described in Table 1.
本实施例所制备的片基 7和 8， 其为含发光薄膜的片基（分别为正 面和背面有孔发光膜片基） ， 制备方法包括在聚氯乙烯发光薄膜打上 直径 150um的孔， 通过热贴合工艺结合在醛基玻片上正面或背面,且孔 洞位置与制备芯片时手工点样的探针点位置一致。  The film bases 7 and 8 prepared in this embodiment are film bases containing light-emitting films (front and back light-emitting film bases, respectively). The preparation method includes punching a 150 μm diameter hole in a polyvinyl chloride light-emitting film, The thermal bonding process is combined on the front or back of the aldehyde-based glass, and the position of the hole is the same as the position of the probe point manually spotted when preparing the chip.
(3 ) 含发光包被物的片基的制备方法 (3) Preparation method of film base containing luminous coating
本实施例所制备的片基 3和 4， 其为背面含发光包被物的片基（罗 丹明-白蛋白包被基片和罗丹明-多肽包被基片）， 制备方法包括将白蛋 白 （上海生物制品研究所）和合成多肽 （合成 EB病毒 VCA抗原， 自 制 )分另1 J与罗丹明 ( 5- ( and-6 ) -carboxytetramethylrhodamine succinimidyl ester, 简称 5 (6) -TAMRA SE, 美国 Anaspec Corporate公司） 用公 知的罗丹明结合多肽技术形成罗丹明 -白蛋白和罗丹明-多肽，然后将它 们分别利用通用的蛋白质包被技术包被在表 1所述氨基玻片背面上。 The substrates 3 and 4 prepared in this embodiment are substrates (rhodamine-albumin-coated substrate and rhodamine-polypeptide-coated substrate) containing a light-emitting coating on the back. The preparation method includes applying albumin (Shanghai Institute of Biological Products) and synthetic peptides (synthesized Epstein-Barr virus VCA antigen, self-made) are divided into 1 J and Rhodamine (5- (and-6) -carboxytetramethylrhodamine succinimidyl ester, referred to as 5 (6) -TAMRA SE, Anaspec, USA) Corporate company) The known rhodamine-binding peptide technology is used to form rhodamine-albumin and rhodamine-polypeptide, and they are respectively coated on the back of the amino slide described in Table 1 using a general protein coating technique.
(4) 含发光涂料的片基的制备方法 (4) Preparation method of film base containing luminescent paint
本实施例所用发光涂料为含荧光物质的白漆。  The luminescent paint used in this embodiment is a white paint containing a fluorescent substance.
本实施例制备的片基 5和 6， 其为含发光涂料的片基（正面有含荧 光物质涂层片基和背面有含荧光物质涂层片基） ， 制备方法包括将载 玻片正面或氨基玻片背面覆涂发光涂料。 所形成的涂层厚度在 30 m 左右。  The substrates 5 and 6 prepared in this embodiment are substrates containing a luminescent coating (the substrates are coated with a fluorescent substance on the front and the substrates are coated with a fluorescent substance on the back). The preparation method includes: The back of the amino slide is coated with a luminescent paint. The thickness of the coating formed is around 30 m.
本实施例所制备的片基和基片列于表 2 中， 其中参照基片为表 1 中的氨基玻片， 检出信号为相对信号值。 表 2  The substrates and substrates prepared in this embodiment are listed in Table 2. The reference substrate is the amino slide in Table 1. The detection signal is a relative signal value. Table 2
Figure imgf000021_0001
Figure imgf000021_0001
*： 对照基片， 为表 1中的氨基玻片  *: The control substrate is the amino slide in Table 1.
2)基片的制备方法 2) Preparation method of substrate
本发明实施例的基片为多片基池基片， 包栝片基及片基池隔离结 构。 其中片基池的隔离结构， 是用高疏水有机硅涂料 （成都晨光化工 设计院） 涂在上述片基上， 并干燥成膜 （膜厚小于 0.25mm) 后形成的 (参考我们的另一项发明专利申请 CN03117397.7) 。 每 1个基片含 8 个片基池，每个片基池尺寸为 4.5mmX4.5mm，片基池间隔离结构宽度 为 4.5mm。 The substrate according to the embodiment of the present invention is a multi-chip base substrate, including a base substrate and a base substrate isolation structure. The isolation structure of the substrate pool is a highly hydrophobic organic silicon coating (Chengdu Chenguang Chemical Design Institute) formed by coating on the above substrate and drying to form a film (film thickness less than 0.25mm) (refer to our other invention patent application CN03117397.7). Each substrate contains 8 substrate pools. The size of each substrate pool is 4.5mm × 4.5mm, and the width of the isolation structure between the substrate pools is 4.5mm.
根椐本实施例制备方法， 也可先在基质 （或原料片基） 上形成隔离 结构， 然后进行着色处理， 形成基片。  According to the preparation method of this embodiment, an isolation structure may be formed on the substrate (or raw material substrate) first, and then a coloring process is performed to form a substrate.
3 ) 鉴定 3) Identification
制备的基片其背景信号增强的确定如下：按公知的芯片点样方法将 HCV抗原和 HIV1+2抗原混合物（各 2 mg/ml)点到基片上的片基池中， 每个片基池内点 4个点形成 2 X 2方阵。然后以 4号样品（HCV抗体和 HIV1+2抗体都为阴性的血清对照物）为弱目标样品、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 2) 。 本实施 例制备的片基及基片， 其弱目标信号-背景比均小于 0.25、 大部分小于 0.10、 部分甚至小于 0.05。 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 well-known chip spotting method. The 4 points in the pool form a 2 X 2 square matrix. Then take sample No. 4 (serum control with negative HCV antibody and HIV 1 + 2 antibody) as the weak target sample, rhodamine-labeled goat anti-human anti-antibody (Jackson ImmunoRresearch Laboratories, USA) as the marker, The chip detection method detected negative targets and substrate background signals (Table 2). The weak target signal-to-background ratio of the film base and the substrate prepared in this embodiment are all less than 0.25, most are less than 0.10, and some are even less than 0.05.
实施例 2: 着色片基和着色基片的制备 Example 2: Preparation of colored substrates and colored substrates
本实施例中， 所述着色基质、 有色涂料和有色薄膜中均分别含 有着色剂和可结合探针的物质。 众所周知， 着色剂选自下述一种或多 种物质： 染料、 颜料、 消光剂。 染料、 颜料、 消光剂在塑料、 涂料等 材料着色中是一个公知的概念， 具有确定的内容。 所述颜料的例子有： 包括炭黑、 金属盐在内的黑颜料， 包括二氧化钛在内的白颜料， 和包 括黄颜料、 红颜料、 蓝颜料、 绿颜料、 金属颜料在内的彩色颜料， 等 等。 通常， 有色涂料中的消光剂是指能降低涂层表面光泽度的材料。 有色涂料的一个大类为色漆， 例如， 下述一种或多种色漆： 珍珠黑、 魔力黑、 珍珠白、 珍珠蓝、 亚光黑、 枣红、 猩红、 中蓝。 众所周知， 油漆中往往含有可结合探针的物质， 例如硝化纤维素、 聚苯乙烯、 聚 丙烯酸脂、 聚砜、 聚醚砜、 聚氯乙烯、 氨基树脂、 聚多糖、 聚氨基酸。 所述基质可选自于改性或未改性的玻璃、 塑料、 金属。 本实施例中优 选为玻璃 （载玻片） 。 1 )着色片基的制备 In this embodiment, the colored substrate, colored paint, and colored film each contain a colorant and a substance that can bind a probe. As is known, the colorant is selected from one or more of the following: dyes, pigments, and matting agents. Dyes, pigments, and matting agents are a well-known concept in the coloring of materials such as plastics and coatings, and have certain contents. Examples of the pigment include: black pigments including carbon black and metal salts, white pigments including titanium dioxide, and color pigments including yellow pigment, red pigment, blue pigment, green pigment, metal pigment, and the like Wait. Generally, matting agents in colored coatings refer to materials that can reduce the gloss of the surface of the coating. A large class of colored paints are colored paints, for example, one or more of the following colored paints: pearl black, magic black, pearl white, pearl blue, matte black, bayonet, scarlet, and medium blue. As we all know, paints often contain substances that can bind probes, such as nitrocellulose, polystyrene, polyacrylate, polysulfone, polyethersulfone, polyvinyl chloride, amino resins, polysaccharides, and polyamino acids. The substrate may be selected from modified or unmodified glass, plastic, and metal. In this embodiment, glass (glass slide) is preferred. 1) Preparation of colored tablets
本实施例着色片基的制备， 包括三种方法： 1 ) 制备含着色基 质的着色基片； 2) 制备含有色涂料的着色基片； 3 ) 制备含有色薄膜 的着色基片。 基于这些方法， 还可衍生出一些其它方法 （例如由它们 组合而成的方法） 。  The preparation of the colored substrate in this embodiment includes three methods: 1) preparing a colored substrate containing a colored substrate; 2) preparing a colored substrate containing a colored paint; 3) preparing a colored substrate containing a colored film. Based on these methods, some other methods can also be derived (for example, methods combined from them).
( 1 ) 有色基质着色片基的制备方法 (1) Preparation method of colored substrate colored tablet
本实施例所制备的片基 9 为含有色基质的着色片基 （黑色塑料片 基） 。 其制备方法为， 将着色剂 （槽法碳黑） 加入用于酶标板制备的 热塑性聚苯乙烯后， 经通常的模压成型制得， 尺寸为 75 X 25 X limn。  The base 9 prepared in this embodiment is a colored base (black plastic base) containing a color base. The preparation method is as follows: the colorant (carbon black) is added to the thermoplastic polystyrene used for the preparation of the microtiter plate, and then the size is 75 X 25 X limn.
(2) 有色涂料着色片基的制备方法 (2) Preparation method of colored paint base
本实施例所用涂料分别为珍珠黑喷漆 （上海启阜实业发展有限公 司） 、 珍珠绿喷漆 （上海启阜实业发展有限公司） 、 哑光黑 （上海启 阜实业发展有限公司） 和川洋自动白喷漆 （成都市红光涂料厂） ， 涂 料中的着色剂分别为以下颜料： 槽法碳黑、 氧化钛、 等。 珍珠黑和珍 珠绿涂料中加入有 （珠光物质作） 消光剂。 其制备方法为， 将涂料喷 涂在载玻片顶面（或背面） ， 干燥后形成着色涂层 （厚度在 30 μ ηι左 右） ， 它们的粗糙度 Ra在 0.4— 0.5 m之间 （成都市计量监督检定测 试所检测） 。 显然， 结合片基 10和 13的制备， 还可制备双面着色涂 层片基。 本实施例所制备部分含有色涂料的着色片基分别记为片基 10 (珍珠黑喷漆 /载玻片）、 片基 11 (珍珠绿喷漆 /载玻片）、 片基 12 (白 喷漆 /载玻片）和 13 (环氧基玻片 /哑光黑喷漆） 。 其中， 片基 10、 11、 12为正面着色片基， 片 13为背面着色片基。  The paints used in this example are pearl black spray paint (Shanghai Qifu Industrial Development Co., Ltd.), pearl green spray paint (Shanghai Qifu Industrial Development Co., Ltd.), matte black (Shanghai Qifu Industrial Development Co., Ltd.) and Chuanyang Auto White. Spray paint (Chengdu Hongguang Coating Factory), the colorants in the paint are the following pigments: channel black, titanium oxide, etc. Pearl black and precious pearl green paints are added with a (matte substance) matting agent. The preparation method is that the coating is sprayed on the top surface (or the back surface) of the slide glass, and a colored coating layer (thickness of about 30 μηι) is formed after drying, and their roughness Ra is between 0.4 and 0.5 m (measured in Chengdu) Supervised verification testing). Obviously, in combination with the preparation of the substrates 10 and 13, a double-sided colored coating substrate can also be prepared. Part of the colored film base containing the color paint prepared in this embodiment is referred to as film base 10 (pearl black spray paint / slide), film base 11 (pearl green spray paint / slide), and film base 12 (white spray paint / slide). Glass slides) and 13 (epoxy glass slides / matte black spray paint). Among them, the bases 10, 11, and 12 are front-side colored bases, and the sheet 13 is a back-side colored base.
(3 )有色薄膜着色基片的制备方法 (3) Preparation method of colored film colored substrate
本实施例所用有色薄膜为黑色聚苯乙烯膜 （厚度约 45um， 自制）， 着色膜的着色剂为槽法碳黑。 然后将其分别热贴合在载玻片正面和氨 基脲基玻片背面。 本实施例所制备部分含有色薄膜的着色基片分别记 为片基 14 (正面贴着色膜） 和片基 15 (背面贴着色膜片基） 。 本实施例所制备的着色背景片基和基片列于表 3中。 本实施例中， 基片含有着色片基及任选存在的隔离结构。 表 3 The colored film used in this embodiment is a black polystyrene film (thickness about 45 um, self-made), and the colorant of the coloring film is channel black. It was then thermally bonded to the front of the glass slide and the back of the semicarbazide-based glass, respectively. Part of the colored substrate containing the colored film prepared in this embodiment is referred to as a base 14 (a front-side colored film) and a base 15 (a back-side colored film base). The colored background substrates and substrates prepared in this example are listed in Table 3. In this embodiment, the substrate includes a colored substrate and an optional isolation structure. table 3
Figure imgf000024_0001
Figure imgf000024_0001
注： 表中表面粗糙度和吸光率的值是片基上多个点检测的平均值。 Note: The values of surface roughness and absorbance in the table are the average value of the detection of multiple points on the substrate.
2) 基片的制备方法 2) Preparation method of substrate
本实施例基片的制备方法与实施例 1中基片的制备方法相同。 实施例 3: 定点着色片基和基片的制备  The method for preparing the substrate in this embodiment is the same as the method for preparing the substrate in Example 1. Example 3: Preparation of fixed-point colored film substrates and substrates
1 ) 定点着色基片的制备方法  1) Preparation method of fixed-point colored substrate
本实施例定点着色基片的制备方法，是将有色涂料用点样器点至透 明基质（表 1所示的载玻片或活化玻片）正面或 /和背面形成着色点（着 色点位置对应于拟固定探针点的位置） 。 众所周知， 有色涂料的一个 大类为色漆， 例如， 下述一种或多种色漆： 珍珠黑、 魔力黑、 珍珠蓝、 亚光黑、 枣红、 猩红、 中蓝、 珍珠白。 通常， 有色涂料中的消光剂是 指能降低涂层表面光泽度的材料。 众所周知， 油漆中往往含有可结合 探针的物质， 例如硝化纤维素、 聚苯乙烯、 聚丙烯酸脂、 聚砜、 聚醚 砜、 聚氯乙烯、 氨基树脂、 聚多糖、 聚氨基酸。 着色可在下述之一步 骤进行： 直接对基质进行定点着色再、 对片基进行定点着色、 对基片 片基池内的基片进行定点着色。  In this embodiment, the method for preparing a fixed-point colored substrate is to spot the colored paint with a spotter onto a transparent substrate (the glass slide or activated glass shown in Table 1) to form colored spots on the front or / and the back (corresponding to the colored spot positions At the position of the probe point to be fixed). It is well known that a large category of colored paints are colored paints, for example, one or more of the following colored paints: pearl black, magic black, pearl blue, matte black, bayonet, scarlet, medium blue, pearl white. Generally, matting agents in pigmented coatings are materials that reduce the gloss of the surface of the coating. As we all know, paints often contain probe-binding substances, such as nitrocellulose, polystyrene, polyacrylate, polysulfone, polyethersulfone, polyvinyl chloride, amino resins, polysaccharides, and polyamino acids. Coloring can be performed in one of the following steps: direct spot coloring of the substrate, then spot coloring of the substrate, and spot coloring of the substrate in the substrate substrate pool.
本实施例中，将黑色涂料和白色涂料用点样器分别点至载玻片正面 制备的基片记作基片 16和 17;将黑色涂料和白色涂料用点样器分别点 至环氧基玻片背面制备的基片记作基片 18和 19。当在片基背面进行定 点着色时， 着色点直径可略大于探针样点直径。 其中所用黑色涂料为 珍珠黑油漆 （含消光剂， 上海启阜实业发展有限公司） ， 所用白色涂 料为珍珠白油漆 （含消光剂， 上海启阜实业发展有限公司） 。 In this embodiment, the substrates prepared by spotting the black paint and the white paint on the front surface of the glass slide are respectively referred to as the substrates 16 and 17; The substrates prepared to the back of the epoxy glass slide are referred to as substrates 18 and 19. When spot coloring is performed on the back of the substrate, the diameter of the colored dots may be slightly larger than the diameter of the probe-like dots. The black paint used is pearl black paint (containing matting agent, Shanghai Qifu Industrial Development Co., Ltd.), and the white paint used is pearl white paint (containing matting agent, Shanghai Qifu Industrial Development Co., Ltd.).
2) 基片的制备方法 2) Preparation method of substrate
本实施例基片的制备方法与实施例 1中基片的制备方法相同。  The method for preparing the substrate in this embodiment is the same as the method for preparing the substrate in Example 1.
3) 鉴定方法 3) Identification method
将上述制备的黑色涂料定点着色片基或基片进行扫描。 扫描仪为共 聚焦激光扫描仪（Afymetrix公司 GMS 418)，扫描激发光波长 532nm， 发射光波长 570mn， 激光强度和增益分别为 60/69， 读取的信号经处理 软件（JAGUARII )处理， 然后取平均值后得到结果。 未着色点的信号 值在 20— 100之间， 而着色点的信号值小于 20。 实施例 4: 背景信号增强芯片探针板的制备  Scan the spotted colored base or substrate of the black paint prepared above. The scanner is a confocal laser scanner (Afymetrix GMS 418), scanning the excitation light wavelength 532nm, the emission light wavelength 570mn, the laser intensity and gain are 60/69, the read signal is processed by processing software (JAGUARII), and then taken Results are obtained after averaging. The signal value of the uncolored points is between 20 and 100, and the signal value of the colored points is less than 20. Example 4: Preparation of a background signal enhancement chip probe card
本实施例背景信号增强芯片探针板的制备， 包括 3种方法： 1 )从实 施例 1制备的背景信号增强基片点样制备； 2) .从未着色片基点样、 然 后对芯片探针板背景着色制备； 3) .从着色片基点样、 然后对芯片探针 板背景着色制备。 基于这些方法， 还可衍生出一些其它方法。 本实施 例所用探针分别为： HCV抗原溶液 （1.5mg/ml) 、 HIV1+2 抗原溶液 ( 1.5mg/ml) 、 以及根椐我们的另一项发明 （PCT/CN2004/000077)制 备的 HCV抗原 -氧化硅溶液 （1.5mg抗原 /ml) 和 HIV1+2抗原-氧化硅 溶液 （1.5mg抗原 /ml) 。  The preparation of the background signal enhancement chip probe board of this embodiment includes three methods: 1) spot preparation of the background signal enhancement substrate prepared from Example 1; 2) spotting the unstained film base, and then chip probe Plate background coloring preparation; 3). Spot from the stained film base, and then prepare the chip probe plate background coloring. Based on these methods, some other methods can be derived. The probes used in this example are: HCV antigen solution (1.5mg / ml), HIV1 + 2 antigen solution (1.5mg / ml), and HCV prepared from another of our inventions (PCT / CN2004 / 000077) Antigen-silica solution (1.5mg antigen / ml) and HIV1 + 2 antigen-silica solution (1.5mg antigen / ml).
1 ) 从着色基片制备背景信号增强芯片探针板的制备方法： 1) A method for preparing a background signal enhancement chip probe card from a colored substrate:
本实施例所用着色基片为实施例 1制备的背景信号增强基片 （基片 1-8) 。 然后， 将上述探针按常规的点样法点样至片基池中。 四种探针 每种点 3个样点， 形成 4x3阵列。 然后用或不用封密液 （例如牛奶封 密液） 封闭。 本方法制得的芯片探针板， 对应于所用基片， 分别命名 为芯片探针板 1、 2、 3、 4、 5、 6、 7、 8、 9和 10。 2) 从未着色基片点样、 然后对芯片探针板背景着色制备背景信号增强 芯片探针板的方法 The colored substrate used in this embodiment is the background signal enhancement substrate (substrates 1-8) prepared in Example 1. Then, the above-mentioned probe is spotted into the substrate pool according to a conventional spotting method. Each of the four probes has 3 samples, forming a 4x3 array. It is then sealed with or without sealing fluid (such as milk sealing fluid). The chip probe boards prepared by this method are named chip probe boards 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 corresponding to the substrates used. 2) Method for preparing a background signal enhancement chip probe card by spotting an uncolored substrate and then coloring the background of the chip probe card
首先按实施例 1所述基片制备方法， 将表 1所示的活化玻片 （非着 色片基） 制备为 8 片基池未着色基片。 然后， 将上述探针按常规的点 样法点样至片基池中。 四种探针每种点 3个样点， 形成 4x3阵列。 再 将罗丹明-白蛋白用作发光物包被在片基正面（罗丹明-白蛋白按公知的 罗丹明结合多肽技术制备） 。 除白蛋白以外， 其它可用以形成发光物 的有吸咐活性的多肽（例如蛋白质 A、 蛋白质 G、 生物素、 亲和素、 抗 原、 抗体、 抗抗体、 多肽、 DNA、 等等） 也可按此方法使用。  First, according to the substrate preparation method described in Example 1, the activated glass slides (non-colored film bases) shown in Table 1 were prepared into 8 base pool uncolored substrates. Then, the above-mentioned probe is spotted into the substrate pool according to a conventional spotting method. Each of the four probes has 3 samples, forming a 4x3 array. Rhodamine-albumin was used as a luminescent substance and coated on the front surface of the substrate (rhodamine-albumin was prepared according to the well-known rhodamine-binding peptide technology). In addition to albumin, other peptides (such as protein A, protein G, biotin, avidin, antigen, antibody, anti-antibody, peptide, DNA, etc.) that can be used to form a luminescent substance can also be used. This method is used.
本实施例以氨基玻片、 醛基玻片、 环氧基玻片、 氨基脲玻片和 PVP 包被玻片为非着色片基， 分别制备的背景信号增强芯片探针板被分别 命名为芯片探针板 11、 12、 13、 14和 15。  In this embodiment, an amino slide, an aldehyde-based slide, an epoxy-based slide, a semicarbazide slide, and a PVP-coated slide are used as non-colored slides. The background signal enhancement chip probe boards prepared separately are named as chips. Probe boards 11, 12, 13, 14, and 15.
3 ) 从着色片基点样、 然后对芯片探针板背景着色制备 3) Spot from the base of the stained film, and then color the chip probe card background
本实施例所用着色基片为实施例 1制备的背景信号增强基片 6和基 片 8。 然后， 将上述探针按常规的点样法点样至片基池中。 四种探针每 种点 3个样点， 形成 4x3阵列。 再将罗丹明 -白蛋白用作发光物包被在 片基正面。 制备的背景信号增强芯片探针板被分别命名为芯片探针板 16和 17。  The colored substrates used in this embodiment are the background signal enhancement substrate 6 and the substrate 8 prepared in Example 1. Then, the above-mentioned probe is spotted into the substrate pool according to a conventional spotting method. Each of the four probes has 3 samples, forming a 4x3 array. Rhodamine-albumin was used as a luminescent substance and coated on the front side of the substrate. The prepared background signal enhancement chip probe cards are named chip probe cards 16 and 17, respectively.
4) 鉴定 4) Identification
本实施例参考芯片探针板，是将上述未着色基片按上述方法点样后， 用牛奶封闭液封闭制成。  In this embodiment, a chip probe board is referred to, and the uncolored substrate is spotted according to the above method, and then is sealed with a milk blocking solution.
将参考芯片探针板和上述制备的背景信号增强芯片探针板进行扫 描。 扫描仪为共聚焦激光扫描仪（Afymetrix公司 GMS 418) ， 扫描激 发光波长 532nm， 发射光波长 570nm， 激光强度和增益分别为 60/69， 读取的信号经处理软件 （ZoCSoft lmageBoost) 处理， 然后取平均值后 得到结果。 参考芯片探针板的背景信号值在 20-100之间， 本实施例制 备的背景信号增强芯片探针板的背景信号值要大得多， 均大于 5000、 甚至大于 10000。 实施例 5: 着色芯片探针板的制备 Scan the reference chip probe card and the background signal enhancement chip probe card prepared above. The scanner is a confocal laser scanner (Afymetrix Corporation GMS 418), which scans the excitation light wavelength of 532nm, the emission light wavelength of 570nm, the laser intensity and gain are 60/69, and the read signal is processed by processing software (ZoCSoft lmageBoost), and then Take the average and get the result. The background signal value of the reference chip probe card is between 20 and 100. The background signal value of the background signal enhancement chip probe card prepared in this embodiment is much larger, both being greater than 5000, or even greater than 10,000. Example 5: Preparation of a colored chip probe card
本实施例着色芯片探针板的制备， 是从着色片基点样制备。 本实施 例所用探针与实施例 4所用探针同。  The preparation of the colored chip probe card in this embodiment is prepared by spotting the colored chip base. The probe used in this example is the same as the probe used in Example 4.
本实施例所用着色基片为实施例 2制备的着色基片（基片 9一 15) 。 然后， 将上述探针按常规的点样法点样至片基池中。 四种探针每种点 3 个样点， 形成 4X 3阵列。 再后用或不用封密液 （例如牛奶封密液） 封 闭。 本方法制得的芯片探针板， 按所用基片命名次序， 分别命名为芯 片探针板 18 (黑色塑料片基芯片探针板） 、 19 (珍珠黑喷漆 /载玻片片 基芯片探针板） 、 20 (珍珠绿喷漆 /载玻片片基芯片探针板） 、 21 (白 喷漆 /载玻片片基芯片探针板） 、 22 (环氧基玻片 /哑光黑喷漆片基芯片 探针板） 、 23 (正面贴着色膜片基芯片探针板） 、 和 24 (背面贴着色 膜片基片基芯片探针板） 。 实施例 6: 定点着色芯片探针板的制备  The coloring substrate used in this embodiment is the coloring substrate (substrate 9-15) prepared in Example 2. Then, the above-mentioned probe is spotted into the substrate pool according to a conventional spotting method. Each of the four probes has 3 samples, forming a 4X 3 array. Seal with or without sealant (such as milk sealant). The chip probe boards prepared by this method are named as chip probe boards 18 (black plastic chip-based chip probe boards) and 19 (pearl black spray paint / slide chip-based chip probes) according to the naming order of the used substrates. Board), 20 (pearl green spray paint / slide-based chip probe board), 21 (white spray paint / slide-based chip probe board), 22 (epoxy glass / matte black spray paint base Chip probe board), 23 (color-coated chip-based chip probe board on the front), and 24 (color-coated chip-based chip probe board on the back). Example 6: Preparation of fixed-point colored chip probe cards
本实施例定点着色芯片探针板的制备， 包括 2种方法： 1 )是从定点 着色基片点样制备； 2) 是从非着色片基点样制备。 基于这些方法， 还可衍生出一些其它方法。 本实施例所用探针与实施例 4所用探针同。  The preparation of the spotted colored chip probe card in this embodiment includes two methods: 1) the spotted colored substrate is spotted and prepared; and 2) the non-stained sliced spotted substrate is prepared. Based on these methods, some other methods can be derived. The probe used in this example is the same as the probe used in Example 4.
1 ) 从定点着色片基制备着色芯片探针板的方法 1) Method for preparing colored chip probe card from fixed-point colored film base
本实施例所用定点着色基片为实施例 3制备的着色基片 16-19。 然 后， 将上述探针按常规的点样法点样至片基池中着色点位置上。 四种 探针每种点 3个样点， 形成 4x3阵列。 再后用或不用封密液 （例如牛 奶封密液） 封闭。 本方法制得的芯片探针板， 对应于所用基片命名次 序， 分别命名为芯片探针板 25 (黑色涂料定点着色载玻片片基芯片探 针板） 、 26 (白色涂料定点着色载玻片片基芯片探针板） 、 27 (黑色 涂料定点着色环氧基玻片片基芯片探针板） 和 28 (黑色涂料定点着色 环氧基玻片片基芯片探针板） 。  The fixed-point colored substrates used in this embodiment are the colored substrates 16-19 prepared in Example 3. Then, the above-mentioned probe is spotted on the spot of colored spots in the substrate pool according to a conventional spotting method. Each of the four probes has 3 samples, forming a 4x3 array. Then block it with or without a sealant (such as milk sealant). The chip probe boards prepared by this method are named chip probe boards 25 (black paint fixed-point colored glass slide-based chip probe boards) and 26 (white paint fixed-point colored glass Chip-based chip probe board), 27 (fixed-point colored epoxy glass slide-based chip probe board with black paint) and 28 (fixed-point colored epoxy glass-based chip probe board with black paint).
2) 从未着色基片点样制备着色芯片探针板的方法 2) Method for preparing colored chip probe card from spotting of uncolored substrate
方法为： 将探针与所述有色涂料混合后再点样至基质上， 干燥后用 或不用封密液 （例如牛奶封密液） 封闭。 本实施例所用涂料分别为珍 珠黑油漆（含消光剂， 上海启阜实业发展有限公司）和珍珠白油漆（含 消光剂， 上海启阜实业发展有限公司） 。 本实施例所用基质为表 1 中 的载玻片。本实施例制备的芯片探针板被分别命名为芯片探针板 29 (黑 色探针芯片探针板） 和 30 (定点白色探针芯片探针板） 。 实施例 7: 低弱目标信号 -背景比芯片试剂盒的制备 The method is as follows: the probe is mixed with the colored paint, and then spotted on the substrate; Or seal without sealing fluid (such as milk sealing fluid). The paints used in this embodiment are pearl black paint (containing matting agent, Shanghai Qifu Industrial Development Co., Ltd.) and pearl white paint (containing matting agent, Shanghai Qifu Industrial Development Co., Ltd.). The substrate used in this example is the glass slide in Table 1. The chip probe boards prepared in this embodiment are named chip probe boards 29 (black probe chip probe boards) and 30 (fixed-point white probe chip probe boards), respectively. Example 7: Preparation of low-weak target signal-background ratio chip kit
本实施例低弱目标信号-背景比芯片试剂盒，包括芯片探针板和任选 存在的标记系统， 以及任选存在的本发明的着色系统。 其制备包括 2 种方法： 1 ) 从背景信号增强芯片探针板或定点着色芯片探针板制备； 2) 从未着色芯片探针板制备。 基于这些方法， 还可衍生出一些其它 方法。  The low-weak target signal-background ratio chip kit of this embodiment includes a chip probe board and an optional labeling system, and an optional coloring system of the present invention. Its preparation includes 2 methods: 1) Preparation from a background signal enhanced chip probe card or a fixed-point colored chip probe card; 2) Preparation from an uncolored chip probe card. Based on these methods, some other methods can be derived.
1 ) 从背景信号增强芯片探针板或定点着色芯片探针板制备低弱目标信 号 -背景比芯片试剂盒的方法 1) Method for preparing low-weak target signal-background ratio chip kit from background signal enhancement chip probe board or fixed-point colored chip probe board
本实施例芯片试剂盒的这一制备方法， 是将背景信号增强芯片探针 板或定点着色芯片探针板与标记系统组合成合乎低弱目标信号-背景比 芯片标准 （小于 0.80、 优选小于 0.50、 更优选小于 0.25) 的芯片试剂 盒。 本实施例所用标记系统分别为罗丹明标记系统和 CY3标记系统， 所用标记物分别为罗丹明标记羊抗人二抗和 CY3标记羊抗人二抗 （羊 抗人二抗的标记方法为公知的标记方法） 。 根椐这些标记系统， 本实 施例选用的芯片探针板包括： 实施例 4制备的背景信号增强芯片探针 板 （芯片探针板 1-17) 和实施例 6制备的定点着色芯片探针板 （定点 黑色芯片探针板 25、 27、 28) 。 所制备的芯片试剂盒分别按此顺序命 名为芯片试剂盒 1、 2、 3、 4、 5、 6、 7、 8、 9、 10、 11、 12、 13、 14、 15、 16、 17、 18、 19、 和 20。  This preparation method of the chip kit of this embodiment is a combination of a background signal enhancement chip probe board or a fixed-point coloring chip probe board and a labeling system to meet a low-weak target signal-background ratio chip standard (less than 0.80, preferably less than 0.50 , More preferably a chip kit of less than 0.25). The labeling systems used in this embodiment are the rhodamine labeling system and the CY3 labeling system, respectively. The labels used are rhodamine-labeled goat anti-human secondary antibody and CY3-labeled goat anti-human secondary antibody. Marking method). Based on these marking systems, the chip probe card used in this embodiment includes the background signal enhancement chip probe card (chip probe card 1-17) prepared in Example 4 and the fixed-point colored chip probe card prepared in Example 6. (Fixed-point black chip probe boards 25, 27, 28). The prepared chip kits were named chip kits 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 respectively in this order. , 19, and 20.
2) 从未着色芯片探针板制备低弱目标信号 -背景比芯片试剂盒的方法 本实施例芯片试剂盒的这一制备方法， 是将未着色芯片探针板与标 记系统和着色系统组合成合乎本发明检测方法的弱目标信号-背景比芯 片标准 （小于 0.80、 优选小于 0.50、 更优选小于 0.25) 的芯片试剂盒。 本实施例所用未着色芯片探针板是以表 1 中的环氧基玻片和氨基脲玻 片为非着色片基， 按实施 1 的隔离结构形成方法制成非着色基片， 然 后按常规点样方法和封闭方法制备。 所用探针溶液为包被用抗人乙肝 表面抗体（HBsAb) )溶液（2mg/ml， 北京人民医院肝病研究所） 。 2) Method for preparing a low-weak target signal-background ratio chip kit from an uncolored chip probe card This method of preparing the chip kit of this embodiment is a combination of an uncolored chip probe card with a labeling system and a coloring system A chip kit with a weak target signal-background ratio chip standard (less than 0.80, preferably less than 0.50, more preferably less than 0.25) in accordance with the detection method of the present invention. The uncolored chip probe card used in this embodiment uses the epoxy glass slide and the semicarbazide glass in Table 1 as non-colored substrates, and is made into a non-colored substrate according to the isolation structure forming method of Embodiment 1. Spotting method and closed method. The probe solution used was an anti-human hepatitis B surface antibody (HBsAb) coating solution (2 mg / ml, Institute of Liver Diseases, Beijing People's Hospital).
本实施例的这一制备， 又有 2种方法： 1 ) .不含标记配基的芯片试 剂盒的制备； 2)含标记配基的芯片试剂盒的制备。  In this preparation of this embodiment, there are two more methods: 1). Preparation of a chip kit containing no labeled ligand; 2) Preparation of a chip kit containing labeled ligand.
( 1 ) 不含标记配基的芯片试剂盒的制备方法 (1) Preparation method of chip kit without labeled ligand
例如， 将发光剂或发光物作为标记系统的标记物质和着色系统的着 色物质， 然后与未着色芯片探针板一起形成试剂盒。 本实施例中所用 发光剂分别为罗丹明和 CY3， 其用量根据所要检测的样品种类优化。 所制备的芯片试剂盒分别为： 芯片试剂盒 21 (基于环氧基玻片的未着 色芯片探针板， 罗丹明发光剂） 、 芯片试剂盒 22 (基于氨基脲玻片的 未着色芯片探针板， 罗丹明发光剂）和芯片试剂盒 23 (基于氨基脲玻 片的未着色芯片探针板， CY3发光剂） 。  For example, a luminescent agent or a luminescent substance is used as a labeling substance of a labeling system and a coloring substance of a coloring system, and then a kit is formed with an uncolored chip probe card. The luminescent agents used in this embodiment are rhodamine and CY3, respectively, and their amounts are optimized according to the type of sample to be detected. The prepared chip kits are: chip kit 21 (unstained chip probe board based on epoxy glass slide, rhodamine luminescent agent), chip kit 22 (unstained chip probe based on semicarbazide slide) Plate, rhodamine luminescent agent) and chip kit 23 (unstained chip probe board based on semicarbazide glass, CY3 luminescent agent).
(2) 含标记配基的芯片试剂盒的制备方法 (2) Preparation method of chip kit containing labeled ligand
例如， 将标记配基及活性分子按优选的比例混合成混合物， 再将优 选量的发光剂与此混合物反应并分别结合到标记配基及活性分子上， 其纯化或未经纯化的产物便是标记物-着色物混合物， 然后与未着色芯 片探针板一起形成试剂盒。 本实施例中所用发光剂分别为罗丹明和 CY3 , 所用标记物分别为罗丹明和 CY3 标记的抗人乙肝表面抗体 (HBsAb) (标记用抗人乙肝表面抗体， 北京人民医院肝病研究所） ， 所用着色物分别为罗丹明和 CY3标记的人白蛋白。 所制备的芯片试剂 盒分别为： 芯片试剂盒 24 (基于环氧基玻片的未着色芯片探针板， 罗 丹明化抗人乙肝表面抗体和罗丹明化白蛋白）和芯片试剂盒 25 (基于 环氧基玻片的未着色芯片探针板， CY3化抗人乙肝表面抗体和 CY3化 白蛋白） 。  For example, the labeled ligand and active molecule are mixed into a mixture at a preferred ratio, and then a preferred amount of luminescent agent is reacted with this mixture and bound to the labeled ligand and active molecule, respectively. The purified or unpurified product is The marker-colorant mixture is then combined with the uncolored chip probe plate to form a kit. The luminescent agents used in this example are rhodamine and CY3, and the labels used are rhodamine and CY3 labeled anti-human hepatitis B surface antibody (HBsAb) (anti-human hepatitis B surface antibody for labeling, Institute of Liver Diseases, Beijing People's Hospital). The compounds were rhodamine and CY3 labeled human albumin, respectively. The prepared chip kits are: Chip kit 24 (unstained chip probe board based on epoxy slides, rhodamine anti-human hepatitis B surface antibody and rhodamine chemical albumin) and chip kit 25 (based on Unstained chip probe plate of epoxy glass slide, CY3 anti-human hepatitis B surface antibody and CY3 albumin).
同理， 标记物和着色物亦可分别制备后混合， 甚至不混合。 3 )鉴定 In the same way, the marker and the color can be prepared separately and mixed, or not even mixed. 3) Identification
本实施例所用弱目标样品分别为上述阴性样品 3 (其也是 HBs Ag 阴性血清） 。  The weak target samples used in this example are the above-mentioned negative samples 3 (which are also HBs Ag negative serum).
芯片试剂盒 20的鉴定实验： 将阴性样品 3稀释至 1/20分别加入芯 片试剂盒的反应器中， 加样量均为 15 μ 1。 反应 30分钟后洗涤 5次， 洗涤液每次加入量为 25 μ 1。 标记物加入量为 15 μ 1， 反应后洗涤 5次， 干燥后进行扫描。  Identification test of chip kit 20: Dilute negative sample 3 to 1/20 and add them to the reactor of chip kit, respectively, and the sample volume is 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 was washed 5 times after the reaction, and scanned after drying.
芯片试剂盒 21、 22和 23的鉴定实验： 将阴性样品 3稀释至 1/10， 然后与芯片试剂盒中的发光剂的溶液 （优选浓度） 等体积混合后室温 反应 30分钟， 再将此制备物分别加入芯片试剂盒的反应器中， 加样量 均为 15 μ 1。 反应 30分钟后洗涤 5次。 干燥后进行扫描。  Identification experiments of chip kits 21, 22, and 23: Dilute negative sample 3 to 1/10, and then mix with the luminescent agent solution (preferred concentration) in the chip kit in equal volumes and react at room temperature for 30 minutes, then prepare this The samples were respectively added to the reactor of the chip kit, and the loading amount was 15 μ1. After 30 minutes of reaction, it was washed 5 times. Scan after drying.
芯片试剂盒 24和 25的鉴定实验：将阴性样品 3稀释至 1/20分别加 入芯片试剂盒的反应器中， 加样量均为 15 μ 1。 反应 30分钟后洗涤 5 次，洗涤液每次加入量为 25 μ 1。然后加入上述标记物-发光物的溶液 (优 选浓度） ， 加入量为 15 μ 1， 反应后洗涤 5次， 干燥后进行扫描。  Identification experiments of chip kits 24 and 25: Dilute negative sample 3 to 1/20 and add them to the reactor of the chip kit, respectively, and the sample volume is 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. Then add the above-mentioned marker-luminescence solution (preferred concentration) in an amount of 15 μl, wash 5 times after the reaction, and scan after drying.
扫描仪为共聚焦激光扫描仪（Afymetrix公司 GMS 418) ， 扫描激 发光波长 532nm， 发射光波长 570nm， 激光强度和增益分别为 60/69， 读取的信号分别经处理软件 ZoCSoft ImageBoost,然后取平均值后得到 结果。本实施例中， 芯片背景信号值均大于 10000， 而阴性样品目标信 号值均小于 2000， 则它们的弱目标信号-背景比均小于 0.2， 个别小于 0.08, 甚至有的试剂量小于 0.03。 实施例 8: 着色芯片试剂盒的制备  The scanner is a confocal laser scanner (Afymetrix GMS 418). The scanning light wavelength is 532nm, the emission light wavelength is 570nm, the laser intensity and gain are 60/69, and the read signals are processed by the software ZoCSoft ImageBoost and averaged Get the result after value. In this embodiment, the background signal values of the chips are all greater than 10,000, and the target signal values of the negative samples are less than 2000. Then their weak target signal-background ratios are all less than 0.2, some are less than 0.08, and even some reagents are less than 0.03. Example 8: Preparation of a colored chip kit
本实施例着色芯片试剂盒包括芯片探针板和任选存在的标记系统， 其制备方法： 通过将着色芯片探针板与标记系统进行色差组合。 制备 方法的典型方法为下述二方法。 基于这些方法， 还可制备出一些其它 着色芯片试剂盒。  The colored chip kit of this embodiment includes a chip probe card and an optional labeling system, and a preparation method thereof: The color difference chip probe card and the labeling system are combined by color difference. The typical method of the preparation method is the following two methods. Based on these methods, some other colored chip kits can also be prepared.
1 )深色芯片试剂盒的制备方法 1) Preparation method of dark chip kit
其为选择深色芯片探针板（芯片探针板）与发光标记系统组合而成， 选择标准是使标记结果与背景有最大的信号反差。 本实施例所用标记 系统为荧光标记系统， 所用标记物为罗丹明标记羊抗人二抗， 所用标 记方法为公知的标记方法。 本实施例选用的深色芯片探针板选自实施 例 5中制备的深色芯片探针板 [芯片探针板 18 (黑色塑料片基芯片探针 板） 、 19 (珍珠黑喷漆 /载玻片片基芯片探针板） 、 20 (珍珠绿喷漆 /载 玻片片基芯片探针板） 、22(环氧基玻片 /哑光黑喷漆片基芯片探针板）、 23 (正面贴着色膜片基芯片探针板） 、 和 24 (背面贴着色膜片基片基 芯片探针板） ]， 分别以这些芯片探针板制备的芯片试剂盒分别按此顺 序命名为芯片试剂盒 26 (黑色塑料片基芯片试剂盒） 、 27 (珍珠黑喷 漆 /载玻片片基芯片试剂盒） 、 28 (珍珠绿喷漆 /载玻片片基芯片试剂 盒） 、 29 (环氧基玻片 /哑光黑喷漆片基芯片试剂盒） 、 30 (正面贴黑 色膜片基芯片试剂盒） 、 和 31 (背面贴黑色膜片基片基芯片试剂盒） 。 It is a combination of selecting a dark chip probe card (chip probe card) and a light-emitting marking system. The selection criterion is to maximize the signal contrast between the marking result and the background. Marking used in this example The system is a fluorescent labeling system. The label used is rhodamine-labeled goat anti-human secondary antibody. The labeling method used is a well-known labeling method. The dark chip probe board used in this embodiment is selected from the dark chip probe board prepared in Example 5 [chip probe board 18 (black plastic chip-based chip probe board), 19 (pearl black spray paint / glass Chip-based chip probe board), 20 (Pearl green spray paint / slide chip-based chip probe board), 22 (Epoxy glass slide / matte black spray-painted chip-based chip probe board), 23 (front side sticker Stained film-based chip probe board), and 24 (Stained film-based chip probe board on the back)], and the chip kits prepared from these chip probe boards are named chip kits 26 in this order. (Black Plastic Chip Kit), 27 (Pearl Black Spray Paint / Slide Chip Kit), 28 (Pearl Green Spray Paint / Slide Chip Kit), 29 (Epoxy Slide / Matte black spray chip chip kit), 30 (black film chip chip kit on the front), and 31 (black film chip chip kit on the back).
2) 浅色芯片试剂盒的制备方法 2) Preparation method of light-color chip kit
其为选择的浅色芯片探针板与标记系统组合而成，选择标准是使标 记结果与背景有最大的信号反差。 本实施例所用标记系统为金-银标记 系统 （参考中国专利申请号为 00807744.4的 《鉴定和 /或定量靶化合物 的方法》 ） ） ， 所用标记物为金标记羊抗人二抗， 所用标记方法为公 知的标记方法。 本实施例选用的浅色芯片探针板选自实施例 5 中制备 的浅色芯片探针板 [芯片探针板 21 (白喷漆 /载玻片片基芯片探针板）]。 以其制备的芯片试剂盒命名为芯片试剂盒 32 (白喷漆 /载玻片片基芯片 试剂盒） 。 实施例 9: 定点着色芯片试剂盒的制备  It is a combination of the selected light chip probe card and the marking system. The selection criterion is to maximize the signal contrast between the marking result and the background. The labeling system used in this embodiment is a gold-silver labeling system (refer to the "Method for Identifying and / or Quantifying Target Compounds" of Chinese Patent Application No. 00807744.4). The labeling method used is gold-labeled goat anti-human secondary antibody. It is a well-known marking method. The light-colored chip probe board used in this embodiment is selected from the light-colored chip probe board [chip probe board 21 (white spray paint / slide-based chip probe board) prepared in Example 5]. The chip kit was named Chip Kit 32 (white spray paint / slide-based chip kit). Example 9: Preparation of spot dye chip kit
本实施例定点着色芯片试剂盒包括芯片探针板和标记系统。 制备方 法为： 其为选择定点着色芯片探针板与标记系统组合而成， 选择标准 是使标记结果与背景有最大的信号反差。  The fixed-point colored chip kit of this embodiment includes a chip probe board and a labeling system. The preparation method is as follows: It is a combination of selecting a fixed-point colored chip probe card and a labeling system, and the selection criterion is to maximize the signal contrast between the labeling result and the background.
本实施例所用标记系统为荧光标记系统， 所用标记物为罗丹明标记 羊抗人二抗， 所用标记方法为公知的标记方法。 本实施例选用的定点 着色芯片探针板为定点深色芯片探针板， 选自实施例 6 中制备的定点 着色芯片探针板 [芯片探针板 25 (黑色涂料定点着色载玻片片基芯片 探针板） 、 27 (黑色涂料定点着色环氧基玻片片基芯片探针板） 、 28 (黑色涂料定点着色环氧基玻片片基芯片探针板） 和 29 (黑色探针芯 片探针板） ]。 分别以这些芯片探针板制备的芯片试剂盒分别按此顺序 命名为芯片试剂盒 32 (黑色涂料定点着色载玻片片基芯片试剂盒） 、 33 (黑色涂料定点着色环氧基玻片片基芯片试剂盒） 、 34 (黑色涂料 定点着色环氧基玻片片基芯片试剂盒）和 35 (黑色探针芯片试剂盒） 。 The labeling system used in this embodiment is a fluorescent labeling system, the labeling substance is rhodamine-labeled goat anti-human secondary antibody, and the labeling method used is a well-known labeling method. The fixed-point colored chip probe board used in this embodiment is a fixed-point dark chip probe board, which is selected from the fixed-point colored chip probe board prepared in Example 6 [chip probe board 25 (black paint fixed-point colored slide glass base Chip probe board), 27 (black paint fixed-point colored epoxy glass slide-based chip probe board), 28 (Black paint fixed-point colored epoxy based glass slide chip probe board) and 29 (black probe chip probe board)]. The chip kits prepared with these chip probe boards were named chip kits 32 (black paint fixed-point stained glass slide chip chip kit) and 33 (black paint fixed-point stained epoxy glass slide chip kits) in this order. Chip Kits), 34 (Black Paint Spotted Epoxy Stained Slides Chip Kit) and 35 (Black Probe Chip Kit).
实施例 6中制备的定点浅色芯片探针板 [芯片探针板 26 (白色涂料 定点着色载玻片片基芯片探针板）和 30 (定点白色探针芯片探针板） ] 也可以按与上述试剂盒制备方法相同的方法与深色标记系统 （例如金- 银标记系统） 一起制成试剂盒。 实施例 10: 低弱目标信号-背景比芯片检测 （1 )  The fixed-point light-colored chip probe board prepared in Example 6 [Chip Probe Board 26 (White paint fixed-point colored slide glass-based chip probe board) and 30 (Fixed-point white probe chip probe board)] can also be pressed The same method as the above kit preparation method is used together with a dark labeling system (such as a gold-silver labeling system) to make a kit. Embodiment 10: Low-weak target signal-background ratio chip detection (1)
本实施例所用低弱目标信号-背景比芯片试剂盒为实施例 7 制备的 部分低弱目标信号 -背景比芯片试剂盒 [芯片试剂盒 1、 2、 3、 4、 5、 6、 7、 8、 9、 10、 11、 12、 13、 14、 15、 16、 17、 18、 19、 20和 21] ， 所 用样品如上所述 (样品 1-4)， 所用弱目标样品为其中的阴性样品。 本实 施例所用参照芯片试剂盒为以表 1 中的环氧基玻片为片基按常规方法 制备的含相同探针的荧光标记芯片试剂盒 （记为芯片试剂盒 0) 。  The low-weak target signal-background ratio chip kit used in this example is part of the low-weak target signal-background ratio chip kit prepared in Example 7. [Chip kits 1, 2, 3, 4, 5, 6, 7, 8, , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21], the samples used are as described above (samples 1-4), and the weak target sample used is the negative sample among them. The reference chip kit used in this embodiment is a fluorescently labeled chip kit (referred to as chip kit 0) containing the same probe and prepared in the conventional manner with the epoxy glass slide in Table 1 as a base.
实验时前述 4种样品 （稀释至 1/20至 1/1000之间） 分别加入芯片 试剂盒的反应器中， 加样量均为 15 μ 1。 反应 30分钟后洗涤 5次。 标 记物加入量为 15 μ 1， 反应后洗涤 5次。 干燥后进行扫描。 扫描仪为共 聚焦激光扫描仪（Afymetrix公司 GMS 418)，扫描激发光波长 532nm， 发射光波长 570nm， 激光强度和增益分别为 60/69， 读取的信号分别经 处理软件 ZoCSoft ImageBoost (针对低弱目标信号-背景比芯片试剂盒， 其中目标读数均为其绝对读数与背景值之差）和 JAGUARII (针对参 照芯片试剂盒） 处理， 然后取平均值后得到结果。 使用上述低弱目标 信号 -背景比芯片试剂盒所得检测结果（阴、 阳性）与样品阴阳性一致。 使用参照芯片试剂盒进行的检测， 1号样品在稀释至 100倍时为阴性结 果； 而使用多个低弱目标信号 -背景比芯片试剂盒进行的检测， 1 号样 品在稀释至 500倍时仍为阳性结果。 下表 4给出部分检测结果。 其它 低弱目标信号 -背景比芯片试剂盒得出的检测结果与这些检测结果类 同。 表 4 During the experiment, the aforementioned four samples (diluted to 1/20 to 1/1000) were respectively added to the reactor of the chip kit, and the amount of each sample was 15 μ1. After 30 minutes of reaction, it was washed 5 times. The labeling amount was 15 μ1, and the reaction was washed 5 times after the reaction. Scan after drying. The scanner is a confocal laser scanner (Afymetrix GMS 418). The scanning light wavelength is 532nm and the emission light wavelength is 570nm. The laser intensity and gain are 60/69 respectively. The read signals are processed by software ZoCSoft ImageBoost ( Target signal-background ratio chip kit, where the target readings are the difference between their absolute readings and background values) and JAGUARII (for the reference chip kit), and then average the results to get the results. The detection results (negative, positive) obtained by using the above-mentioned low-weak target signal-background ratio chip kit are consistent with the sample negative. For the test using the reference chip kit, sample 1 was negative when diluted to 100 times. For the test using multiple low-target signal-background ratio chip kits, the sample 1 was still diluted to 500 times. Is a positive result. Table 4 below gives some test results. The detection results of other low-weak target signal-background ratio chip kits are similar to these detection results. Table 4
Figure imgf000033_0001
实施例 11: 低弱目标信号-背景比芯片检测 （2)
Figure imgf000033_0001
Example 11: Low-weak target signal-background ratio chip detection (2)
本实施例所用低弱目标信号-背景比芯片试剂盒为实施例 7 制备的 部分低弱目标信号 -背景比芯片试剂盒 [芯片试剂盒芯片试剂盒 21 (基 于环氧基玻片的未着色芯片探针板， 罗丹明） 、 芯片试剂盒 22 (基于 氨基脲玻片的未着色芯片探针板， 罗丹明） 和芯片试剂盒 23 (基于氨 基脲玻片的未着色芯片探针板， CY3)〗 ， 所用样品为乙肝表面抗原检 测用 ELISA试剂盒 （北京天坛生物制品股分有限公司） 中的阴性对照 物和阳性对照物， 所用弱目标样品为其中的阴性对照物。 本实施例所 用参照芯片试剂盒为实施例 10中的荧光标记芯片试剂盒（芯片试剂盒 0) 。  The low-weak target signal-background ratio chip kit used in this example is part of the low-weak target signal-background ratio chip kit prepared in Example 7. [Chip kit chip kit 21 (unstained chip based on epoxy glass slides) Probe Board, Rhodamine), Chip Kit 22 (Unstained Chip Probe Board Based on Semicarbazide, Rhodamine) and Chip Kit 23 (Unstained Chip Probe Board Based on Semicarbazide, CY3) The sample used was the negative control and positive control in the ELISA kit (Beijing Tiantan Biological Products Co., Ltd.) for the detection of hepatitis B surface antigen, and the weak target sample was used as the negative control. The reference chip kit used in this example is the fluorescently labeled chip kit (Chip Kit 0) in Example 10.
操作方法： 使用低弱目标信号-背景比芯片试剂盒检测时， 将 2种样 品稀释至 1/20至 1/1000之间， 分别加入芯片试剂盒中的发光剂 （优化 浓度的罗丹明或 CY3 ) ， 室温反应 30分钟后加入芯片反应器中， 加样 量均为 15 μ 1。 反应完成后洗涤 5次。 干燥后进行扫描。  Operation method: When using low-weak target signal-background ratio chip kit for detection, dilute 2 samples to 1/20 to 1/1000, and add the luminescent agent (optimized concentration of rhodamine or CY3) in the chip kit. ). After reacting at room temperature for 30 minutes, add it to the chip reactor, and the sample volume is 15 μ1. After the reaction was completed, washing was performed 5 times. Scan after drying.
使用参照芯片试剂盒时， 将 2种样品稀释至 1/20至 1/10000之间， 按实施例 10所述方法进行操作。  When using the reference chip kit, dilute the two samples to between 1/20 and 1/10000, and proceed as described in Example 10.
扫描仪为共聚焦激光扫描仪 （Afymetrix公司 GMS 418或成都光电 所 Scan-2) ， 扫描激发光波长 532nm， 发射光波长 570nm， 激光强度 和增益分别为 60/69， 读取的信号分别经处理软件 ZoCSoft lmageBoos t (针对低弱目标信号-背景比芯片试剂盒） 和 JAGUARII (针对参照 芯片试剂盒） 处理， 然后取平均值后得到结果。 使用低弱目标信号-背 景比芯片试剂盒进行的检测， 弱目标信号 -背景比小于 0.1， 且阴、 阳性 结果与所用样品一致。 使用参照芯片试剂盒进行的检测， 阳性对照在 稀释至 50倍时为阴性结果；而使用低弱目标信号-背景比芯片试剂盒进 行的检测， 阳性对照在稀释至 1000倍时仍为阳性结果。 实施例 12: 低弱目标信号-背景比芯片检测 （3) The scanner is a confocal laser scanner (Afymetrix GMS 418 or Chengdu Optoelectronics Institute Scan-2). The scanning light wavelength is 532nm and the emission light wavelength is 570nm. The laser intensity and gain are 60/69, and the read signals are processed separately. Software ZoCSoft lmageBoos t (For low-weak target signal-background ratio chip kit) and JAGUARII (for reference chip kit), and then average the results to get the results. Detection using low-weak target signal-background ratio chip kit, the weak target signal-background ratio is less than 0.1, and the negative and positive results are consistent with the sample used. For the tests performed with the reference chip kit, the positive control was negative when diluted to 50 times; for the tests performed with the low target signal-background ratio chip kit, the positive control was still positive when diluted to 1000 times. Example 12: Low-weak target signal-background ratio chip detection (3)
本实施例所用低弱目标信号-背景比芯片试剂盒为实施例 7 制备的 部分低弱目标信号 -背景比芯片试剂盒 [芯片试剂盒 24 (基于环氧基玻 片的未着色芯片探针板， 罗丹明化抗人乙肝表面抗体和罗丹明化白蛋 白） 和芯片试剂盒 25 (基于环氧基玻片的未着色芯片探针板， CY3化 抗人乙肝表面抗体和 CY3化白蛋白） ] ， 所用样品及弱目标样品与实 施例 11 中的相同。 本实施例所用参照芯片试剂盒为实施例 10中的荧 光标记芯片试剂盒 （芯片试剂盒 0) 。  The low-weak target signal-background ratio chip kit used in this example is part of the low-weak target signal-background ratio chip kit prepared in Example 7. [Chip Kit 24 (Unstained chip probe board based on epoxy glass slides) , Rhodamine anti-hepatitis B surface antibody and rhodamine albumin) and chip kit 25 (unstained chip probe board based on epoxy slides, CY3 anti-human hepatitis B surface antibody and CY3 albumin)] The samples used and the weak target samples are the same as in Example 11. The reference chip kit used in this example is the fluorescently labeled chip kit (Chip Kit 0) in Example 10.
操作方法： 使用低弱目标信号-背景比芯片试剂盒检测时， 将 2种样 品稀释至 1/20至 1/10000之间， 分别加入芯片试剂盒的反应器中， 加 样量均为 15 μ 1。反应完成后洗涤 5次。优化浓度的标记物 -发光物加入 量为 15 μ 1， 反应后洗涤 5次， 洗涤液每次加入量为 25 μ 1， 干燥后进 行扫描。 使用参照芯片试剂盒时， 操作方法与实施例 11中参照芯片试 剂盒操作方法同。  Operation method: When using the low-weak target signal-background ratio chip kit for detection, dilute the two samples to between 1/20 and 1/10000, and add them to the reactor of the chip kit. 1. After the reaction was completed, washing was performed 5 times. The optimal concentration of the labeling substance-the amount of luminescent substance was 15 μ1, and the solution was washed 5 times after the reaction, and the washing solution was added 25 μ1 each time. After drying, scanning was performed. When using the reference chip kit, the operation method is the same as that of the reference chip kit in Example 11.
扫描仪为共聚焦激光扫描仪 （Afymetrix公司 GMS 418) ， 扫描激 发光波长 532nm， 发射光波长 570nm， 激光强度和增益分别为 60/69， 读取的信号分别经处理软件 ZoCSoft ImageBoost (针对低弱目标信号- 背景比芯片试剂盒） 和 JAGUARII (针对参照芯片试剂盒） 处理， 然 后取平均值后得到结果。 使用低弱目标信号 -背景比芯片试剂盒进行的 检测， 弱目标信号 -背景比小于 0.1， 且阴、 阳性结果与所用样品一致。 使用参照芯片试剂盒进行的检测， 阳性对照在稀释至 50倍时为阴性结 果； 而使用低弱目标信号 -背景比芯片试剂盒进行的检测， 阳性对照在 稀释至 500倍时仍为阳性结果。 实施例 13: 着色芯片检测 The scanner is a confocal laser scanner (Afymetrix GMS 418). The scanning light wavelength is 532nm, the emission light wavelength is 570nm, the laser intensity and gain are 60/69, and the read signals are processed by software ZoCSoft ImageBoost (for low and strong The target signal-background ratio chip kit) and JAGUARII (for the reference chip kit) are processed, and then the average value is obtained to obtain the result. Detection using low-weak target signal-background ratio chip kit, the weak target signal-background ratio is less than 0.1, and the negative and positive results are consistent with the sample used. For the tests performed with the reference chip kit, the positive control was negative when diluted to 50 times. For the tests performed with the low target signal-background ratio chip kit, the positive control was still positive when diluted to 500 times. Example 13: Color chip detection
本实施例所用样品如上所述（样品 1-4号） ， 所用弱目标样品为其 中的阴性样品。 本实施例的检测， 分别为深色芯片检测和浅色芯片检  The samples used in this example are as described above (samples 1-4), and the weak target sample used is the negative sample among them. The detection in this embodiment is respectively a dark chip detection and a light chip detection.
1 )深色芯片检测 1) Dark chip detection
所用芯片试剂盒分别为实施例 8制备的深色芯片试剂盒 26 (黑色塑 料片基芯片试剂盒） 、 27 (珍珠黑喷漆 /载玻片片基芯片试剂盒） 、 28 (珍珠绿喷漆 /载玻片片基芯片试剂盒） 、 29 (环氧基玻片 /哑光黑喷 漆片基芯片试剂盒）、 30 (正面贴黑色膜片基芯片试剂盒）、和 31 (背 面贴黑色膜片基片基芯片试剂盒） 。 所用参照芯片试剂盒为以表 1 中 的氨基玻片为片基按常规方法制备的荧光标记芯片试剂盒。  The chip kits used were the dark chip kits 26 (black plastic chip-based chip kits), 27 (pearl black spray paint / slide chip-based chip kits), 28 (pearl green spray paint / loaded) prepared in Example 8, respectively. Slide-based chip kit), 29 (epoxy slide / matte black spray-painted chip-based chip kit), 30 (front-mounted black film-based chip kit), and 31 (back-mounted black film-based chip kit) Chip-based kits). The reference chip kit used was a fluorescently labeled chip kit prepared in accordance with conventional methods using the amino slides in Table 1 as a base.
实验时前述 4种样品 （稀释至 1/20至 1/100之间）分别加入芯片试 剂盒的反应器中， 加样量均为 15ul。 反应 30分钟后洗涤 5次。 标记物 加入量为 15ul， 反应后洗涤 5次， 干燥后进行扫描。 扫描仪为共聚焦 激光扫描仪（Afymetrix公司 GMS 418) ， 扫描激发光波长 532nm， 发 射光波长 570i m， 激光强度和增益分别为 60/69， 读取的信号经处理软 件 JAGUARII处理， 然后取平均值后得到结果。 使用深色芯片试剂盒 进行的检测， 阴、 阳性结果与所用样品一致。 使用参照芯片试剂盒进 行的检测， 阳性对照 1在稀释至 100倍时为阴性结果； 而使用深色芯 片试剂盒进行的检测， 阳性对照在稀释至 200倍时仍为阳性结果。  During the experiment, the aforementioned four samples (diluted to 1/20 to 1/100) were respectively added to the reactor of the chip reagent box, and the sample volume was 15 ul. After 30 minutes of reaction, it was washed 5 times. The labeling amount was 15 ul, washed 5 times after the reaction, and scanned after drying. The scanner is a confocal laser scanner (Afymetrix GMS 418). The scanning light wavelength is 532nm and the emission light wavelength is 570im. The laser intensity and gain are 60/69. The read signals are processed by the processing software JAGUARII and then averaged. Get the result after value. The detection using dark chip kits showed negative and positive results consistent with the samples used. For the test using the reference chip kit, the positive control 1 was negative when diluted to 100 times; for the test using the dark chip kit, the positive control was still positive when diluted to 200 times.
2) 浅色芯片检测 2) Light chip detection
所用芯片试剂盒为实施例 8制备的浅色芯片试剂盒 [芯片试剂盒 32 (白喷漆 /载玻片片基芯片试剂盒） ]。  The chip kit used was the light-colored chip kit prepared in Example 8 [chip kit 32 (white spray paint / slide-based chip kit)].
实验时按照金-银标记法（参考中国专利申请 00807744.4)操作。在 每一个生物芯片探针板的 4个反应器中均分别加有 1 : 100稀释的样品。 加样量均为 15 μ 1。 反应 30分钟后洗涤 5次。 然后分别加入标记物、 标记放大物、 反应中止物等， 洗涤 5 次后干燥， 然后进行扫描。 扫描 仪为 EPSON 1260扫描仪 （EPSON公司） ， 扫描照射光线为白光， 信 号光线亦为白光，读取的信号经处理软件（ZoCSoft lmageBoost)处理， 然后取平均值后得到阴性或阳性反应结果。 所得阳性样品标记结果为 黑色沉淀 （可用肉眼观察） ， 阴、 阳性结果与所用样品一致。 实施例 14: 定点着色芯片检测 The experiment was performed according to the gold-silver labeling method (refer to Chinese patent application 00807744.4). In each of the four reactors of the biochip probe plate, a 1: 100 diluted sample was added. The sample volume was 15 μ1. After 30 minutes of reaction, it was washed 5 times. Then add the label, label amplification, reaction stopper, etc., wash 5 times, dry, and then scan. The scanner is an EPSON 1260 scanner (EPSON company). The scanning light is white and the signal light is white. The read signal is processed by processing software (ZoCSoft lmageBoost). Then take the average to get negative or positive results. The positive results obtained are marked as black precipitates (observable with the naked eye), and the negative and positive results are consistent with the samples used. Example 14: Spot color chip detection
本实施例所用样品、弱目标样品同实施例 13。本实施例所用芯片试 剂盒分别为实施例 9制备的定点着色芯片试剂盒 [芯片试剂盒 32 (黑 色涂料定点着色载玻片片基芯片试剂盒） 、 33 (黑色涂料定点着色环 氧基玻片片基芯片试剂盒） 、 34 (黑色涂料定点着色环氧基玻片片基 芯片试剂盒） 和 35 (黑色探针芯片试剂盒） ]。  The sample and weak target sample used in this example are the same as in Example 13. The chip kits used in this example are the fixed-point colored chip kits prepared in Example 9 [chip kit 32 (black paint fixed-point colored slide glass-based chip kit), 33 (black paint fixed-point colored epoxy glass slides) Chip-based chip kits), 34 (black paint fixed-point stained epoxy-based glass slide chip-based kits) and 35 (black probe chip kits)].
实验时前述 4种样品 （稀释至 1/20至 1/100之间） 分别加入芯片试 剂盒的反应器中， 加样量均为 15 μ 1。 反应 30分钟后洗涤 5次。 标记 物加入量为 15 μ 1， 反应后洗涤 5次， 干燥后进行扫描。 扫描仪为共聚 焦激光扫描仪 （Afymetrix公司 GMS 418) ， 扫描激发光波长 532nm, 发射光波长 570nm， 激光强度和增益分别为 60/69， 读取的信号经处理 软件 JAGUARII处理， 然后取平均值后得到结果。 使用定点着色芯片 试剂盒进行的检测， 阴、 阳性结果与所用样品一致。  During the experiment, the aforementioned four samples (diluted to 1/20 to 1/100) were respectively added to the reactor of the chip reagent cartridge, and the sample loading amounts were all 15 μ1. After 30 minutes of reaction, it was washed 5 times. The labeling amount was 15 μ1. After the reaction, it was washed 5 times and dried and then scanned. The scanner is a confocal laser scanner (Afymetrix Corporation GMS 418). The scanning light wavelength is 532nm, the emission light wavelength is 570nm, the laser intensity and gain are 60/69, and the read signal is processed by the processing software JAGUARII, and then averaged. After getting the results. The detection using the fixed-point staining chip kit, the negative and positive results are consistent with the samples used.

Claims

权利要求书 Claim
1、 一种样品、 特别是生物样品的芯片检测方法， 其包括芯片试剂 盒制备过程和样品检测信号形成过程， 该方法的特征在于包含对芯片进 行着色以使背景与目标之间的色差最大化的步骤， 所述着色包含加入着 色剂或含着色剂的着色物， 并且针对的是芯片反应器中的探针点或 /和包 围探针点的区域， 但不包括对探针或 /和探针捕获物的标记。 1. A chip detection method for a sample, particularly a biological sample, comprising a chip kit preparation process and a sample detection signal formation process, the method is characterized by including coloring the chip to maximize the color difference between the background and the target Step, the coloring includes adding a colorant or a colorant containing the colorant, and is directed to the probe point in the chip reactor or / and the area surrounding the probe point, but does not include the probe or / and the probe Mark of the needle catch.
2、根据权利要求 1所述的检测方法，其中所述背景与目标之间的色 差最大化包括下述之任一种选择: A. 当标记物质为发光剂时， 使所述芯 片背景或 /和探针点在白光下为深色、 优选方案为黑色； B. 当标记物质 为在白光下深色、 优选黑色的非发光剂时， 使所述芯片背景或 /和探针点 在白光下为浅色、优选方案为白色； C 当标记物质为在白光下浅色、优 选白色的非发光剂时， 使所述芯片背景或 /和探针点在白光下为深色、 优 选为黑色。  2. The detection method according to claim 1, wherein maximizing the color difference between the background and the target includes any one of the following options: A. When the labeling substance is a luminescent agent, make the chip background or / And the probe point is dark under white light, preferably black; B. when the labeling substance is a dark, preferably black non-luminescent agent under white light, the chip background or / and the probe point is under white light It is light-colored, preferably white; C When the labeling substance is a light-colored, preferably white, non-luminous agent under white light, the chip background or / and probe dots are dark-colored, preferably black under white light.
3、 根据权利要求 1或 2所述的检测方法， 其中所述着色剂选自下述 一种或多种物质： 染料、 颜料和消光剂。  3. The detection method according to claim 1 or 2, wherein the colorant is selected from one or more of the following: a dye, a pigment, and a matting agent.
4、 根据权利要求 1一 3之一所述的检测方法， 其中所述着色物选自 含所述着色剂的下述一种或多种材料： 有色膜、 有色薄片、 有色包被和 有色涂料。  4. The detection method according to any one of claims 1 to 3, wherein the colorant is selected from one or more of the following materials containing the colorant: a colored film, a colored sheet, a colored coating, and a colored paint .
5、根据权利要求 4所述的制备方法，其中所述有色涂料还含有可结 合探针的物质。  The preparation method according to claim 4, wherein the colored paint further contains a substance capable of binding a probe.
6、根据权利要求 5所述的制备方法，其中所述可结合探针的物质包 括下述一种或多种有机物及其衍生物： 硝化纤维素、 聚苯乙烯、 聚丙烯 酸脂、 聚砜、 聚醚砜、 聚氯乙烯、 氨基树脂、 聚多糖、 聚氨基酸。  6. The method according to claim 5, 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.
7、 根据权利要求 4一 6之一所述的检测方法， 其中所述有色涂料含 消光剂。  7. The detection method according to any one of claims 4 to 6, wherein the colored paint contains a matting agent.
8、 根据权利要求 4一 7之一所述的检测方法， 其中所述有色涂料选 自于色漆。  8. The detection method according to any one of claims 4 to 7, wherein the colored paint is selected from a colored paint.
9、 根据权利要求 4一 8之一所述的检测方法， 其中所述着色包括将 探针与所述有色涂料混合后再点样至片基上制作芯片。  9. The detection method according to any one of claims 4 to 8, wherein the coloring comprises mixing a probe with the colored paint and then spotting it on a substrate to make a chip.
10、一种样品、特别是生物样品的芯片检测方法， 其包括芯片试剂 盒制备过程和样品检测信号形成过程， 该方法的特征在于使得检测信号 的读取在下述条件下进行： 芯片弱目标信号 -背景比小于 0.80、 优选小于 0.50、 更优选小于 0.25。 10. A chip detection method for a sample, particularly a biological sample, comprising a chip reagent The method of cartridge preparation and sample detection signal formation is characterized in that the detection signal is read under the following conditions: chip weak target signal-background ratio is less than 0.80, preferably less than 0.50, more preferably less than 0.25.
11、根据权利要求 10所述的检测方法， 其中所述条件是通过对芯片 进行着色获得的。  The detection method according to claim 10, wherein the condition is obtained by coloring a chip.
12、根据权利要求 11所述的检测方法， 其中所述着色选自下述一种 或多种着色： A. 在芯片试剂盒的制备过程中对芯片背景进行加强信号的 着色； B. 在样品检测信号形成过程中对芯片背景进行加强信号的着色； C. 在芯片试剂盒的制备过程中对探针点进行降低弱目标信号的着色； . 在样品检测信号形成过程中对探针点进行降低弱目标信号的着色。  12. The detection method according to claim 11, wherein the coloring is selected from one or more of the following: A. coloring the chip background to enhance the signal during the preparation of the chip kit; B. in the sample Colorize the background of the chip to strengthen the signal during the formation of the detection signal; C. Color the probe points to reduce the weak target signal during the preparation of the chip kit; Reduce the probe points during the formation of the sample detection signal Coloring of weak target signals.
13、根据权利要求 12所述的检测方法， 其中所述加强信号的着色包 括在所述背景中弓 I入发光剂或含发光剂的发光物。  13. The detection method according to claim 12, wherein the coloring of the enhanced signal includes a luminescent agent or a luminescent agent containing a luminescent agent in the background.
14、根据权利要求 13所述的检测方法， 其中所述发光剂选自于下述 一种或多种可发射信号光线的物质： 包括荧光物质在内的激发发光物质 和包括化学发光物质和电化学发光物质在内的自主发光物质。  14. The detection method according to claim 13, wherein the luminescent agent is selected from one or more of the following substances capable of emitting signal light: an excited luminescent substance including a fluorescent substance, and a chemiluminescent substance and an electroluminescent substance. Autoluminescent substances including chemiluminescent substances.
15、根据权利要求 13所述的检测方法， 其中所述发光物选自于下述 一种或多种： 含发光剂的涂料、薄膜、 片及发光剂与活性分子的复合物， 所述活性分子可固定在芯片样品点周围区域上。  15. The detection method according to claim 13, wherein the luminescent substance is selected from one or more of the following: a luminescent agent-containing coating, a film, a sheet, and a complex of the luminescent agent and an active molecule, the activity Molecules can be immobilized on the area around the chip sample spot.
16、根据权利要求 12— 15之一所述的检测方法， 其中所述降低弱目 标信号的着色包括对探针点引入深色着色剂或含深色着色剂的深色着色 物， 所述深色着色剂选自下述一种或多种颜色为深色、 优选为黑色的物 质： 染料、 有色颜料、 消光剂。  16. The detection method according to any one of claims 12 to 15, wherein the coloring of reducing a weak target signal comprises introducing a dark colorant or a dark colorant containing a dark colorant to the probe point, and the dark The coloring agent is selected from one or more of the following materials, which are dark and preferably black: dyes, colored pigments, and matting agents.
17、根据权利要求 16所述的检测方法， 其中所述深色着色物选自下 述一种或多种： 含所述深色着色剂的涂料、 薄膜、 片及所述着色剂与活 性分子的复合物， 所述活性分子在样品检测信号形成过程中可固定在芯 片样品点中除探针捕获的样品目标物以外的物质上。 .  17. The detection method according to claim 16, wherein the dark colorant is selected from one or more of the following: coatings, films, sheets containing the dark colorant, and the colorant and active molecules The active molecule may be fixed to a substance other than the sample target captured by the probe in the chip sample spot during the formation of the sample detection signal. .
18、根据权利要求 17所述的检测方法， 其中所述着色包括将探针与 含所述深色着色剂的涂料混合后再点样至片基上制作芯片。  18. The detection method according to claim 17, wherein the coloring comprises mixing a probe with a coating containing the dark colorant, and then spotting the chip onto a substrate.
19、根据权利要求 11一 18之一所述的检测方法，其中所述着色包括 在所述样品检测信号的形成过程中进行的着色。  The detection method according to any one of claims 11 to 18, wherein the coloring includes coloring performed during the formation of the sample detection signal.
20、根据权利要求 19所述的检测方法， 其中所述着色包含将样品与 所述发光剂或发光物反应后再加入反应器、 并使其中包围探针点的区域 上的检测信号增强 200%以上、 优选 500%以上。 20. The detection method according to claim 19, wherein the coloring comprises combining a sample with After the luminescent agent or luminescent substance is reacted, the reactor is added, and the detection signal on the area surrounding the probe point is increased by 200% or more, preferably 500% or more.
21、根据权利要求 19所述的检测方法， 其中所述着色包含将样品加 入反应器后再加入所述发光剂或发光物进行反应、 并使其中包围探针点 的区域上的检测信号增强 200%以上、 优选 500 %以上。  21. The detection method according to claim 19, wherein the coloring comprises adding a sample to the reactor and then adding the luminescent agent or luminescent substance for reaction, and enhancing the detection signal on the area surrounding the probe point by 200. % Or more, preferably 500% or more.
22、一种平面芯片片基， 其特征在于包含着色剂或含着色剂的着色 物。  22. A flat chip substrate, characterized by comprising a colorant or a colorant containing a colorant.
23、 根据权利要求 22所述的芯片片基， 其为按权利要求 1-21之一所 述芯片检测方法进行着色而制成。  23. The chip substrate according to claim 22, which is made by coloring according to the chip detection method according to any one of claims 1-21.
24、根据权利要求 23所述的芯片片基， 其中所述着色为体着色、 面 着色或定点着色。  24. The chip substrate according to claim 23, wherein the coloring is bulk coloring, surface coloring, or spot coloring.
25、根据权利要求 24所述的芯片片基， 其含玻璃基质和所述有色涂 料涂层。  25. The chip substrate according to claim 24, comprising a glass substrate and the colored paint coating.
26、根据权利要求 25所述的芯片片基，其中所述有色涂料为黑色的 漆或白色的漆。  26. The chip substrate according to claim 25, wherein the colored paint is a black paint or a white paint.
27、 根据权利要求 22— 26之一所述的芯片片基， 其表面粗糙度 Pfa 在 0.02— 3.0 μ m之间、 优选方案在 0.25— 3.0 μ m之间。  27. The chip substrate according to claim 22, wherein a surface roughness Pfa is between 0.02 and 3.0 μm, and a preferred solution is between 0.25 and 3.0 μm.
28、 一种芯片基片， 其包含着色平面片基及任选存在的隔离结构， 所述着色平面片基含着色剂或含着色剂的着色物。  28. A chip substrate comprising a colored planar substrate and an optional isolation structure, wherein the colored planar substrate contains a colorant or a colorant containing a colorant.
29、根据权利要求 28所述的芯片基片，其中所述着色平面片基为根 据权利要求 22 27之一所述的芯片片基。  The chip substrate according to claim 28, wherein the colored flat substrate is a chip substrate according to one of claims 22 to 27.
30、 一种芯片探针板， 其包含平面片基、 固定在所述片基上的探针 点及任选存在的隔离结构， 该芯片探针板的特征在于： A. 所述平面片基 含着色剂或含着色剂的着色物； 或 /和 B. 所述探针点含着色剂或含着色 剂的着色物， 但不含标记物质。  30. A chip probe board comprising a flat substrate, a probe point fixed on the substrate, and an optional isolation structure, the chip probe board is characterized by: A. the planar substrate A colorant or a colorant containing colorant; or / and B. The probe point contains a colorant or a colorant-containing colorant, but does not contain a labeling substance.
31、 根据权利要求 30所述的芯片探针板， 其为按权利要求 1-21之一 所述芯片检测方法着色而制备的芯片探针板。  31. The chip probe card according to claim 30, which is a chip probe card prepared by coloring according to the chip detection method according to one of claims 1-21.
32、根据权利要求 31所述的芯片探针板， 其包含如权利要求 22— 29 之一所述的芯片基片。  32. The chip probe card according to claim 31, comprising a chip substrate according to any one of claims 22-29.
33、根据权利要求 31或 32所述的芯片探针板，其探针点含所述着色 剂或着色物。 33. The chip probe card according to claim 31 or 32, wherein a probe point thereof contains the colorant or colorant.
34、 一种芯片试剂盒， 其包括芯片探针板和任选存在的标记系统， 该芯片试剂盒的特征在于： A.所述芯片探针板中的平面片基含着色剂或 含着色剂的着色物;或 /和 B.所述芯片探针板中的探针点含着色剂或含着 色剂的着色物，但不含标记物质； 或 /和 C.其还含着色系统， 所述着色系 统含着色剂或含着色剂的着色物。 34. A chip kit comprising a chip probe card and an optional labeling system, the chip kit is characterized by: A. The flat film base in the chip probe card contains a colorant or a colorant Or. And / or B. the probe point in the chip probe card contains a colorant or a colorant containing colorant, but does not contain a labeling substance; or / and C. it also contains a coloring system, said The coloring system contains a colorant or a colorant containing a colorant.
35、根据权利要求 34所述的芯片试剂盒，其为按权利要求 1一 9之一 所述的芯片检测方法制备的芯片试剂盒。  35. The chip kit according to claim 34, which is a chip kit prepared according to the chip detection method according to any one of claims 1 to 9.
36、 根据权利要求 34所述的芯片试剂盒， 其弱目标信号-背景比小 于 0.80、 优选小于 0.50、 更优选小于 0.25。  36. The chip kit according to claim 34, wherein the weak target signal-background ratio is less than 0.80, preferably less than 0.50, more preferably less than 0.25.
37、 根据权利要求 36所述的芯片试剂盒， 其为按权利要求 10— 21 之一所述的芯片检测方法制备的芯片试剂盒。  37. The chip kit according to claim 36, which is a chip kit prepared according to the chip detection method according to any one of claims 10-21.
38、根据权利要求 35— 37之一所述的芯片试剂盒，其包含权利要求 30— 33之一所述的芯片探针板。  38. The chip kit according to any one of claims 35 to 37, comprising the chip probe card according to one of claims 30 to 33.
39、 根据权利要求 35— 37之一所述的芯片试剂盒， 其标记系统和 / 或着色系统含所述发光剂或 /和所述发光物。  39. The chip kit according to any one of claims 35 to 37, wherein a marking system and / or a coloring system contains the luminescent agent or / and the luminescent substance.
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