Standard chip and preparation method and application thereof

Abstract

The invention discloses a standard chip and a preparation method and application thereof. The standard chip comprises a first chip substrate, a first compound and a signal body, the first compound is fixed on at least partial area of at least one surface of the first chip substrate, the signal body comprises a signal group and a signal carrier, the signal group is connected with the signal carrier, and the signal carrier is connected with the first compound. The signal carrier is attached to the first compound. The standard chip provided by the invention has the advantages of high signal strength, slow attenuation, long service life, no need of additional biochemical reagents to generate signals, simplicity in preparation and capability of realizing repeated production, and can be used for gene sequencing instrument performance evaluation or equipment debugging.

Description

Technical Field

[0001] The present invention relates to the technical field of biochips, and in particular to a standard chip and a preparation method and application thereof. Background Art

[0002] During the production process of gene sequencers, the system needs to be debugged to meet the requirements of sequencing, including instrument platform leveling and optical system calibration, such as laser power parameter debugging. If sequencing chips are used for system debugging, a series of problems will arise, such as: the signal strength of the sequencing chip decays rapidly with the number of uses, which makes the debugging process of the gene sequencer produce an uncontrollable variable, resulting in difficulty in obtaining the same debugging results even if the optical system under the same conditions is debugged, thus affecting the judgment of the debugging results. In addition, sequencing chips usually require matching fluid systems and reagents for biochemical reactions to obtain sequencing signals for system debugging, which increases the complexity of debugging. Not only that, for gene sequencers that have not yet been completed, the method of debugging using sequencing chips may not be implemented because they are not yet equipped with structures or devices for fluid and reagent reactions, greatly reducing the scope of use of sequencing chips for system debugging.

[0003] Therefore, there is an urgent need to provide a standard chip with high signal intensity, slow decay, no need for additional biochemical reagents to generate signals, and reproducible production. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems in the related art to a certain extent. To this end, the present invention provides a standard chip and a preparation method and application thereof. The standard chip has high signal strength, slow decay, long service life, does not require additional biochemical reagents to generate signals, and can achieve repeatable production. It can provide reliable debugging parameters for the production and debugging of gene sequencing instruments, so that the instruments have unified standards when leaving the factory, and greatly simplify the debugging process of the sequencing hardware system.

[0005] To this end, the first aspect of the present invention provides a standard chip, which includes a first chip substrate, a first compound and a signal body, wherein the first compound is fixed to at least a partial area of ​​at least one surface of the first chip substrate, and the signal body includes a signal group and a signal carrier, wherein the signal group is connected to the signal carrier, and the signal carrier is connected to the first compound.

[0006] Compared with the sequencing chip, the standard chip provided by the present invention has a more diverse surface modification process. At the same time, the signal generation method when using the standard chip is also different from that of the sequencing chip. The sequencing chip needs to undergo a sequencing biochemical reaction before signal collection, while the standard chip can fix a group with a signal on the surface after modification and packaging. After one modification, the signal can be collected multiple times without the need for a biochemical process. In addition, the signal intensity generated by the standard chip provided by the present invention is much higher than that generated by the sequencing chip, and it can be reused without significant signal attenuation, which can provide a stable reference for system debugging.

[0007] According to an embodiment of the present invention, the first compound has a first functional group, the signal carrier has a second functional group, and the first functional group and the second functional group are mutually chemically reactive functional groups.

[0008] According to an embodiment of the present invention, the first functional group includes at least one selected from halogen, nitro, ester, thiol, epoxy, amino, carboxyl, hydroxyl, azido, alkenyl, and alkynyl.

[0009] According to an embodiment of the present invention, the first compound is a thiol or a silane molecule.

[0010] According to a preferred embodiment of the present invention, the molecular formula of the silane molecule is X a Si(Y b )M c Z, where

[0011] X is H, F, Cl, Br, I, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy;

[0012] Y is independently selected from C 1-10 alkyl;

[0013] M c Independently selected from C 1-50 Alkylene, -(OCH2CH2) c -、-(CH(OH)CH2-) c ;

[0014] Z is independently selected from the following groups which are unsubstituted or substituted: 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, C 3-20 Cycloalkyl, 3-20 membered heterocyclic group, C 6-20 Aryl, 5-20 membered heteroaryl;

[0015] The C 1-20 Alkyl, C 2-20Alkenyl, C 2-20 Alkynyl, C 3-20 Cycloalkyl, 3-20 membered heterocyclic group, C 6-20 Aryl, 5-20 membered heteroaryl, at least one R a Substitution, the R a Each is independently selected from halogen, nitro, ester, amino, carboxyl, hydroxyl, azido, epoxy, and thiol;

[0016] The a, b, and c are all integers, and their values ​​satisfy: 0≤a≤3, 0≤b≤3, and a+b=3; 1≤c≤50.

[0017] According to an embodiment of the present invention, the molar ratio of the second functional group to the signal group is 1:1-1:50.

[0018] According to an embodiment of the present invention, the second functional group includes at least one selected from halogen, thiol, aldehyde, hydroxyl, carboxyl, amino, alkenyl, alkynyl, and azido.

[0019] According to an embodiment of the present invention, the signal carrier includes at least one selected from polymer 1, antigen-antibody complex, nanoparticle, and nucleic acid molecule.

[0020] According to an embodiment of the present invention, the signal carrier includes a polymer 1, and the polymer is selected from at least one of polyethylene glycol, polypropylene, polyacrylic acid, and polymethyl methacrylate.

[0021] According to an embodiment of the present invention, in the first chip substrate region where the first compound is fixed, the density of the polymer 1 is 0.001-1 per μm 2 .

[0022] According to an embodiment of the present invention, the size of the polymer 1 is 5nm-500nm.

[0023] According to an embodiment of the present invention, the signal carrier includes an antigen-antibody complex. In the first chip substrate region where the first compound is fixed, the density of the antigen-antibody complex is 0.001-5 per μm. 2 .

[0024] According to an embodiment of the present invention, the signal group is connected to the antibody in the antigen-antibody complex, and the first compound is connected to the antigen in the antigen-antibody complex.

[0025] According to an embodiment of the present invention, the signal carrier includes nanoparticles, and the nanoparticles include at least one selected from silica nanoparticles, zirconium oxide nanoparticles, cerium oxide nanoparticles, aluminum oxide nanoparticles, gold nanoparticles, DNA nanospheres, RNA nanospheres, magnetic beads bound to DNA, and magnetic beads bound to RNA.

[0026] According to an embodiment of the present invention, in the first chip substrate region where the first compound is fixed, the density of the nanoparticles is 0.001-1 / μm 2 .

[0027] According to an embodiment of the present invention, the signal carrier includes a nucleic acid molecule, and the nucleic acid molecule is a single-stranded nucleic acid molecule or a double-stranded nucleic acid molecule.

[0028] According to an embodiment of the present invention, the length of the nucleic acid molecule is 1-200 bp.

[0029] According to a specific embodiment of the present invention, the length of the nucleic acid molecule is 20-100 bp.

[0030] According to a specific embodiment of the present invention, the length of the nucleic acid molecule is 30-80 bp.

[0031] According to a specific embodiment of the present invention, the length of the nucleic acid molecule is 40-70 bp.

[0032] According to an embodiment of the present invention, in the first chip substrate region where the first compound is fixed, the density of the nucleic acid molecules is 0.001-5 / μm 2 .

[0033] According to an embodiment of the present invention, the signal group is connected to any nucleic acid chain in the nucleic acid molecule.

[0034] According to an embodiment of the present invention, the particle size or dimension of the signal carrier is 5nm-500nm.

[0035] According to an embodiment of the present invention, the signal group is a fluorescent group.

[0036] According to an embodiment of the present invention, the excitation wavelength of the fluorescent group is 300-800 nm, and the emission wavelength is 300-800 nm.

[0037] According to an embodiment of the present invention, the fluorescent group includes at least one selected from cy3, cy5, HEX, atto, and FAM.

[0038] According to an embodiment of the present invention, a metal layer is provided between the first compound and the first chip substrate.

[0039] According to an embodiment of the present invention, the metal of the metal layer includes at least one selected from Au and Al.

[0040] According to an embodiment of the present invention, the thickness of the metal layer is less than or equal to 1 μm.

[0041] According to an embodiment of the present invention, the first chip substrate has a regularly arranged geometric structure, and the first compound is fixed on the surface of the geometric structure.

[0042] According to an embodiment of the present invention, the geometric structure is a depression or a protrusion.

[0043] According to an embodiment of the present invention, the geometric structure is a cylinder, a cone, a truncated cone, a prism, a pyramid, a truncated cone or an irregular shape.

[0044] According to an embodiment of the present invention, the geometric structure is a cylinder, the cross-sectional diameter of the cylinder is 300nm-500nm, the height of the cylinder is 300nm-500nm, and the center distance between adjacent cylinders is 600nm-1000nm.

[0045] According to an embodiment of the present invention, the geometric structure is arranged in a polygonal arrangement or a honeycomb arrangement.

[0046] According to an embodiment of the present invention, the standard chip further includes a second chip substrate, and the second chip substrate is fixed to a side of the first chip substrate having the signal body.

[0047] According to an embodiment of the present invention, the signal body is fixed to at least a partial area on the surface of the second chip substrate on the side opposite to the first chip substrate, the signal body includes a signal group and a signal carrier, the signal group is connected to the signal carrier, and the signal body is the same as or different from the signal fixed on the first chip substrate.

[0048] According to an embodiment of the present invention, the standard chip has a channel arranged in the length direction, and the signal body is at least combined with the surface of the first chip substrate in the area where the channel is located.

[0049] According to an embodiment of the present invention, the standard chip is used for gene sequencer performance evaluation and / or equipment debugging.

[0050] A second aspect of the present invention provides a method for preparing a standard chip, comprising the following steps:

[0051] Step 1: fixing a first compound on at least a partial area of ​​at least one surface of a first chip substrate;

[0052] Step 2: Connect a signal body to the first compound to obtain a standard chip, wherein the signal body includes a signal group and a signal carrier, the signal group is connected to the signal carrier, and the signal carrier is fixed on the first compound.

[0053] The preparation method of the standard chip provided by the present invention is simpler than that of the sequencing chip and can shorten the chip preparation process.

[0054] According to an embodiment of the present invention, the first compound has a first functional group, the signal carrier has a second functional group, and the first functional group and the second functional group are mutually chemically reactive functional groups.

[0055] According to an embodiment of the present invention, the first functional group is selected from at least one of halogen, thiol, nitro, ester, epoxy, amino, carboxyl, hydroxyl, azido, alkenyl, and alkynyl.

[0056] According to an embodiment of the present invention, the first compound is a thiol or a silane molecule.

[0057] According to an embodiment of the present invention, the molecular formula of the silane molecule is X a Si(Y b )M c Z, where

[0058] X is H, F, Cl, Br, I, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy;

[0059] Y is independently selected from C 1-10 alkyl;

[0060] M c Independently selected from C 1-50 Alkylene, -(OCH2CH2) c -、-(CH(OH)CH2-) c ;

[0061] Z is independently selected from the following groups which are unsubstituted or substituted: 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, C 3-20 Cycloalkyl, 3-20 membered heterocyclic group, C 6-20 Aryl, 5-20 membered heteroaryl;

[0062] The C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, C 3-20 Cycloalkyl, 3-20 membered heterocyclic group, C 6-20Aryl, 5-20 membered heteroaryl, at least one R a Substitution, the R a Each is independently selected from halogen, nitro, ester, amino, carboxyl, hydroxyl, azido, epoxy, and thiol;

[0063] The a, b, and c are all integers, and their values ​​satisfy: 0≤a≤3, 0≤b≤3, and a+b=3; 1≤c≤50.

[0064] According to an embodiment of the present invention, the second functional group is selected from at least one of halogen, thiol, aldehyde, hydroxyl, carboxyl, amino, alkenyl, alkynyl, and azido.

[0065] According to an embodiment of the present invention, the method for fixing the first compound in step one includes at least one of chemical vapor deposition, physical vapor deposition, atomic layer deposition, magnetron sputtering, solution method, spin coating, and spray coating.

[0066] According to an embodiment of the present invention, the first compound is a silane molecule, and the silane molecule is fixed on the first chip substrate by chemical vapor deposition.

[0067] According to an embodiment of the present invention, the reaction temperature of the chemical vapor deposition method is 25-100° C., and the reaction time is 2-48 hours.

[0068] According to an embodiment of the present invention, the silane molecules include trimethoxyaminosilane and triethoxyaminosilane.

[0069] According to an embodiment of the present invention, the volume ratio of the trimethoxyaminosilane to the triethoxyaminosilane is 1:1-1:20.

[0070] According to an embodiment of the present invention, the signal carrier includes at least one selected from polymer 1, nanoparticles, nucleic acid molecules, and antigen-antibody complexes.

[0071] According to an embodiment of the present invention, when the signal carrier is polymer 1, the method for introducing the signal body includes: applying the polymer 1 modified with the signal group to the surface of the first chip substrate fixed with the first compound obtained in step 1, so that the polymer is bound to the surface of the first chip substrate.

[0072] According to an embodiment of the present invention, the introduction of the signal body is carried out at 25° C.-100° C., and the reaction time is 1 h-48 h.

[0073] According to an embodiment of the present invention, the polymer 1 is selected from at least one of polyethylene glycol, polypropylene, polyacrylic acid, and polymethyl methacrylate.

[0074] According to an embodiment of the present invention, the size of the polymer 1 is 5nm-500nm.

[0075] According to an embodiment of the present invention, in the first chip substrate region where the first compound is fixed, the density of the polymer 1 is 0.001-1 per μm 2 .

[0076] According to an embodiment of the present invention, when the signal carrier is a nanoparticle, the method for introducing the signal body includes: applying the nanoparticle modified with the signal group to the surface of the first chip substrate fixed with the first compound obtained in step one, so that the nanoparticle is bound to the surface of the first chip substrate.

[0077] According to an embodiment of the present invention, the introduction of the signal body is carried out at 25-100° C., and the reaction time is 1-48 hours.

[0078] According to an embodiment of the present invention, the nanoparticles are selected from at least one of silica nanoparticles, zirconium oxide nanoparticles, cerium oxide nanoparticles, aluminum oxide nanoparticles, gold nanoparticles, DNA nanospheres, RNA nanospheres, magnetic beads combined with DNA, and magnetic beads combined with RNA.

[0079] According to an embodiment of the present invention, the particle size of the nanoparticles is 5nm-500nm.

[0080] According to an embodiment of the present invention, in the first chip substrate region where the first compound is fixed, the density of the nanoparticles is 0.001-1 / μm 2 .

[0081] According to an embodiment of the present invention, when the signal carrier is a nucleic acid molecule, the method for introducing the signal body includes:

[0082] Method 1: When the nucleic acid molecule is a double-stranded nucleic acid molecule, the double-stranded nucleic acid molecule modified with the signal group is applied to the surface of the first chip substrate fixed with the first compound obtained in step 1, so that the double-stranded nucleic acid molecule is bound to the surface of the first chip substrate;

[0083] Or method two, when the nucleic acid molecule is a single-stranded nucleic acid molecule, a first single-stranded nucleic acid molecule not modified with a signal group is applied to the surface of the first chip substrate fixed with the first compound obtained in step one, so that the first single-stranded nucleic acid molecule is bound to the surface of the first chip substrate; a second single-stranded nucleic acid molecule is added, wherein the second single-stranded nucleic acid molecule is bound to a signal group and contains a base that is complementary to a part of the first single-stranded nucleic acid molecule, so that the second single-stranded nucleic acid molecule is connected to the first single-stranded nucleic acid molecule.

[0084] According to an embodiment of the present invention, the length of the nucleic acid molecule is 1-200 bp.

[0085] According to an embodiment of the present invention, the introduction of the signal body is carried out at 25-100° C., and the reaction time is 1-48 hours.

[0086] According to an embodiment of the present invention, in the first chip substrate region where the first compound is fixed, the density of the nucleic acid molecules is 0.001-5 / μm 2 .

[0087] According to an embodiment of the present invention, when the signal carrier is an antigen-antibody complex, the method for introducing the signal body includes: applying the antigen-antibody complex modified with the signal group to the surface of the first chip substrate fixed with the first compound obtained in step one, so that the antigen-antibody complex is bound to the surface of the first chip substrate.

[0088] According to an embodiment of the present invention, the introduction of the signal body is carried out at 25-100° C., and the reaction time is 1-48 hours.

[0089] According to an embodiment of the present invention, the signal group in step 2 includes at least one selected from cy3, cy5, HEX, atto, and FAM.

[0090] According to an embodiment of the present invention, in step 2, the signal carrier is fixed on the first compound by using at least one of a solution method, a spin coating method, a screen printing method, and a spray coating method.

[0091] According to an embodiment of the present invention, in step 2, the signal carrier is fixed on the first compound by physical adsorption or chemical coupling.

[0092] According to an embodiment of the present invention, the physical adsorption includes at least one of hydrogen bonding, van der Waals force, and like dissolves like effect.

[0093] According to an embodiment of the present invention, the chemical coupling includes at least one of hydroxyl-carboxyl coupling, amino-carboxyl coupling, thiol-thiol coupling, and click chemistry.

[0094] According to an embodiment of the present invention, the preparation method further comprises cleaning the surface of the first chip substrate before fixing the first compound.

[0095] According to an embodiment of the present invention, the surface cleaning includes at least one of solution cleaning, plasma activation, and mechanical polishing.

[0096] According to an embodiment of the present invention, the solution cleaning includes at least one of acid reagent or alkaline reagent cleaning, organic reagent cleaning, and aqueous solution ultrasound.

[0097] According to an embodiment of the present invention, the gas used for the plasma activation includes at least one selected from the group consisting of air, oxygen, nitrogen, argon, hydrogen, and carbon tetrafluoride.

[0098] According to an embodiment of the present invention, the mechanical polishing includes performing mechanical polishing using a polishing agent composed of at least one of diamond, silica sol, calcium carbonate, cerium oxide, and zirconium oxide.

[0099] According to an embodiment of the present invention, the preparation method further comprises performing chip packaging after performing step one.

[0100] According to an embodiment of the present invention, the chip packaging method includes: fixing the second chip substrate to the side of the first chip substrate having the first compound, to form a channel having two ends running through in a preset direction.

[0101] According to an embodiment of the present invention, the chip packaging method includes at least one selected from thermal bonding, laser bonding, and chemical and physical bonding.

[0102] According to an embodiment of the present invention, the chemical and physical bonding includes at least one selected from UV curing, thermal curing, and pressure-sensitive bonding.

[0103] According to an embodiment of the present invention, the first chip substrate and the second chip substrate are made of the same material or different materials.

[0104] According to an embodiment of the present invention, the first chip substrate and the second chip substrate are independently selected from at least one of blank or patterned glass, quartz, silicon-based material, and polymer 2.

[0105] According to an embodiment of the present invention, the silicon-based material includes at least one selected from silicon dioxide, polycrystalline silicon, and single crystal silicon.

[0106] According to an embodiment of the present invention, the polymer 2 includes at least one selected from HMDS, PE, and PP.

[0107] The third aspect of the present invention provides use of the standard chip described in the first aspect in performance evaluation or equipment debugging of a gene sequencer.

[0108] A fourth aspect of the present invention provides a debugging method for a sequencer, wherein the sequencer includes an optical imaging system, and the debugging method includes:

[0109] (1) using the optical imaging system to image the standard chip and obtain imaging data;

[0110] (2) debugging the sequencer based on the imaging data obtained in step (1);

[0111] Wherein, the standard chip is the standard chip described in the first aspect.

[0112] The standard chip provided by the present invention is stable and reliable, and can provide reliable debugging parameters for instrument production debugging, so that the instruments have unified standards when leaving the factory, provide a debugging benchmark for mass production of instruments and greatly simplify the debugging process of the sequencing hardware system.

[0113] According to an embodiment of the present invention, debugging the sequencer in step (2) includes debugging a carrier, and the carrier is used to fix the chip.

[0114] According to an embodiment of the present invention, the debugging of the carrier is specifically as follows: exciting the signal group to generate an optical signal through the optical imaging system, and imaging the standard chip; acquiring a fluorescence image of the standard chip; and adjusting the setting parameters of the carrier based on the fluorescence image and a preset image clarity.

[0115] According to an embodiment of the present invention, debugging the sequencer in step (2) includes debugging the optical imaging system.

[0116] According to an embodiment of the present invention, the optical imaging system includes an excitation light source, and the debugging of the optical imaging system includes debugging of laser power parameters.

[0117] According to an embodiment of the present invention, the debugging of the laser power parameters includes: exciting the signal group to generate an optical signal through the optical imaging system; and adjusting the laser power parameters until the optical signal intensity reaches the optical signal intensity.

[0118] A fifth aspect of the present invention provides a method for evaluating the performance of a sequencer, wherein the sequencer comprises a stage and an optical imaging system, and the method comprises the following steps:

[0119] (1) using the optical imaging system to image the standard chip and obtain imaging data, wherein the optical imaging system includes an optical imaging lens, and the imaging data includes a position change of the optical imaging lens during the imaging process, as well as the optical signal intensity of each sequencing channel and a fluorescent panoramic image;

[0120] (2) evaluating the setting of the stage based on the position change of the optical imaging lens during the imaging process, and evaluating the photographing area of ​​the sequencer based on the optical signal intensity and the fluorescent panoramic image;

[0121] Wherein, the standard chip is the standard chip described in the first aspect.

[0122] Compared with the prior art, the present invention has the following beneficial effects:

[0123] (1) Compared with sequencing chips, the standard chip provided by the present invention can adopt more diverse surface modification processes. At the same time, the signal generation method when using the standard chip is also different from that of the sequencing chip. The sequencing chip needs to undergo sequencing biochemical reactions before signal collection, while the standard chip can fix groups with signals on the surface after modification and packaging. After one modification, multiple signals can be collected without the need for a biochemical process.

[0124] (2) The signal intensity generated by the standard chip provided by the present invention is much higher than that generated by the sequencing chip, and can be reused without significant signal attenuation, thus providing a stable reference for system debugging;

[0125] (3) The preparation method of the standard chip provided by the present invention is simpler than that of the sequencing chip, and the chip preparation process can be shortened;

[0126] (4) The standard chip provided by the present invention is stable and reliable, and can provide reliable debugging parameters for instrument production debugging, so that the instruments have unified standards when leaving the factory, provide a debugging benchmark for mass production of instruments and greatly simplify the debugging process of the sequencing hardware system.

[0127] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0128] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0129] Figure 1 The schematic diagram of the preparation process of the fluorescent polymer chip provided in Example 1 and the fluorescent nanoparticle chip provided in Example 2 of the present invention is shown;

[0130] Figure 2 The imaging brightness diagram provided by Example 1 of the present invention is shown;

[0131] Figure 3 Shows Figure 2 Statistical graph of fluorescence signal brightness at the middle horizontal line;

[0132] Figure 4 shows an imaging brightness diagram provided by Example 2 of the present invention;

[0133] Figure 5 Shows Figure 4 Statistical graph of fluorescence signal brightness at the middle horizontal line;

[0134] Figure 6 A schematic diagram of the preparation process of the fluorescent probe chip provided in Example 3 of the present invention is shown;

[0135] Figure 7 shows an imaging brightness diagram provided by Example 3 of the present invention;

[0136] Figure 8 Shows Figure 7 Statistical graph of fluorescence signal brightness at the middle horizontal line;

[0137] Fig. 9 A schematic diagram of the preparation process of the patterned fluorescent polymer chip provided in Example 4 of the present invention is shown;

[0138] Fig.10 shows an imaging brightness diagram provided by Example 4 of the present invention;

[0139] Fig.11 Shows Fig.10 Statistical graph of fluorescence signal brightness at the middle horizontal line;

[0140] Fig.12 The imaging brightness diagram after the instrument is debugged by 20 laser irradiations provided in Example 4 of the present invention is shown;

[0141] Fig.13 Shows Fig.12 Statistical graph of fluorescence signal brightness at the middle horizontal line;

[0142] Fig.14 The imaging brightness diagram provided by Comparative Example 1 of the present invention is shown;

[0143] Fig.15 Shows Fig.14 Statistical graph of fluorescence signal brightness at the middle horizontal line. DETAILED DESCRIPTION

[0144] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0145] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0146] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0147] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0148] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0149] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0150] Definitions and explanations of terms

[0151] Unless otherwise specified, the definitions of groups and terms recorded in the specification and claims of this application, including their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, definitions of specific compounds in examples, etc., can be arbitrarily combined and combined with each other. The group definitions and compound structures after such combination and combination shall fall within the scope recorded in the specification of this application.

[0152] The term "halogen" is to be understood as meaning fluorine, chlorine, bromine, iodine.

[0153] The term "nitro" is understood to mean -NO2.

[0154] The term "ester group" should be understood as -COOR (R is generally an alkyl group or other non-H group).

[0155] The term "mercapto" is understood to mean -SH. The term "epoxy" is understood to mean a three-membered ring consisting of two carbon atoms and one oxygen atom.

[0156] The term "amino" is to be understood as -NH2. The term "carboxyl" is to be understood as -COOH.

[0157] The term "hydroxyl" is to be understood as -OH.

[0158] The term "azido" is understood to mean -N3.

[0159] The term "C 1-20“Alkyl” is understood as meaning a straight-chain or branched, saturated, monovalent hydrocarbon radical having 1 to 20 carbon atoms. The alkyl radical is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, etc. “C 1-10 "Alkyl" is understood to mean a straight-chain or branched saturated monovalent hydrocarbon radical having 1 to 10 carbon atoms. 1-6 "Alkyl" is understood to mean a straight-chain or branched saturated monovalent hydrocarbon radical having 1 to 6 carbon atoms.

[0160] The term "C 1-6 "Haloalkyl" is understood to mean a linear or branched saturated monovalent hydrocarbon radical having 1 to 6 carbon atoms, which contains one or more halogen substituents.

[0161] The term "C 1-6 "Alkoxy" is understood to be a straight-chain or branched alkyl group having 1 to 6 carbon atoms, which is attached via an oxygen bond at any carbon atom in the alkyl group. For example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, sec-butoxy, tert-butoxy, etc.

[0162] The term "alkenyl" is understood to mean an unbranched or branched monovalent hydrocarbon chain containing one or more carbon-carbon double bonds. 2-20 “Alkenyl” is understood as preferably meaning a linear or branched, monovalent hydrocarbon radical which contains one or more double bonds and has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms.

[0163] The term "alkynyl" is understood to mean an unbranched or branched monovalent hydrocarbon chain containing one or more carbon-carbon triple bonds. 2-20 “Alkynyl” is understood as preferably meaning a linear or branched, monovalent hydrocarbon radical which contains one or more triple bonds and has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms.

[0164] The term "C 3-20"Cycloalkyl" is understood to mean a saturated, monovalent monocyclic or bicyclic hydrocarbon ring having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. For example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl.

[0165] The term "3-20 membered heterocyclyl" means a saturated monovalent monocyclic or bicyclic hydrocarbon ring containing 1-10 heteroatoms selected from N, O and S. In particular, the heterocyclyl may include, but is not limited to, a 4-membered ring such as azetidinyl, oxetanyl; a 5-membered ring such as tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl; or a 6-membered ring such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl or trithianyl; or a 7-membered ring such as diazepanyl. Optionally, the heterocyclyl may be benzo-fused. The heterocyclyl may be bicyclic, for example, but not limited to, a 5,5-membered ring such as a hexahydrocyclopenta [c] pyrrole-2 (1H) -yl ring, or a 5,6-membered bicyclic ring such as a hexahydropyrrolo [1,2-a] pyrazine-2 (1H) -yl ring. The ring containing the nitrogen atom may be partially unsaturated, i.e. it may contain one or more double bonds, such as but not limited to 2,5-dihydro-1H-pyrrolyl, 4H-[1,3,4]thiadiazinyl, 4,5-dihydrooxazolyl or 4H-[1,4]thiazinyl, or it may be benzo-fused, such as but not limited to dihydroisoquinolinyl.

[0166] The term "C 6-20 The term "aryl" is understood to mean preferably a monovalent aromatic or partially aromatic monocyclic, bicyclic or tricyclic hydrocarbon ring having 6 to 20 carbon atoms. In particular, a ring having 6 carbon atoms ("C6 aryl"), such as phenyl; or a ring having 9 carbon atoms ("C9 aryl"), such as indanyl or indenyl, or a ring having 10 carbon atoms ("C 10 aryl), such as tetrahydronaphthyl, dihydronaphthyl or naphthyl, or a ring having 13 carbon atoms ("C 13 aryl), such as fluorenyl, or a ring having 14 carbon atoms ("C 14 "aryl"), for example anthracenyl.

[0167] The term "5-20 membered heteroaryl" is understood to include monovalent monocyclic, bicyclic or tricyclic aromatic ring systems having 5-20 ring atoms and containing 1-5 heteroatoms independently selected from N, O and S, for example "5-14 membered heteroaryl". The term "5-14 membered heteroaryl" is understood to include monovalent monocyclic, bicyclic or tricyclic aromatic ring systems having 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ring atoms, in particular 5 or 6 or 9 or 10 carbon atoms, and containing 1 to 5, preferably 1 to 3 heteroatoms each independently selected from N, O and S, which additionally in each case may be benzo-fused. In particular, the heteroaryl group is selected from thienyl, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, thia-4H-pyrazolyl and the like and benzo derivatives thereof, such as benzofuranyl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl and the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and the like and benzo derivatives thereof, such as quinolyl, quinazolinyl, isoquinolyl and the like; or azinyl, indolizinyl, purinyl and the like and benzo derivatives thereof; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl and the like.

[0168] According to an embodiment of the present invention, the present invention provides a standard chip and a preparation method and application thereof. The structure of the standard chip can be composed of a first chip substrate and a second chip substrate, and the surface is modified according to performance requirements.

[0169] According to an embodiment of the present invention, the first chip substrate and the second chip substrate may be a non-patterned substrate, or may be the same or different patterned substrates, for example, a regularly arranged patterned concave or convex structure is formed on the substrate surface, and the specific arrangement may be a polygonal arrangement or a honeycomb arrangement. The geometric structure, i.e., the patterned structure, may be a cylinder, a cone, a truncated cone, a prism, a pyramid, a prism or an irregular shape, and the size of the structure may be adjusted according to actual needs, and the surface of the patterned concave or convex structure may be modified.

[0170] In some embodiments, the geometric structure forms a regular arrangement on the surface of the chip substrate, which can be understood as the geometric structure is arranged in a certain regular pattern to form a pattern with a certain shape. In some embodiments, the surface has at least one array unit, and the array unit has a regularly arranged geometric structure. Specifically, the array unit is a square array unit, that is, the figure formed by the collection of multiple geometric structures is a square. Exemplarily, the geometric structures are arranged in rows and columns, the distance between adjacent rows is equal to the distance between adjacent columns, and the collection of multiple geometric structures includes the same number of rows and columns. The array unit is a rectangular array unit, that is, the figure formed by the collection of multiple geometric structures is a rectangle. Exemplarily, the geometric structures are arranged in rows and columns, the distance between adjacent rows is equal to the distance between adjacent columns, and the collection of multiple geometric structures includes different numbers of rows and columns. The array unit is a honeycomb array unit, that is, the figure formed by the collection of multiple geometric structures is a honeycomb, that is, multiple geometric structures are arranged in a certain regular pattern, and the collection of these geometric structures forms a honeycomb. Of course, for the same surface, two or more array units of square array units, rectangular array units and honeycomb array units can be contained at the same time. In one embodiment, the same surface of the chip substrate may include one or more geometric structure sets, and the geometric structures in each set are arranged according to the same rules. In another embodiment, the same surface of the chip substrate includes one or more geometric structure sets, and the arrangement of the geometric structures in at least one set is different from the arrangement of the geometric structures in another set, that is, the same chip substrate includes a combination of multiple arrangement modes, such as splicing or nesting.

[0171] According to a specific embodiment of the present invention, the first chip substrate and / or the second chip substrate is selected as a patterned substrate, which has a regularly arranged cylindrical recessed structure, the cross-sectional diameter of the cylinder is 300nm~500nm, for example, 300nm, 350nm, 400nm, 450nm or 500nm, the height of the cylinder is 300nm~500nm, for example, 300nm, 350nm, 400nm, 450nm or 500nm, the center distance between adjacent cylinders is 600nm~1000nm, for example, 600nm, 650nm, 700nm, 750nm, 800nm, 850nm, 900nm, 950nm or 1000nm.

[0172] According to an embodiment of the present invention, the first chip substrate and the second chip substrate can be independently prepared by selecting glass, quartz, silicon-based materials or polymer 2. The silicon-based material can be commonly used silicon dioxide, polycrystalline silicon or single crystal silicon, and the polymer 2 can be HMDS, PE or PP. It should be understood that the material of the chip substrate can be reasonably adjusted according to the needs of the actual application scenario.

[0173] According to an embodiment of the present invention, the surface modification of the chip substrate includes surface modification of the first chip substrate, specifically including fixing the first compound to at least a partial area of ​​at least one surface of the first chip substrate, the signal body includes a signal group and a signal carrier, the signal group is connected to the signal carrier, and the signal carrier is fixed on the first compound.

[0174] Specifically, the first compound has a first functional group, the signal carrier has a second functional group, the first functional group and the second functional group are mutually chemically reactive functional groups, and the signal carrier is fixed on the first compound through a chemical reaction between the first functional group and the second functional group.

[0175] According to a specific embodiment of the present invention, the first compound can be fixed on the first compound by chemical vapor deposition, physical vapor deposition, atomic layer deposition, magnetron sputtering, solution method, spin coating, spray coating, etc. Among them, the chemical vapor deposition method with mild and controllable reaction conditions is preferred, the reaction can be carried out at 25-100° C., and the reaction time is 1-48h, which can be reasonably adjusted according to the properties of the first compound and the fixing effect.

[0176] According to a specific embodiment of the present invention, the first functional group may be at least one of halogen, nitro, ester, thiol, epoxy, amino, carboxyl, hydroxyl, azido, alkenyl, and alkynyl, and the second functional group may be at least one of halogen, thiol, aldehyde, hydroxyl, carboxyl, amino, alkenyl, alkynyl, and azido. For example, the first functional group is an amino group, and the second functional group is a carboxyl group that can chemically react with the amino group; the first functional group is a hydroxyl group, and the second functional group is a carboxyl group that can chemically react with the hydroxyl group.

[0177] According to a specific embodiment of the present invention, the first compound having the first functional group is preferably a thiol or a silane molecule. The molecular formula of the silane molecule is X a Si(Y b )M c Z, where

[0178] X is H, F, Cl, Br, I, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy;

[0179] Y is independently selected from C 1-10 alkyl;

[0180] M c Independently selected from C 1-50 Alkylene, -(OCH2CH2) c -、-(CH(OH)CH2-) c ;

[0181] Z is independently selected from the following groups which are unsubstituted or substituted: 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, C 3-20 Cycloalkyl, 3-20 membered heterocyclic group, C 6-20 Aryl, 5-20 membered heteroaryl;

[0182] C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, C 3-20 Cycloalkyl, 3-20 membered heterocyclic group, C 6-20 Aryl, 5-20 membered heteroaryl, at least one R a Substitution, the R a Each is independently selected from halogen, nitro, ester, amino, carboxyl, hydroxyl, azido, epoxy, and thiol;

[0183] a, b, and c are all integers, and their values ​​satisfy: 0≤a≤3, 0≤b≤3, and a+b=3; 1≤c≤50.

[0184] Exemplarily, the silane molecules are preferably trimethoxyaminosilane and triethoxyaminosilane, and the volume ratio between the two is 1:1-1:20, for example 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20.

[0185] According to a specific embodiment of the present invention, the first compound is fixed on the first chip substrate by chemical vapor deposition, wherein the first compound is selected from trimethoxyaminosilane and triethoxyaminosilane, the volume ratio of the two is 1:5, the reaction temperature is 40°C, and the reaction time is 10 hours.

[0186] According to an embodiment of the present invention, the signal body includes a signal carrier and a signal group. The signal body is introduced by connecting the signal carrier to the first compound, and the fixation method is such as physical adsorption or chemical coupling. Wherein physical adsorption includes but is not limited to the use of hydrogen bonding, van der Waals force, and similar dissolving effect, and chemical coupling includes but is not limited to the use of hydroxyl-carboxyl coupling, amino-carboxyl coupling, thiol-thiol coupling, and click chemistry. The reaction can be carried out at 25-100°C and the reaction time is 1-48h. It can be selected and adjusted according to the properties of the signal carrier and the fixation result.

[0187] According to a specific embodiment of the present invention, a signal carrier with a carboxyl functional group is selected and fixed on the chip substrate in a chemical bond manner through an amino-carboxyl coupling reaction with a first compound with an amino group, such as trimethoxyaminosilane and / or triethoxyaminosilane.

[0188] According to an embodiment of the present invention, the signal carrier can be a polymer 1, an antigen-antibody complex, a nanoparticle or a nucleic acid molecule. The signal group can be a fluorescent group, and its excitation wavelength and emission wavelength can be both 300-800nm, and can be cy3, cy5, HEX, atto, FAM, but is not limited to these five signal groups. The molar ratio between the second functional group and the signal group possessed by the signal carrier is 1:1-1:50, so as to achieve different signal brightness requirements, for example, the molar ratio between the second functional group and the signal group possessed by the signal carrier can be 1:1, 1:2, 1:5, 1:8, 1:10, 1:12, 1:15, 1:18, 1:20, 1:22, 1:25, 1:28, 1:30, 1:32, 1:35, 1:38, 1:40, 1:42, 1:45, 1:48, 1:50 and other specific situations.

[0189] According to a specific embodiment of the present invention, when the signal carrier is polymer 1, it is applied to the surface of the first chip substrate fixed with the first compound, so that polymer 1 is bound to the surface of the first chip substrate. Polymer 1 includes but is not limited to polyethylene glycol, polypropylene, polyacrylic acid, and polymethyl methacrylate. The size of polymer 1 is 5nm-500nm. Exemplarily, the size of polymer 1 can be 5nm, 10nm, 15nm, 20nm, 25nm, 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, 400nm, 410nm, 420nm, 430nm, 440nm, 450nm, 460nm, 470nm, 480nm, 490nm, 510nm, 520nm, 530nm, 540nm, 550nm, 560nm, 570nm, 580nm, 590nm, 610nm, The density of the polymer 1 in the first chip substrate region to which the first compound is fixed is 0.001-1 / μm. 2, so that the signals generated by the signal groups on the surface of the standard chip can be distinguished and identified. If the density of polymer 1 is too high, it will increase the difficulty of identifying the signals generated by the signal groups, and even make it impossible to distinguish adjacent signals.

[0190] According to a specific embodiment of the present invention, when the signal carrier is an antigen-antibody complex, it is applied to the surface of the first chip substrate fixed with the first compound, so that the antigen-antibody complex is bound to the surface of the first chip substrate. The signal group is connected to the antibody in the antigen-antibody complex, and the first compound is connected to the antigen in the antigen-antibody complex.

[0191] According to a specific embodiment of the present invention, when the signal carrier is a nanoparticle, it is applied to the surface of the first chip substrate fixed with the first compound, so that the nanoparticle is bound to the surface of the first chip substrate. The nanoparticles include but are not limited to silicon dioxide nanoparticles, zirconium oxide nanoparticles, cerium oxide nanoparticles, aluminum oxide nanoparticles, gold nanoparticles, DNA nanospheres, RNA nanospheres, magnetic beads bound to DNA, and magnetic beads bound to RNA. The particle size of the nanoparticles is 5nm-500nm. Exemplarily, the size of the nanoparticles can be 5nm, 10nm, 15nm, 20nm, 25nm, 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, 210nm, 220nm, 230nm The density of the nanoparticles is 0.001-1 nanoparticles / μm based on the first chip substrate area fixed with the first compound, and the ... 2 , so that the signals generated by the signal groups on the surface of the standard chip can be distinguished and identified. If the density of the nanoparticles is too high, it will increase the difficulty of identifying the signals generated by the signal groups, and even make it impossible to distinguish adjacent signals.

[0192] According to a specific embodiment of the present invention, when the signal carrier is a nucleic acid molecule, there are two ways to fix it on the first compound:

[0193] (1) When the nucleic acid molecule is a double-stranded nucleic acid molecule, applying it to the surface of the first chip substrate on which the first compound is fixed, so that the double-stranded nucleic acid molecule is bound to the surface of the first chip substrate;

[0194] Or (2) when the nucleic acid molecule is a single-stranded nucleic acid molecule, a first single-stranded nucleic acid molecule not modified with a signal group is applied to the surface of a first chip substrate fixed with a first compound, so that the first single-stranded nucleic acid molecule is bound to the surface of the first chip substrate; a second single-stranded nucleic acid molecule is added, wherein the second single-stranded nucleic acid molecule is bound to a signal group and contains a base that is complementary to a portion of the first single-stranded nucleic acid molecule, so that the second single-stranded nucleic acid molecule is connected to the first single-stranded nucleic acid molecule.

[0195] Both of the above methods can achieve the purpose of fixing the signal carrier on the first compound to achieve the introduction of the signal body. In addition, the length of the nucleic acid molecule can be 1-200 bp, such as 20-100 bp, 30-80 bp, 40-70 bp. In the first chip substrate area where the first compound is fixed, the density of the nucleic acid molecule is 0.001-5 strands / μm 2 , so that the signals generated by the signal groups on the surface of the standard chip can be distinguished and identified. If the density of nucleic acid molecules is too high, it will increase the difficulty of identifying the signals generated by the signal groups, and even make it impossible to distinguish adjacent signals.

[0196] According to a specific embodiment of the present invention, the signal carrier may be fixed on the first compound by using a solution method, a spin coating method, a screen printing method or a spray coating method.

[0197] According to an embodiment of the present invention, a metal layer may be further provided between the first compound and the first chip substrate, and the metal layer may be used to reduce surface defects of the chip substrate and / or the cover plate and improve the uniformity of the chip surface. In addition, in the chip preparation process, considering the feasibility of the process, it is preferred to react on the entire surface of the first chip substrate to generate a polymer layer; then the polymer layer of the first chip substrate is processed, and only the polymer in a partial area is retained to meet the need for detecting the optical signal generated by the nucleic acid molecule reaction. In some embodiments, the polymer on the surface of the remaining area is removed by a polishing process. At this time, a metal layer is provided on the surface of the first chip substrate, and during the polishing process, the degree of the polishing reaction is indicated by detecting the physical and chemical properties of the metal layer, thereby indicating the polishing end point. The choice of metal includes but is not limited to gold or aluminum. The thickness of the metal layer is less than or equal to 1 μm, preferably 15 nm to 800 nm, more preferably 20 to 500 nm, and most preferably 20 to 200 nm. Within this thickness range, the metal layer can exert the above-mentioned effects. If the thickness is too thick, such as when the thickness of the metal layer is greater than 1500 nm, the brightness of the optical signal collected by the chip with the metal layer is reduced compared to the chip without the metal layer, thereby affecting the judgment of the signal. Exemplarily, the thickness of the metal layer can be 10nm, 15nm, 20nm, 25nm, 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, 250nm, 300nm, 400nm, 500nm, 600nm, 700nm, 800nm, 900nm, 1000nm and the like.

[0198] According to one embodiment of the present invention, the standard chip includes a second chip substrate, and the second chip substrate is fixed to the side of the first chip substrate having a signal body. The second chip substrate can be a substrate that has not been surface treated, such as a glass substrate, a silicon-based substrate, etc. In some embodiments, a signal body is fixed to at least a portion of the surface of the second chip substrate on the side opposite to the first chip substrate, and the signal body includes a signal group and a signal carrier, the signal group is connected to the signal carrier, and the signal body is the same as or different from the signal body fixed on the first chip substrate. The surface modification method and preferred conditions of the second chip substrate can refer to the first chip substrate, and in order to save space, it will not be repeated here.

[0199] According to an embodiment of the present invention, the process of preparing a standard chip may further include pre-cleaning the surface of the chip substrate, for example, using solution cleaning, plasma activation, mechanical polishing, etc., to remove surface dirt.

[0200] According to an embodiment of the present invention, the chip substrate can be subsequently packaged and integrated so that the designed chip has the function of liquid permeability or sealing protection, realizes the flow reaction of different reagents on the chip during the preparation process of the chip, and withstands multiple cycles (10-10000 times) of reagent flushing. The specific steps include fixing the second chip substrate to the side of the first chip substrate having the first compound to form a channel arranged in the length direction. The channel can achieve the function of physical separation of the chip, and there can be one or more flow channels on a chip. In addition, the chip can form an open chip without bonding and packaging to form a flow channel. The chip packaging step can also be adjusted according to actual needs. For example, the chip packaging can be performed after the first compound is fixed to the chip substrate, and the signal body is introduced later. The chip packaging can also be performed after the signal body is introduced. The order of specific process steps can be reasonably adjusted.

[0201] The standard chip provided in the embodiment of the present application can be used to debug a sequencer or evaluate the performance of the sequencer, wherein the sequencer includes an optical imaging system.

[0202] As an embodiment of the present invention, the debugging method includes: (1) using an optical imaging system to image a standard chip and obtain imaging data; (2) debugging a sequencer based on the imaging data obtained in step (1). In some embodiments, the debugging of the sequencer can be based on the imaging results obtained for the standard chip, and the carrier for fixing the chip is debugged to obtain suitable setting parameters. In one embodiment, the debugging of the carrier is specifically: exciting the signal group through the optical imaging system to generate an optical signal, and imaging the standard chip; obtaining a fluorescent image of the standard chip; adjusting the setting parameters of the carrier based on the fluorescent image and the preset image clarity. In other embodiments, the debugging of the sequencer can be based on the imaging results obtained for the standard chip with different laser power parameters, and the optical imaging system is debugged to obtain suitable laser power parameters. In one embodiment, the debugging of the laser power parameters includes: exciting the signal group through the optical imaging system to generate an optical signal; adjusting the laser power parameters until the optical signal intensity reaches the optical signal intensity.

[0203] As an embodiment of the present invention, a sequencer includes a carrier and an optical imaging system, and a method for evaluating the performance of the sequencer includes the following steps: (1) using the optical imaging system to image a standard chip to obtain imaging data, wherein the optical imaging system includes an optical imaging lens, and the imaging data includes a position change of the optical imaging lens during the imaging process, and an optical signal intensity and a fluorescent panoramic image of each sequencing channel; (2) evaluating the setting of the carrier based on the position change of the optical imaging lens during the imaging process, and evaluating the photographing area of ​​the sequencer based on the optical signal intensity and the fluorescent panoramic image.

[0204] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will appreciate that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in this area or the product instructions are used. Where the manufacturers of reagents or instruments are not indicated, they are all conventional products that can be obtained commercially.

[0205] Example 1 Fluorescent polymer chip

[0206] 1. Preparation of fluorescent polymer chips

[0207] The specific preparation process is as follows Figure 1 As shown:

[0208] (1) Using glass as the first chip substrate, ultrasonic cleaning is performed using a solution, and then air plasma treatment is performed, wherein the solution used in the ultrasonic cleaning step is a surfactant and ethanol;

[0209] (2) using chemical vapor deposition to modify a layer of silane molecules on the substrate, specifically trimethoxyaminosilane and triethoxyaminosilane, with a volume ratio of 1:10, a reaction temperature of 50°C, and a reaction time of 24 hours;

[0210] (3) encapsulating the substrate modified with silane molecules to form a complete chip structure, wherein the upper and lower glass substrates constituting the chip structure can both be glass modified with silane molecules; or one piece of glass modified with silane molecules and the other piece of cleaned blank glass; the encapsulation method can be encapsulation by photosensitive curing method or by pressure-sensitive adhesive encapsulation;

[0211] (4) Fluorescent polymers are modified on the upper and lower surfaces of the channel formed after substrate encapsulation, or fluorescent polymers are modified only on one side of the glass substrate with silane molecules fixed. Polyacrylic acid modified with fluorescent groups is introduced into the channel formed after substrate encapsulation, with a size of 5nm-500nm. The number of fluorescent groups on the polymer surface can be adjusted to achieve different brightness. The ratio of carboxyl groups to fluorescent groups is about 1:20. The polymer is fixed to the substrate in the form of chemical bonds through a condensation reaction between the surface carboxyl groups and the amino groups on the chip substrate. The reaction time is 24h and the reaction temperature is 50°C. The density of the polymer fixed on the substrate can be adjusted by the concentration of the reacted polymer solution to achieve 0.001-1 / μm 2 The modified chip can be placed in an aqueous solution or dry condition at 4°C or at room temperature.

[0212] 2. Performance Testing

[0213] The prepared fluorescent polymer chips were used for sequencing platform testing, and the imaging brightness diagrams are shown in Figure 2. Figure 2 ,Depend on Figure 2 It can be seen that the fluorescent groups on the standard chip have good dispersion and highly uniform size; Figure 2 The brightness of the fluorescence signal at the horizontal line is counted, and the results are as follows Figure 3 As shown by Figure 3 It can be seen that the fluorescent groups on the standard chip have highly uniform signal intensity and extremely low background brightness.

[0214] Example 2 Fluorescent Nanoparticle Chip

[0215] 1. Preparation of fluorescent nanoparticle chips

[0216] The specific preparation process is as follows Figure 1 As shown:

[0217] (1) Using glass as the first chip substrate, ultrasonic cleaning is performed using a solution, and then air plasma treatment is performed, wherein the solution used in the ultrasonic cleaning step is a surfactant and ethanol;

[0218] (2) using a physical vapor deposition method to modify a layer of silane molecules on the substrate, specifically trimethoxyaminosilane and triethoxyaminosilane, the volume ratio of the two is 1:8, the reaction temperature is 60°C, and the reaction time is 15h;

[0219] (3) encapsulating the substrate modified with silane molecules to form a complete chip structure, wherein the upper and lower glass substrates constituting the chip structure can both be glass modified with silane molecules; or one piece of glass modified with silane molecules and the other piece of cleaned blank glass; the encapsulation method can be encapsulation by photosensitive curing method or by pressure-sensitive adhesive encapsulation;

[0220] (4) Fluorescent nanoparticles are modified on the upper and lower surfaces of the channel formed after substrate encapsulation, or fluorescent nanoparticles are modified on only one side of the glass substrate with silane molecules fixed. Nanoparticles modified with fluorescent groups and having carboxyl functional groups - silica nanoparticles with a particle size of 5nm-500nm are introduced into the channel formed after substrate encapsulation. The number of fluorescent groups on the surface of the nanoparticles can be adjusted to achieve different brightness. The ratio of carboxyl groups to fluorescent groups is approximately 1:10. The nanoparticles are fixed to the substrate in the form of chemical bonds through a condensation reaction between the surface carboxyl groups and the amino groups on the chip substrate. The reaction time is 18h and the reaction temperature is 40°C. The density of the nanoparticles fixed on the substrate can be adjusted by the concentration of the reacted nanoparticle solution to achieve 0.001-1 / μm 2 The modified chip can be placed in an aqueous solution or dry condition at 4°C or at room temperature.

[0221] 2. Performance Testing

[0222] The prepared fluorescent nanoparticle chips were used for sequencing platform testing, and the imaging brightness diagrams are shown in Figure 2. Figure 4 ,Depend on Figure 4 It can be seen that the fluorescent groups on the standard chip have good dispersion and highly uniform size; Figure 4 The brightness of the fluorescence signal at the horizontal line is counted. Figure 5 As shown by Figure 5 It can be seen that the fluorescent groups on the standard chip have highly uniform signal intensity and extremely low background brightness.

[0223] Example 3 Fluorescent probe chip

[0224] 1. Preparation of fluorescent probe chip

[0225] The specific preparation process is as follows Figure 6 As shown:

[0226] (1) Using glass as the first chip substrate, ultrasonic cleaning is performed using a solution, and then air plasma treatment is performed, wherein the solution used in the ultrasonic cleaning step is a surfactant and ethanol;

[0227] (2) modifying a layer of silane molecules on the substrate by a solution method, specifically trimethoxyaminosilane and triethoxyaminosilane, with a volume ratio of 1:6, a reaction temperature of 50°C, and a reaction time of 18 h;

[0228] (3) encapsulating the substrate modified with silane molecules to form a complete chip structure, wherein the upper and lower glass substrates constituting the chip structure can both be glass modified with silane molecules; or one piece of glass modified with silane molecules and the other piece of cleaned blank glass; the encapsulation method can be encapsulation by photosensitive curing method or by pressure-sensitive adhesive encapsulation;

[0229] (4) Modifying the nucleic acid fragments on the upper and lower surfaces of the channel formed after the substrate is encapsulated, or modifying the nucleic acid fragments on only one side of the glass substrate with silane molecules fixed. A nucleic acid fragment with a carboxyl functional group is introduced into the channel formed after the substrate is encapsulated, wherein the nucleic acid fragment can be a single-stranded DNA formed by natural bases connected by phosphodiester bonds, or a nucleic acid fragment formed by modified or structurally modified bases. The nucleic acid fragment has a specific sequence and can specifically form a double-stranded structure with the corresponding nucleic acid sequence through hydrogen bonding. The length of the nucleic acid fragment can be 10-200 bp. The carboxyl group on the nucleic acid fragment and the amino group on the chip substrate are fixed to the substrate in the form of a chemical bond through a condensation reaction, the reaction time is 14 hours, and the reaction temperature is 35°C. The density of the nucleic acid fragment fixed on the substrate can be adjusted by the concentration of the reacted nucleic acid fragment solution to achieve 0.001-5 fragments / μm 2density. The modified chip can be placed in the chip preservation solution at 4°C or at room temperature. The chip modified with nucleic acid fragments can hybridize and complement with specific nucleic acid sequences. By marking fluorescent groups on the complementary sequence and exciting it with appropriate wavelengths, the chip surface signal generation and detection can be achieved. The complementary sequence can be marked with a single fluorescent group or with two or more fluorescent groups to achieve the purpose of enhancing the fluorescent signal. Usually, the single-molecule sequencing platform can detect a single fluorescent group, and a single fluorescent-labeled sequence can realize the instrument debugging function, and the complementary sequence can be eluted by a cleaning reagent, and then re-hybridized with the fluorescent-labeled complementary sequence to realize the reuse of the chip.

[0230] 2. Performance Testing

[0231] The prepared fluorescent nanoparticle chips were used for sequencing platform testing, and the imaging brightness diagrams are shown in Figure 2. Figure 7 ,right Figure 7 The brightness of the fluorescence signal at the horizontal line is counted. Figure 8 As shown. Figure 7 , Figure 8 It can be seen that the standard chip can repeatedly generate signals by repeatedly hybridizing fluorescent probes and base biochemical reactions, realizing the repeated use of the chip. By using single-molecule fluorescent labeled nucleic acid probes, imaging has better signal morphology, and the fluorescent group has good dispersion and highly uniform size, which is close to the actual situation of sequencing.

[0232] Example 4 Patterned fluorescent polymer chip

[0233] 1. Preparation of patterned fluorescent polymer chips

[0234] The specific preparation process is as follows Fig. 9 As shown:

[0235] (1) Using silicon as the first chip substrate, the surface is patterned to form circular holes with a diameter of 300-500 nm and a hole depth of 300-500 nm. The circular holes are arranged in a honeycomb pattern, and the center distance between adjacent holes is 600-1000 nm;

[0236] (2) cleaning the first chip substrate by ultrasonic cleaning with a solution and then treating it with air plasma, wherein the solution used in the ultrasonic cleaning step is a surfactant and ethanol;

[0237] (3) using magnetron sputtering to modify a layer of silane molecules on the substrate, specifically trimethoxyaminosilane and triethoxyaminosilane, with a volume ratio of 1:10, a reaction temperature of 50°C, and a reaction time of 20 hours;

[0238] (4) The substrate modified with silane molecules is cut and cleaned to remove the surface photoresist, and is further cleaned by chemical mechanical polishing to ensure that there is no residual glue and residual silane molecules outside the hole. The substrate after cleaning is packaged into a complete chip structure. The packaging method can be packaging by photosensitive curing or packaging by pressure-sensitive bonding adhesive;

[0239] (5) Fluorescent group-modified polymer polyethylene glycol with carboxyl functional groups is introduced into the channel formed after encapsulation of the substrate. The size of the polymer is 5nm-500nm. The number of fluorescent groups on the surface of the polymer can be adjusted to achieve different brightness. The ratio of carboxyl groups to fluorescent groups is about 1:30. The polymer is fixed to the substrate in the form of chemical bonds through a condensation reaction between the surface carboxyl groups and the modified amino groups in the patterned area of ​​the chip substrate. The reaction time is 15h and the reaction temperature is 50°C. The density of the polymer fixed on the substrate can be adjusted by the concentration of the reacted polymer solution to achieve 0.001-1 / μm 2 The modified chip can be placed in an aqueous solution or dry condition at 4°C or at room temperature.

[0240] 2. Performance Testing

[0241] The prepared patterned fluorescent polymer chip was used for sequencing platform testing, and its imaging brightness was shown in the figure below. Fig.10 ,Depend on Fig.10 It can be seen that the fluorescent groups on the standard chip have good dispersion and highly uniform size, and the imaging morphology is good, which is close to the real morphology of sequencing. Fig.10 The brightness of the fluorescence signal at the horizontal line is counted. Fig.11 As shown by Fig.11 It can be seen that the fluorescent groups on the standard chip have highly uniform signal intensity and extremely low background brightness.

[0242] The standard chip prepared in Example 4 was subjected to 20 laser irradiations to debug the instrument. The imaging brightness diagram is shown in FIG. Fig.12 , Fig.12 The left area shows the imaging brightness of the biochip after instrument debugging, and the right area shows the imaging brightness before debugging. Fig.12 It can be seen that after the standard chip has been debugged by laser irradiation 20 times, the brightness of the area used for instrument debugging (left side) and the brightness of the area not irradiated for a long time (right side) are almost the same; Fig.12 The brightness of the fluorescence signal at the horizontal line is counted, and the results are as follows Fig.13 As shown, Fig.13 It shows that the signal brightness of the area where the standard chip is used for instrument debugging (left side) and the area that is not irradiated for a long time (right side) have little attenuation (<5%), indicating that the standard chip has the advantages of being stable, durable and can be reused many times.

[0243] Comparative Example 1

[0244] 1. Preparation of biochip

[0245] The specific preparation process is as follows:

[0246] (1) Using silicon as the first chip substrate, the surface is patterned to form circular holes with a diameter of 300-500 nm and a hole depth of 300-500 nm. The circular holes are arranged in a honeycomb pattern, and the center distance between adjacent holes is 600-1000 nm;

[0247] (2) cleaning the first chip substrate by ultrasonic cleaning with a solution and then treating it with air plasma, wherein the solution used in the ultrasonic cleaning step is a surfactant and ethanol;

[0248] (3) using a chemical vapor deposition method to modify a layer of silane molecules on the substrate, specifically trimethoxyaminosilane and triethoxyaminosilane, with a volume ratio of 1:10, a reaction temperature of 50° C., and a reaction time of 20 h;

[0249] (4) The substrate modified with silane molecules is cut and cleaned to remove the surface photoresist, and is further cleaned by chemical mechanical polishing to ensure that there is no residual glue outside the hole and residual silane molecules of chemical vapor deposition. The substrate after cleaning is packaged into a complete chip structure. The packaging method can be packaging by photosensitive curing or packaging by pressure-sensitive bonding adhesive;

[0250] (5) A polymer with a carboxyl functional group is introduced into the channel formed after the substrate is encapsulated. The polymer can be polyethylene glycol, polypropylene, polyacrylic acid, or polymethyl methacrylate, with a size of 5nm-500nm. The ratio of carboxyl groups to fluorescent groups can be 1:1-1:50. The polymer is fixed to the substrate in the form of a chemical bond through a condensation reaction between the surface carboxyl groups and the modified amino groups in the patterned area on the chip substrate. The reaction time is 15h and the reaction temperature is 50°C. The density of the polymer fixed on the substrate can be adjusted by the concentration of the reacted polymer solution to achieve 0.001-1 pieces / μm 2 The probe is inserted into the channel formed after the substrate is encapsulated, and the probe is fixed to the polymer by chemical bonding.

[0251] 2. Performance Testing

[0252] A nucleic acid library is added to the base channel where the probe is fixed. The nucleic acid library is connected to the probe and fixed in the circular hole structure. After the nucleic acid library is amplified, nucleotides or their analogs containing fluorescent labels are added to perform base extension reaction, and the fluorescent signal is collected. The imaging brightness diagram is as shown in FIG. Fig.14 As shown, Fig.14The left area shows the imaging brightness of the biochip after instrument debugging, and the right area shows the imaging brightness before debugging, indicating that the surface signal of the biochip decays rapidly during the instrument debugging process and cannot be used multiple times. Fig.14 The brightness of the fluorescence signal at the horizontal line is counted, and the results are as follows Fig.15 As shown, Fig.15 The brightness of the biochip was significantly reduced during testing (first use).

[0253] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", "some implementation schemes" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0254] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A standard chip, It is characterized in that It includes a first chip substrate, a first compound and a signal body, wherein the first compound is fixed to at least a partial area of ​​at least one surface of the first chip substrate, and the signal body includes a signal group and a signal carrier, wherein the signal group is connected to the signal carrier, and the signal carrier is connected to the first compound.

2. The standard chip according to claim 1, It is characterized in that The first compound has a first functional group, the signal carrier has a second functional group, and the first functional group and the second functional group are mutually chemically reactive functional groups; Optionally, the first functional group comprises at least one selected from halogen, nitro, ester, thiol, epoxy, amino, carboxyl, hydroxyl, azido, alkenyl, and alkynyl; Optionally, the first compound is a thiol or silane molecule; Optionally, the molecular formula of the silane molecule is X a Si(Y b )M c Z, where X is H, F, Cl, Br, I, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy; Y is independently selected from C 1-10 alkyl; M c Independently selected from C 1-50 Alkylene, -(OCH 2 CH 2 ) c -、-(CH(OH)CH 2 -) c ; Z is independently selected from the following groups which are unsubstituted or substituted: 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, C 3-20 Cycloalkyl, 3-20 membered heterocyclic group, C 6-20 Aryl, 5-20 membered heteroaryl; The C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, C 3-20 Cycloalkyl, 3-20 membered heterocyclic group, C 6-20 Aryl, 5-20 membered heteroaryl, at least one R a Substitution, the R a Each is independently selected from halogen, nitro, ester, amino, carboxyl, hydroxyl, azido, epoxy, and thiol; a, b, and c are all integers, and their values ​​satisfy: 0≤a≤3, 0≤b≤3, and a+b=3; 1≤c≤50; Optionally, the molar ratio of the second functional group to the signal group is 1:1-1:50; Optionally, the second functional group includes at least one selected from halogen, thiol, aldehyde, hydroxyl, carboxyl, amino, alkenyl, alkynyl, and azide.

3. The standard chip according to claim 1, It is characterized in that The signal carrier comprises at least one selected from polymer 1, antigen-antibody complex, nanoparticle, and nucleic acid molecule; Optionally, the signal carrier comprises polymer 1, and the polymer 1 is selected from at least one of polyethylene glycol, polypropylene, polyacrylic acid, and polymethyl methacrylate; Optionally, in the first chip substrate region where the first compound is fixed, the density of the polymer 1 is 0.001-1 per μm 2 ; Optionally, the size of the polymer 1 is 5nm-500nm; Optionally, the signal carrier comprises an antigen-antibody complex, and in the first chip substrate region where the first compound is fixed, the density of the antigen-antibody complex is 0.001-5 per μm 2 ; Optionally, the signal group is linked to the antibody in the antigen-antibody complex, and the first compound is linked to the antigen in the antigen-antibody complex; Optionally, the signal carrier comprises nanoparticles, and the nanoparticles comprise at least one selected from silica nanoparticles, zirconium oxide nanoparticles, cerium oxide nanoparticles, aluminum oxide nanoparticles, gold nanoparticles, DNA nanospheres, RNA nanospheres, magnetic beads bound to DNA, and magnetic beads bound to RNA; Optionally, in the first chip substrate region where the first compound is fixed, the density of the nanoparticles is 0.001-1 / μm 2 ; Optionally, the signal carrier comprises a nucleic acid molecule, and the nucleic acid molecule is a single-stranded nucleic acid molecule or a double-stranded nucleic acid molecule; Optionally, the length of the nucleic acid molecule is 1-200 bp; Optionally, the length of the nucleic acid molecule is 20-100 bp; Optionally, the length of the nucleic acid molecule is 30-80 bp; Optionally, the length of the nucleic acid molecule is 40-70 bp; Optionally, in the first chip substrate region where the first compound is fixed, the density of the nucleic acid molecules is 0.001-5 / μm 2 ; Optionally, the signal group is linked to any nucleic acid strand in the nucleic acid molecule; Optionally, the particle size or size of the signal carrier is 5nm-500nm; Optionally, the signal group is a fluorescent group; Optionally, the excitation wavelength of the fluorescent group is 300-800nm, and the emission wavelength is 300-800nm; Optionally, the fluorescent group includes at least one selected from cy3, cy5, HEX, atto, and FAM.

4. The standard chip according to claim 1, It is characterized in that providing a metal layer between the first compound and the first chip substrate; Optionally, the metal of the metal layer includes at least one selected from Au and Al; Optionally, the thickness of the metal layer is less than or equal to 1 μm; Optionally, the first chip substrate has a regularly arranged geometric structure, and the first compound is fixed on the surface of the geometric structure; Optionally, the geometric structure is a depression or a protrusion; Optionally, the geometric structure is a cylinder, a cone, a truncated cone, a prism, a pyramid, a truncated pyramid or an irregular shape; Optionally, the geometric structure is a cylinder, the cross-sectional diameter of the cylinder is 300nm to 500nm, the height of the cylinder is 300nm to 500nm, and the center distance between adjacent cylinders is 600nm to 1000nm; Optionally, the geometric structure is arranged in a polygonal arrangement or a honeycomb arrangement.

5. The standard chip according to claim 1, It is characterized in that The standard chip further comprises a second chip substrate, wherein the second chip substrate is fixed to a side of the first chip substrate having the signal body; Optionally, the signal body is fixed to at least a part of the surface of the second chip substrate on one side opposite to the first chip substrate, the signal body comprises a signal group and a signal carrier, the signal group is connected to the signal carrier, and the signal body is the same as or different from the signal body fixed on the first chip substrate; Optionally, the standard chip has a channel arranged in the length direction, and the signal body is at least combined with the surface of the first chip substrate in the area where the channel is located; Optionally, the standard chip is used for gene sequencer performance evaluation and / or equipment debugging.

6. A method for preparing a standard chip, It is characterized in that The following steps are involved: Step 1: fixing a first compound on at least a partial area of ​​at least one surface of a first chip substrate; Step 2: Connecting a signal body to the first compound to obtain a standard chip, wherein the signal body includes a signal group and a signal carrier, the signal group is connected to the signal carrier, and the signal carrier is fixed on the first compound; Optionally, the first compound has a first functional group, the signal carrier has a second functional group, and the first functional group and the second functional group are mutually chemically reactive functional groups; Optionally, the first functional group is selected from at least one of halogen, thiol, nitro, ester, epoxy, amino, carboxyl, hydroxyl, azido, alkenyl, and alkynyl; Optionally, the first compound is a thiol or silane molecule; Optionally, the molecular formula of the silane molecule is X a Si(Y b )M c Z, where X is H, F, Cl, Br, I, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy; Y is independently selected from C 1-10 alkyl; M c Independently selected from C 1-50 Alkylene, -(OCH 2 CH 2 ) c -、-(CH(OH)CH 2 -) c ; Z is independently selected from the following groups which are unsubstituted or substituted: 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, C 3-20 Cycloalkyl, 3-20 membered heterocyclic group, C 6-20 Aryl, 5-20 membered heteroaryl; The C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, C 3-20 Cycloalkyl, 3-20 membered heterocyclic group, C 6-20 Aryl, 5-20 membered heteroaryl, at least one R a Substitution, the R a Each is independently selected from halogen, nitro, ester, amino, carboxyl, hydroxyl, azido, epoxy, and thiol; a, b, and c are all integers, and their values ​​satisfy: 0≤a≤3, 0≤b≤3, and a+b=3; 1≤c≤50; Optionally, the second functional group is selected from at least one of halogen, thiol, aldehyde, hydroxyl, carboxyl, amino, alkenyl, alkynyl, and azido; Optionally, the method for fixing the first compound in step 1 comprises at least one of chemical vapor deposition, physical vapor deposition, atomic layer deposition, magnetron sputtering, solution method, spin coating, and spray coating; Optionally, the first compound is a silane molecule, and the silane molecule is fixed on the first chip substrate by chemical vapor deposition; Optionally, the reaction temperature of the chemical vapor deposition method is 25-100° C., and the reaction time is 2-48 h; Optionally, the silane molecules include trimethoxyaminosilane and triethoxyaminosilane; Optionally, the volume ratio of the trimethoxyaminosilane to the triethoxyaminosilane is 1:1-1:20; Optionally, the signal carrier comprises at least one selected from polymer 1, nanoparticles, nucleic acid molecules, and antigen-antibody complexes; Optionally, when the signal carrier is polymer 1, the method for introducing the signal body comprises: applying the polymer 1 modified with the signal group to the surface of the first chip substrate fixed with the first compound obtained in step 1, so that the polymer 1 is bound to the surface of the first chip substrate; Optionally, the introduction of the signal body is carried out at 25°C-100°C, and the reaction time is 1h-48h; Optionally, the polymer 1 is selected from at least one of polyethylene glycol, polypropylene, polyacrylic acid, and polymethyl methacrylate; Optionally, the size of the polymer 1 is 5nm-500nm; Optionally, in the first chip substrate region where the first compound is fixed, the density of the polymer 1 is 0.001-1 per μm 2 ; Optionally, when the signal carrier is a nanoparticle, the method for introducing the signal body comprises: applying the nanoparticle modified with the signal group to the surface of the first chip substrate fixed with the first compound obtained in step 1, so that the nanoparticle is bound to the surface of the first chip substrate; Optionally, the introduction of the signal body is carried out at 25-100° C., and the reaction time is 1-48 h; Optionally, the nanoparticles are selected from at least one of silica nanoparticles, zirconium oxide nanoparticles, cerium oxide nanoparticles, aluminum oxide nanoparticles, gold nanoparticles, DNA nanospheres, RNA nanospheres, magnetic beads combined with DNA, and magnetic beads combined with RNA; Optionally, the particle size of the nanoparticles is 5nm-500nm; Optionally, in the first chip substrate region where the first compound is fixed, the density of the nanoparticles is 0.001-1 / μm 2 ; Optionally, when the signal carrier is a nucleic acid molecule, the method for introducing the signal body includes: Method 1: When the nucleic acid molecule is a double-stranded nucleic acid molecule, the double-stranded nucleic acid molecule modified with the signal group is applied to the surface of the first chip substrate fixed with the first compound obtained in step 1, so that the double-stranded nucleic acid molecule is bound to the surface of the first chip substrate; Or method 2, when the nucleic acid molecule is a single-stranded nucleic acid molecule, applying a first single-stranded nucleic acid molecule without signal group modification to the surface of the first chip substrate fixed with the first compound obtained in step 1, so that the first single-stranded nucleic acid molecule is bound to the surface of the first chip substrate; adding a second single-stranded nucleic acid molecule, wherein the second single-stranded nucleic acid molecule is bound to a signal group and contains a base that is complementary to a part of the first single-stranded nucleic acid molecule, so that the second single-stranded nucleic acid molecule is connected to the first single-stranded nucleic acid molecule; Optionally, the length of the nucleic acid molecule is 1-200 bp; Optionally, the introduction of the signal body is carried out at 25-100° C., and the reaction time is 1-48 h; Optionally, in the first chip substrate region where the first compound is fixed, the density of the nucleic acid molecules is 0.001-5 / μm 2 ; Optionally, when the signal carrier is an antigen-antibody complex, the method for introducing the signal body comprises: applying the antigen-antibody complex modified with the signal group to the surface of the first chip substrate fixed with the first compound obtained in step 1, so that the antigen-antibody complex is bound to the surface of the first chip substrate; Optionally, the introduction of the signal body is carried out at 25-100° C., and the reaction time is 1-48 h; Optionally, the signal group in step 2 includes at least one selected from cy3, cy5, HEX, atto, and FAM; Optionally, in step 2, the signal carrier is fixed on the first compound by using at least one of a solution method, a spin coating method, a screen printing method, and a spray coating method; Optionally, in step 2, the signal carrier is fixed on the first compound by physical adsorption or chemical coupling; Optionally, the physical adsorption includes at least one of hydrogen bonding, van der Waals force, and like dissolves like effect; Optionally, the chemical coupling comprises at least one of hydroxyl-carboxyl coupling, amino-carboxyl coupling, thiol-thiol coupling, and click chemistry.

7. The preparation method according to claim 6, It is characterized in that The preparation method further comprises cleaning the surface of the first chip substrate before fixing the first compound; Optionally, the surface cleaning comprises at least one of solution cleaning, plasma activation, and mechanical polishing; Optionally, the solution cleaning includes at least one of acid reagent or alkaline reagent cleaning, organic reagent cleaning, and aqueous solution ultrasound; Optionally, the gas used for the plasma activation includes at least one selected from the group consisting of air, oxygen, nitrogen, argon, hydrogen, and carbon tetrafluoride; Optionally, the mechanical polishing includes mechanical polishing using at least one polishing agent selected from the group consisting of diamond, silica sol, calcium carbonate, cerium oxide, and zirconium oxide; Optionally, the preparation method further comprises performing chip packaging after performing step 1; Optionally, the chip packaging method includes: fixing a second chip substrate to a side of the first chip substrate having the first compound to form a channel having two ends running through in a preset direction; Optionally, the chip packaging method includes at least one selected from thermal bonding, laser bonding, and chemical and physical bonding; Optionally, the chemical and physical bonding includes at least one selected from UV curing, heat curing, and pressure-sensitive bonding; Optionally, the first chip substrate and the second chip substrate are made of the same material or different materials; Optionally, the first chip substrate and the second chip substrate are independently selected from at least one of blank or patterned glass, quartz, silicon-based material, polymer 2; Optionally, the silicon-based material includes at least one selected from silicon dioxide, polycrystalline silicon, and monocrystalline silicon; Optionally, the polymer 2 includes at least one selected from HMDS, PE, and PP.

8. Use of the standard chip according to any one of claims 1 to 5 in performance evaluation or equipment debugging of a gene sequencer.

9. A method for debugging a sequencer, It is characterized in that The sequencer includes an optical imaging system, and the debugging method includes: (1) using the optical imaging system to image the standard chip and obtain imaging data; (2) debugging the sequencer based on the imaging data obtained in step (1); Wherein, the standard chip is the standard chip according to any one of claims 1 to 5; Optionally, debugging the sequencer in step (2) includes debugging a carrier, and the carrier is used to fix the chip; Optionally, the debugging of the carrier specifically includes: exciting the signal group to generate an optical signal through the optical imaging system, and imaging the standard chip; acquiring a fluorescence image of the standard chip; and adjusting the setting parameters of the carrier based on the fluorescence image and a preset image clarity; Optionally, debugging the sequencer in step (2) includes debugging the optical imaging system; Optionally, the optical imaging system includes an excitation light source, and the debugging of the optical imaging system includes debugging of laser power parameters; Optionally, the adjustment of the laser power parameters includes: exciting the signal group to generate an optical signal through the optical imaging system; and adjusting the laser power parameters until the optical signal intensity reaches the optical signal intensity.

10. A method for evaluating the performance of a sequencer. It is characterized in that The sequencer comprises a stage and an optical imaging system, and the method comprises the following steps: (1) using the optical imaging system to image the standard chip and obtain imaging data, wherein the optical imaging system includes an optical imaging lens, and the imaging data includes a position change of the optical imaging lens during the imaging process, as well as the optical signal intensity of each sequencing channel and a fluorescent panoramic image; (2) evaluating the setting of the stage based on the position change of the optical imaging lens during the imaging process, and evaluating the photographing area of ​​the sequencer based on the optical signal intensity and the fluorescent panoramic image; Wherein, the standard chip is the standard chip described in any one of claims 1-5.

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