Use of compounds, microarray chip substrate, microarray chip and use and method thereof

Abstract

Use of a compound, microarray chip substrate, microarray chip and use and method thereof. The present application provides use of a compound, or a geometric isomer, tautomer, isotopically labeled, hydrate, solvate or salt thereof in chip surface modification. Also provided is a microarray chip substrate modified by the compound, and a microarray chip comprising the microarray chip substrate. The microarray chip substrate of the present application can greatly improve the surface modification function by surface modification, improve nucleic acid synthesis yield, amplify detection signal, and avoid non-specificity and cross-talk. It has a wide application prospect.

Description

Technical Field

[0001] This application relates generally to the field of biotechnology, and in particular to the use of a compound, a microarray chip substrate, a microarray chip, and the use and methods thereof. Background Technology

[0002] Microarray chips, also known as biochips, are a novel type of biosensor that integrates biomolecular recognition technology at a microscale. These chips immobilize a large number of biomolecules, such as DNA, RNA, or proteins, within specific tiny regions, forming a highly dense molecular array. Through specific techniques, such as optical or electronic scanning, the interactions between these molecules and target molecules in the sample can be detected. This technology is characterized by high throughput, high sensitivity, high parallelism, and high automation. It is widely used in genomics, proteomics, drug development, and other fields for gene expression analysis, mutation detection, and protein-protein interaction research. The fabrication process of microarray chips involves the interdisciplinary integration of microelectronics, biology, and physics, representing a typical example of the combination of biotechnology and information technology. Its emergence has greatly improved the efficiency and precision of biological experiments, driving the rapid development of life sciences. Based on function, they include gene sequencing chips, expression profiling chips, disease diagnostic chips, drug screening chips, sample preparation chips, biochemical reaction chips, and result detection chips; based on working method, there are passive and active chips; and based on chip structure and working principle, they are divided into microarray chips and microfluidic chips. Microarray chips are composed of biomolecules arranged in an array. Their analytical applications are based on affinity interactions between biomolecules, such as the binding of antigens and antibodies and the complementary base interactions of nucleic acid molecules; therefore, they can also be called affinity-based biochips. Microfluidic chips, on the other hand, are characterized by various microchannel network structures used to control and detect microfluidics containing biochemical components. Furthermore, with technological advancements, the application scope of microarray chips is constantly expanding, and their application prospects in disease diagnosis, personalized medicine, and other fields are very broad. Summary of the Invention

[0003] Currently, microarray chips, whether applied to nucleic acid synthesis and detection or protein synthesis and detection, face challenges such as insufficient detection efficiency and low synthesis yield per unit area. Surface modification is the optimal approach to address these issues. Surface modification achieves the blocking of non-specific adsorption, and the surface-modified materials can significantly increase the functional group density per unit area, thereby improving detection sensitivity and coupling efficiency.

[0004] Based on this, this application provides the use of the compound represented by formula (I), or its geometric isomers, tautomers, isotopic labels, hydrates, solvates, or salts thereof, in chip surface modification, wherein formula (I) is:

[0005]

[0006] Wherein, X1 is CH or N, X2 is CH2, C=O or SS, and when X2 is the terminal group of the compound, it is CH3 or CH3C=O; R1 and R5 are each independently -COOH or -OH or other negatively charged groups in aqueous solution, R3 is -NH2 or -C(=O)OR, R6 is -NH2, -C(=O)OR or -OH, where R is a carbon chain with C≤5, R2 and R4 are each independently benzene, biphenyl, anthraquinone or other superhydrophobic conjugable groups, n≥1, n2≥3.

[0007] On the other hand, this application also provides a microarray chip substrate, which is modified with a compound of formula (I), or its geometric isomers, tautomers, isotopic markers, hydrates, solvates, or salts thereof, wherein formula (I) is:

[0008]

[0009] Wherein, X1 is CH or N, X2 is CH2, C=O or SS, and when X2 is the terminal group of the compound, it is CH3 or CH3C=O; R1 and R5 are each independently -COOH or -OH or other negatively charged groups in aqueous solution, R3 is -NH2 or -C(=O)OR, R6 is -NH2, -C(=O)OR or -OH, where R is a carbon chain with C≤5, R2 and R4 are each independently benzene, biphenyl, anthraquinone or other superhydrophobic conjugable groups, n≥1, n2≥3.

[0010] In another aspect, this application also provides a microarray chip comprising the microarray chip substrate described herein.

[0011] In another aspect, this application also provides a kit comprising a microarray chip substrate or a microarray chip as described herein.

[0012] On the other hand, this application also provides the use of the microarray chip substrate or the microarray chip or the kit described herein in in situ nucleic acid synthesis, SNP detection, single-molecule immunoassay or single-cell sequencing.

[0013] On the other hand, this application also provides a method for modifying a microarray chip substrate, comprising:

[0014] Hydrophilic materials are deposited or coated between microarrays on a microarray chip substrate, and hydrophobic materials are deposited or coated on the microarray.

[0015] The compound described herein was uniformly coated on the chip surface and then oscillated uniformly on a shaker at a frequency of -30° to +30° for 1-10 hours to form a self-assembled film.

[0016] On the other hand, this application also provides a method for in situ synthesis of nucleic acids, comprising:

[0017] Nucleic acid monomers are linked to the microarray chip substrate or the microarray chip described herein;

[0018] Addressable microelectrode arrays are used to guide the molecular assembly of specific nucleic acid base sequences on the microarray chip substrate or on the microarray chip.

[0019] On the other hand, this application also provides a method for SNP detection, comprising:

[0020] The nucleic acid single-stranded probe is printed onto the microarray chip substrate or the microarray chip described herein, and the -OH of the compound shown in formula (I) is bound to the 5' phosphate of the nucleic acid single-stranded probe under the catalysis of polymerase to complete the preparation of the SNP detection chip;

[0021] The nucleic acid to be tested is broken into fragments of 150-800bp, and the broken nucleic acid fragments are hybridized with the single-stranded nucleic acid probe on the SNP detection chip.

[0022] On the other hand, this application also provides a method for immune detection, comprising:

[0023] The probe molecules are printed onto the microarray chip substrate or the microarray chip described in this article to complete the fabrication of the immune detection chip;

[0024] The test sample containing the target molecule is slowly passed through or covered on the surface of the microarray. After the reaction, an antibody with a fluorescent label is slowly passed through or covered on the surface of the microarray. The reaction is then observed by fluorescence color development.

[0025] The purpose of this application is to provide a microarray chip and its surface modification scheme for in situ nucleic acid synthesis, SNP detection, single-molecule immunoassay, and single-cell sequencing. Specifically, the chip structure is a microporous or micropillar array with high-density modified functional groups. The molecular backbone between the functional groups is closely packed to isolate non-specific adsorption, and the array has extreme hydrophobicity to isolate crosstalk.

[0026] To achieve this objective, the present application adopts the following technical solution:

[0027] This application provides a microarray chip and its surface modification scheme for in situ nucleic acid synthesis, SNP detection, single-molecule immunoassay, and single-cell sequencing. The chip microarrays are deposited or coated with hydrophilic materials such as SiO2 and polydopamine, while SiNx or resin-based hydrophobic materials are deposited on the arrays. The modification material consists of amphiphilic molecules with oppositely charged molecules, with -COOH and -NH2 functional groups arranged closely on both sides of the molecule, allowing for alternating close packing between molecular chains to form a dense molecular layer. Simultaneously, the hydrophilic groups such as -COOH and -NH2 on the molecular backbone are alternately arranged with hydrophobic groups such as aromatic groups. During assembly on the array, the hydrophilic functional groups are exposed for modification, while the hydrophobic functional groups are exposed between the arrays to prevent crosstalk and non-specific binding.

[0028] Compared with the prior art, this application has the following beneficial effects:

[0029] (1) Compared with traditional microarray chips, the functional group density per unit area is greatly increased in this application. When used for nucleic acid in situ synthesis, SNP detection, single-molecule immunoassay or single-cell sequencing, it can increase the yield of in situ synthesis, increase the detection limit and amplify the detection sensitivity.

[0030] (2) Compared with traditional microarray chips, the molecular backbone of this application is densely packed, which seals the non-specific adsorption and binding on the array, thereby improving the specificity of detection and the purity and accuracy of synthesis.

[0031] (3) This application uses single-molecule self-assembly, which avoids multiple differential modifications. The method is simple and has good uniformity, which can realize large-scale production and has low cost.

[0032] (4) The modification method of this application produces extremely different hydrophilic and hydrophobic behaviors between and on microarrays, which can avoid crosstalk problems in many applications such as single-cell sequencing, single-molecule immunoassay, molecular hybridization, and digital PCR (dPCR).

[0033] (5) Compared with existing modification schemes, this application has excellent biocompatibility and will not have a negative impact on biological macromolecules.

[0034] Other features and advantages of this application will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the application. Other advantages of this application can be realized and obtained by means of the embodiments described in the description and the accompanying drawings. Attached Figure Description

[0035] The accompanying drawings are used to provide an understanding of the technical solutions of this application and constitute a part of the specification. They are used together with the embodiments of this application to explain the technical solutions of this application and do not constitute a limitation on the technical solutions of this application.

[0036] Figure 1This is the mass spectrum of the compound of formula (II) in Example 1 of this application.

[0037] Figure 2 The infrared spectrum of the compound of formula (II) in Example 1 of this application is shown.

[0038] Figure 3 The results are observed under an atomic force microscope (AFM) of the nanosheet structure self-assembled from the compound of formula (II) in Example 1 of this application.

[0039] Figure 4 This is a comparison of DNA synthesis yield in the DNA in situ synthesis chip of Example 1 of this application. The control group was modified with sucrose, while the experimental group was modified with a compound of formula (II).

[0040] Figure 5 The results are observed under an atomic force microscope (AFM) of the nanosheet structure self-assembled from the compound of formula (III) in Example 2 of this application.

[0041] Figure 6 The above figure shows the comparison results of SNP gene chip detection signals in Example 2 of this application. The top figure shows the untreated chip, and the bottom figure shows the chip modified with the compound of formula (III).

[0042] Figure 7 The results are observed under an atomic force microscope (AFM) of the nanosheet structure self-assembled from the compound of formula (IV) in Example 3 of this application.

[0043] Figure 8 The images show a comparison of the antibody conjugation efficiency of the immunoassay chip in Example 3 of this application. The left image shows the chip modified with the compound of formula (IV), and the right image shows the unmodified chip.

[0044] Figure 9 The comparison results of SNP detection signals in Example 4 of this application are shown. The left figure shows the control group blocked with conventional BSA, and the right figure shows the experimental group modified with the compound of formula (III).

[0045] Figure 10 This is the result of comparing crosstalk between dPCR detection units in Example 5 of this application. The left figure is the control group, and the right figure is the experimental group modified with the compound of formula (II).

[0046] Detailed Explanation

[0047] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this application pertains. When a quantity, concentration, or other value or parameter is expressed as a range, preferred range, or preferred upper and lower numerical limits, it should be understood that this is equivalent to specifically disclosing any range by combining any pair of upper or preferred values ​​with any lower or preferred value, regardless of whether the range is specifically disclosed. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within that range.

[0048] When used with a numerical variable, the terms "about" or "approximately" usually mean that the value of the variable and all values ​​of the variable are within the experimental error (e.g., within a 95% confidence interval for the mean) or within ±10% of the specified value, or a wider range.

[0049] The expression "comprising," or similar expressions such as "including," "containing," and "having," is open-ended and does not exclude additional unlisted elements, steps, or components. The expression "consisting of," excludes any unspecified elements, steps, or components. The expression "substantially consisting of," limits the scope to the specified elements, steps, or components, plus optional elements, steps, or components that do not materially affect the essential and novel features of the claimed subject matter. It should be understood that the expression "comprising" encompasses both the expressions "substantially consisting of" and "consisting of."

[0050] The expression "at least one" or "one or more" indicates 1, 2, 3, 4, 5, 6, 7, 8, 9 or more kinds.

[0051] Unless otherwise specified, "nM" in this article refers to "n mol / L", "μM" refers to "μmol / L", and "mM" refers to "m mol / L".

[0052] This application provides a microarray chip that can be used for in situ nucleic acid synthesis, SNP detection, single-molecule immunoassay, and single-cell sequencing. The substrate of this microarray chip, through surface modification, can significantly increase the functional groups on the surface, thereby improving nucleic acid synthesis yield, amplifying detection signals, and avoiding nonspecificity and crosstalk. It has broad application prospects.

[0053] This application provides the use of the compound represented by formula (I), or its geometrical isomers, tautomers, isotopic labels, hydrates, solvates, or salts thereof, in chip surface modification, where formula (I) is:

[0054]

[0055] Wherein, X1 is CH or N, X2 is CH2, C=O or SS, and when X2 is the terminal group of the compound, it is CH3 or CH3C=O; R1 and R5 are each independently -COOH or -OH or other negatively charged groups in aqueous solution, R3 is -NH2 or -C(=O)OR, R6 is -NH2, -C(=O)OR or -OH, where R is a carbon chain with C≤5, R2 and R4 are each independently benzene, biphenyl, anthraquinone or other superhydrophobic conjugable groups, n≥1, n2≥3.

[0056] This application provides compounds of formula (I), or their geometric isomers, tautomers, isotopic labels, hydrates, solvates, or salts thereof, wherein formula (I) is:

[0057]

[0058] Wherein, X1 is CH or N, X2 is CH2, C=O, or SS, and when X2 is the terminal group of the compound, it is CH3 or CH3C=O; R1 and R5 are each independently -COOH or -OH or other negatively charged groups in aqueous solution, R3 is -NH2 or -C(=O)OR, R6 is -NH2, -C(=O)OR, or -OH, where R is a carbon chain with C≤5, R2 and R4 are each independently benzene, biphenyl, anthraquinone, or other superhydrophobic conjugable groups, n≥1, n2≥3. Both ends are n2, n2≥3, so the number of repeating units at both ends is the same to achieve self-assembly and construction of amphiphilic compounds.

[0059] In some implementations, n2 is a multiple of 4.

[0060] In some implementations, n is 1.

[0061] In some implementations, R1 is -COOH, R5 is -OH, R3 is -NH2, and R6 is -OH.

[0062] In some implementations, R1 is -COOH, R5 is -COOH, R3 is -NH2, and R6 is -NH2.

[0063] In some implementations, R2 and R4 are benzene, biphenyl, or anthraquinone.

[0064] In some implementations, R1 is -COOH, R5 is -OH, R3 is -NH2, and R6 is -OH, R2 and R4 are benzene, biphenyl, or anthraquinone, and n is 1.

[0065] In some implementations, R1 is -COOH, R5 is -COOH, R3 is -NH2, and R6 is -NH2, R2 and R4 are benzene, biphenyl, or anthraquinone, and n is 1.

[0066] In some implementations, equation (I) is:

[0067]

[0068]

[0069] In formulas (I-1), (I-2), and (I-3), the definitions of each group are the same as in formula (I).

[0070] In some embodiments, the compound represented by formula (I) is

[0071]

[0072] On the other hand, this application also provides a microarray chip substrate, which is modified with a compound of formula (I), or its geometric isomers, tautomers, isotopic markers, hydrates, solvates or salts thereof, wherein formula (I) is:

[0073]

[0074] Wherein, X1 is CH or N, X2 is CH2, C=O, or SS, and when X2 is the terminal group of the compound, it is CH3 or CH3C=O; R1 and R5 are each independently -COOH or -OH or other negatively charged groups in aqueous solution, R3 is -NH2 or -C(=O)OR, R6 is -NH2, -C(=O)OR, or -OH, where R is a carbon chain with C≤5, R2 and R4 are each independently benzene, biphenyl, anthraquinone, or other superhydrophobic conjugable groups, n≥1, n2≥3. Both ends are n2, n2≥3, so the number of repeating units at both ends is the same to achieve self-assembly and construction of amphiphilic compounds.

[0075] In some implementations, n2 is a multiple of 4.

[0076] In some implementations, n is 1.

[0077] In some implementations, R1 is -COOH, R5 is -OH, R3 is -NH2, and R6 is -OH.

[0078] In some implementations, R1 is -COOH, R5 is -COOH, R3 is -NH2, and R6 is -NH2.

[0079] In some implementations, R2 and R4 are benzene, biphenyl, or anthraquinone.

[0080] In some implementations, R1 is -COOH, R5 is -OH, R3 is -NH2, and R6 is -OH, R2 and R4 are benzene, biphenyl, or anthraquinone, and n is 1.

[0081] In some implementations, R1 is -COOH, R5 is -COOH, R3 is -NH2, and R6 is -NH2, R2 and R4 are benzene, biphenyl, or anthraquinone, and n is 1.

[0082] In some implementations, equation (I) is:

[0083]

[0084] In formulas (I-1), (I-2), and (I-3), the definitions of each group are the same as in formula (I).

[0085] In some embodiments, the compound represented by formula (I) is

[0086]

[0087] In some embodiments, the material of the microarray chip substrate is selected from one or more of glass, metal, plasmonic materials, nitrocellulose, cellulose acetate, silicone wafers, nylon films, polypropylene films, micro-magnetic beads, latex microspheres, resin microspheres, and plastics. In some embodiments, the glass is selected from: lead glass, bismuth glass, zinc glass, barium glass, calcium glass, soda-lime glass, tellurium glass, lead silicate glass, alkaline silicate glass, alkali metal aluminum silicate glass, and alkali metal aluminum borosilicate glass; the metal is selected from: transition metals (selected from iron, cobalt, nickel, copper, molybdenum, vanadium, zinc, and manganese), noble metals, alkaline earth metals, and rare earth metals and their salts; the latex microspheres are selected from monodisperse triblock polymer latex microspheres, monodisperse polystyrene latex microspheres, and monodisperse polymethyl methacrylate latex microspheres; the resin microspheres are selected from phenolic resin, polystyrene resin, and polymethyl methacrylate resin microspheres; the plasmonic materials are selected from plasma gold, plasma silver, plasma copper, plasma aluminum nanomaterials, magnetic plasmonic materials, graphene, and semiconductor materials.

[0088] In another aspect, this application also provides a microarray chip comprising the microarray chip substrate described herein.

[0089] In some implementation schemes, the chip provided in this application may be a gene sequencing chip, expression profiling chip, disease diagnosis chip, drug screening chip, sample preparation chip, biochemical reaction chip, or result detection chip, etc.

[0090] Disease diagnosis includes products and services that obtain clinical diagnostic information and thereby determine disease or bodily function by testing human samples (e.g., blood, body fluids, tissues, etc.) outside the human body. Testing may include, but is not limited to, in vitro diagnostic tests on proteins such as antigen-antibody pairs, enzyme-enzyme substrate pairs, ligand-receptor pairs (cells), nucleic acids, hormones, or small molecules. Any in vitro diagnostic test that can utilize the microarray chip substrate or microarray chip described in this application may be included in this application.

[0091] In another aspect, this application also provides a kit comprising a microarray chip substrate or a microarray chip as described herein.

[0092] In some embodiments, the kit includes reagents for processing samples to release and process internal nucleic acids or proteins, including reagents for nucleic acid extraction, thermal lysis, physical lysis, reverse transcription amplification of nucleic acids, modification with terminal labeling groups, and / or single-stranding of nucleic acids. In some embodiments, the labeling group is selected from fluorescent groups, biotinylated groups, isotope labeling, magnetic labeling, chemiluminescent labeling, redox active moieties, optical dyes, or phosphate groups.

[0093] In some embodiments, the fluorescent group includes, but is not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, etc., or their analogues), Alexa. Series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750, etc. or their analogues), IRDye series dyes and their derivatives (including IRDye 680, IRDye 750, IRDye 800) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP), etc.).

[0094] In some implementations, isotope labeling includes, but is not limited to, 212 Bi、 131 I, 111 In、 90 Y、 186 Re、211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F.

[0095] In some embodiments, the chemiluminescent label is selected from aminobenzoyl hydrazide labeling, isoaminobenzoyl hydrazide labeling, aromatic acridine ester labeling, imidazole labeling, acridine ester salt labeling, oxalate labeling, luciferin labeling, luciferase labeling, and jellyfish luminescent protein labeling.

[0096] In some embodiments, the phosphate group is selected from inorganic phosphate compounds and organic phosphate compounds. Inorganic phosphate compounds include phosphoric oxyacids such as phosphoric acid, phosphorous acid, hypophosphoric acid, orthophosphoric acid, pyrophosphoric acid, and their salts. Organic phosphate compounds contain at least one phosphate group attached to an organic moiety and include unsaturated organic phosphate compounds such as phosphate monomers; and saturated organic phosphate compounds, and water-soluble polymers with phosphate groups. Water-soluble polymers with phosphate groups are addition polymers containing at least two phosphate groups independently located at the side or terminal positions of the water-soluble polymer backbone. Water-soluble polymers with phosphate groups can be homopolymers or copolymers, and have a degree of polymerization of at least 2. In some embodiments, the phosphate group is selected from phosphonate groups, thiophosphate groups, phosphate diester groups, morpholinophosphate groups, piperazine phosphate groups, and phosphoramide groups.

[0097] In some embodiments, the labeling groups include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine esters and their derivatives (such as acridine sulfonamide), dioxane and its derivatives, rofen and its derivatives, and peroxazone and its derivatives.

[0098] On the other hand, this application also provides the use of the microarray chip substrate or the microarray chip or the kit described herein in in situ nucleic acid synthesis, SNP detection, single-molecule immunoassay, single-cell sequencing, molecular hybridization or digital PCR.

[0099] On the other hand, this application also provides a method for modifying a microarray chip substrate, comprising:

[0100] Hydrophilic materials are deposited or coated between microarrays on a microarray chip substrate, and hydrophobic materials are deposited or coated on the microarray.

[0101] The compound described herein was uniformly coated on the chip surface and then oscillated uniformly on a shaker at a frequency of -30° to +30° for 1-10 hours to form a self-assembled film.

[0102] In some embodiments, the compounds described herein self-assemble to form a film with a hydrophilic portion on one side and a hydrophobic portion on the other.

[0103] In some embodiments, the hydrophobic portion of the compound described herein is bonded to a hydrophobic material on a microarray, such that the hydrophilic portion of the compound described herein is exposed on the microarray; and the hydrophilic portion of the compound described herein is bonded to a hydrophilic material between the microarrays, such that the hydrophobic portion of the compound described herein is exposed between the microarrays.

[0104] In some implementations, the hydrophilic material is SiO2 or polydopamine, and the hydrophobic material is SiNx or resin-based hydrophobic materials.

[0105] On the other hand, this application also provides a method for in situ synthesis of nucleic acids, comprising:

[0106] Nucleic acid monomers are linked to the microarray chip substrate or the microarray chip described herein;

[0107] Addressable microelectrode arrays can be used to guide the molecular assembly of specific nucleic acid base sequences on a microarray chip substrate or microarray chip.

[0108] In some implementation schemes, the method for in situ synthesis of nucleic acids provided in this application includes:

[0109] Microarray fabrication: The glass underwent standard pre-cleaning, followed by Mo deposition as Gate 1, then a PVX layer. A 30nm thick IGZO layer was deposited by magnetic sputtering as the channel material, and then a SiO2 layer was spin-coated. Al was deposited on the SiO2 to prepare Gate 2, followed by ILD deposition as the dielectric layer. Wet etching was performed using a metal mask to form the source (S) and drain (D). Metal electrodes (20nm Cr / 40nm Au) were deposited on the source and drain using a sputter. The sensing area was exposed by electron beam lithography, and finally Au was deposited using a sputter, followed by SiNx deposition.

[0110] Surface modification: The compound described in this article is uniformly coated on the chip surface, and then the chip is uniformly oscillated at -30° to +30° at a period of 15 min for 1-5 h to form a self-assembled film layer.

[0111] Nucleic acid synthesis: After ultrasonic cleaning with PBS, a current-response switch is used. This chip integrates a high-density TFT array, where each electrode can be independently controlled. When the electrode is energized, it produces acid, removing the protecting groups from DNA monomers and connecting them to form an addressable microelectrode array to guide the molecular assembly of specific DNA base sequences. This responds to digital commands and allows for independent control of the synthesis of different types and sequences of DNA molecules.

[0112] In some implementations, the compounds described herein are:

[0113] in It can be replaced with other conjugable hydrophobic groups, but C≤15, X2 is CH2, C=O or SS, CH2 or C=O is preferred, and when X2 is the end group of the compound, it is CH3 or CH3C=O.

[0114] In some implementations, the nucleic acid molecule can be any nucleic acid molecule composed of natural nucleotides and / or non-natural nucleotides, such as DNA molecules, RNA molecules, or derivatives thereof.

[0115] On the other hand, this application also provides a method for SNP detection, comprising:

[0116] The nucleic acid single-stranded probe is printed onto the microarray chip substrate or the microarray chip described in this article. Under the catalysis of polymerase, the -OH of the compound shown in formula (I) binds to the 5' phosphate of the nucleic acid single-stranded probe to complete the preparation of the SNP detection chip.

[0117] The nucleic acid to be tested is broken into fragments of 150-800bp, and the broken nucleic acid fragments are hybridized with single-stranded nucleic acid probes on an SNP detection chip.

[0118] In some implementation schemes, the SNP detection method provided in this application includes:

[0119] Microarray fabrication: The glass is pre-cleaned according to standard procedures, a layer of SiO2 is deposited on the surface, and then SiNx is deposited on top of it. Periodic and regular dense array is etched out by exposure. After the SiNx is etched at the intervals between the microarray units, the SiO2 film is fully exposed.

[0120] Surface modification: The compound described in this article is uniformly coated on the chip surface, and then the chip is uniformly oscillated on a shaker at a frequency of -30° to +30° for 15 min intervals for 3-10 h to form a self-assembled film layer.

[0121] SNP detection: Single-stranded DNA probes are printed onto corresponding microarrays with a purity OD260 / 280 between 1.6 and 1.8. Under enzymatic catalysis, -OH groups bind to the 5' phosphate group, completing chip fabrication. The analyte is then broken into 150-800 bp fragments, and hybridization is performed at 42°C.

[0122] In some implementations, the compounds described herein are:

[0123]

[0124] Wherein, X2 is CH2 or C=O, and when X2 is an end group of the compound, it is CH3 or CH3C=O.

[0125] In some implementations, the sample is processed prior to detection. Processing methods include column extraction of the target nucleic acid, magnetic bead extraction, thermal lysis extraction, ultrasonic extraction, or physical grinding extraction. In some implementations, the nucleic acid is optionally reverse transcribed using MMLV reverse transcriptase, recombinant MMLV reverse transcriptase, PrimeScript reverse transcriptase, or ProtoScript reverse transcriptase.

[0126] On the other hand, this application also provides a method for immune detection, comprising:

[0127] The probe molecules are printed onto the microarray chip substrate or the microarray chip described in this article to complete the fabrication of the immune detection chip;

[0128] The test sample containing the target molecule is slowly passed through or covered on the surface of the microarray. After the reaction, an antibody with a fluorescent label is slowly passed through or covered on the surface of the microarray. The reaction is then observed by fluorescence color development.

[0129] In some implementation schemes, the immunoassay method provided in this application includes:

[0130] Microarray fabrication: The glass is pre-cleaned according to standard procedures, a layer of SiO2 is deposited on the surface, and then SiNx is deposited on top of it. Periodic and regular dense array is etched out by exposure. After the SiNx is etched at the intervals between the microarray units, the SiO2 film is fully exposed.

[0131] Surface modification: The compound described in this article is uniformly coated on the chip surface, and then the chip is uniformly oscillated on a shaker at a frequency of -30° to +30° for 15 min intervals for 3-10 h to form a self-assembled film layer.

[0132] Antibody conjugation: The antibody was printed onto the corresponding microarray, which was then activated with DIC:NHS at a 1:1 ratio and incubated overnight at 4°C and 80% humidity. The microarray was washed sequentially with 10xPBS, 1xPBS, and ddH2O to remove any unconjugated antibodies.

[0133] Immunoassay: The test sample (blood, saliva, or other antigen-containing liquids) is slowly passed through the chip or covered on the microarray surface, reacting for 5-30 minutes. The sample is then washed sequentially with 10xPBS, 1xPBS, and ddH2O to remove unbound or non-specifically bound samples. Next, antibodies with different epitopes and fluorescent labels are slowly passed through the chip or covered on the microarray surface, reacting for 5-30 minutes. The sample is then washed sequentially with 10xPBS, 1xPBS, and ddH2O. Observation is then performed using fluorescence color development.

[0134] In some implementations, the compounds described herein are:

[0135]

[0136] Where n≥2, n²≥3, and R² is... Equivalent conjugable hydrophobic groups, wherein 6≤C≤24; X1 is CH2.

[0137] In some implementations, the protein used for immunoassay can be, for example, an antigen, antibody, ligand, receptor, enzyme, or enzyme substrate. It can detect the target molecule through a specific interaction with it. For an antigen and its antibody, the target molecule can be one of the antigen and its antibody, and the probe molecule can be the other. The same rule applies to ligands and their receptors, enzymes and their substrates, and so on.

[0138] In some implementations, the sample to be tested can be blood, serum, urine, saliva or semen, or tissue or cells.

[0139] This application describes several embodiments, but these descriptions are exemplary and not restrictive, and it will be apparent to those skilled in the art that many more embodiments and implementations are possible within the scope of the embodiments described herein. Although many possible combinations of features are shown in the accompanying drawings and discussed in the detailed description, many other combinations of the disclosed features are also possible. Unless specifically limited, any feature of any embodiment may be used in combination with any other feature in any other embodiment, or may substitute for any other feature in any other embodiment.

[0140] This application includes and contemplates combinations of features known to those skilled in the art. The embodiments and features disclosed in this application can also be combined with any conventional features to form a unique inventive scheme as defined by the claims. Any feature of any embodiment can also be combined with features from other inventive schemes to form another unique inventive scheme as defined by the claims. Therefore, it should be understood that any feature shown and / or discussed in this application can be implemented individually or in any suitable combination. Therefore, the embodiments are not limited except by the limitations imposed by the appended claims and their equivalents. Furthermore, various modifications and changes can be made within the scope of the appended claims.

[0141] Furthermore, in describing representative embodiments, the specification may have presented methods and / or processes as a specific sequence of steps. However, the method or process should not be limited to the specific order of steps described herein, to the extent that it does not depend on such a specific order. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of steps set forth in the specification should not be construed as a limitation of the claims. Moreover, the claims concerning the method and / or process should not be limited to the steps performed in the written order, and those skilled in the art will readily understand that these orders can be varied and still remain within the spirit and scope of the embodiments of this application.

[0142] Experimental methods in the following embodiments without specific conditions are generally determined according to national standards. Experimental materials in the following embodiments without specified sources are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, then generally accepted international standards, conventional conditions, or conditions recommended by the manufacturer are followed. Unless otherwise defined or stated, all technical and scientific terms used in this application have the same meaning as those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to the methods of this application. Example

[0143] Example 1. Improved Synthesis Yield in DNA In-situ Synthesis Chip

[0144] 1. Microarray Fabrication: The glass undergoes standard pre-cleaning, followed by Mo deposition as Gate 1, then a PVX (inorganic protective layer) layer. A 30nm thick IGZO (indium gallium zinc oxide) layer is deposited by magnetic sputtering as the channel material, and then a layer of SiO2 is spin-coated. Al is deposited on the SiO2 to prepare Gate 2, followed by the deposition of an ILD (Inter-Layer Dielectric) layer (SiO2 or SiNx) as the dielectric layer. Wet etching is performed using a metal mask to form the source (S) and drain (D) electrodes. Metal electrodes (20nm Cr / 40nm Au) are deposited on the source and drain using a sputter. The sensing area is exposed by electron beam lithography, and finally Au is deposited using a sputter, followed by SiNx deposition.

[0145] 2. Surface modification: The microarray was modified using a compound of formula (II) as follows:

[0146]

[0147] The preparation method of the compound of formula (II) is as follows: Under an anhydrous and oxygen-free environment, the reactant monomers (COOHCH=CH2, CH2=CHC) are reacted... 10 H7, CH2=CH2, CHOH=CH2, NH2CH=CH2) are dissolved in DMF or tetrahydrofuran and added sequentially from left to right. The molar ratio of substrate to newly added monomer is 1:1, 1:1.2, and 1:1.2, increasing by 20% for every two steps to ensure excess. The reaction is carried out at 100-150℃ for at least 1.5 hours. Catalysts include Cr and Mo. After the reaction is complete, the product is filtered and purified by column chromatography. After purification, the next monomer is added, and the above steps are repeated until all units have completed polymerization.

[0148] The polymerized compounds were validated using mass spectrometry and infrared spectroscopy. The mass spectrometry was performed using Bruker Autoflex. TM The Speed ​​LRF instrument is used to spread the sample solution (concentration 1.5 mg / mL) evenly on the target. After thorough evaporation and drying, a matrix layer is spread evenly. A solid-state fast ultraviolet laser with an adjustable pulse frequency of 1 Hz to 2000 Hz and a wavelength of 355 nm is used to focus on the target sample for accurate positioning. Infrared spectroscopy is performed using a PerkinElmer Spectrum™ 400 instrument. A small amount of extract is deposited on the ATR crystal, and after the solvent evaporates, the resulting oil film is tested for spectral analysis. After background testing, 25 μl of extract is spread as small droplets along the length of the ATR crystal using a micropipette for infrared spectroscopy.

[0149] The results are as follows Figure 1 (mass spectrometry) and Figure 2 As shown in the infrared spectrum.

[0150] The compound of formula (II) has a molecular weight of 6351, derived from... Figure 1 It can be seen that the molecular weight confirmed by mass spectrometry characterization of the mass-charge ratio is consistent with it. From Figure 2 The infrared spectrum shows that the compound contains the corresponding -NH2, -COOH, -OH and naphthalene, proving that the molecular structure of the compound is correct.

[0151] The synthesized compound of formula (II) was allowed to self-assemble into a nanosheet structure, which was then observed under atomic force microscopy (AFM, Bruker Dimension). The structure of the nanosheets was observed below, and the results are as follows: Figure 3 As shown

[0152] The negatively charged ends of the compound of formula (II) are arranged alternately with -COOH and -OH, and the hydrophobic end is biphenyl, which improves hydrophobicity and prevents crosstalk caused by the diffusion of acidification environment during synthesis. Sucrose (used as control group) or surface modification molecules of formula (II) (experimental group) are uniformly coated on the chip surface and shaken uniformly at -30° to +30° with a period of 15 min for 3 h to form a self-assembled film layer.

[0153] 3. DNA Synthesis: After ultrasonic cleaning with PBS, an electric current is used as the reaction switch. The chip integrates a high-density TFT array, and each electrode can be independently controlled. When the electrode is energized, acid is generated, removing the protecting groups of DNA monomers and connecting them to form an addressable microelectrode array to guide the molecular assembly of specific DNA base sequences. This responds to digital commands and independently controls the synthesis of DNA molecules. The sequence of the synthesized DNA molecule is shown in SEQ ID NO:1.

[0154] 4. Results: such as Figure 4 As shown, the DNA synthesis yield of the chip modified with the compound of formula (II) is significantly higher than that of the chip modified with sucrose.

[0155] Example 2. Amplification of SNP gene chip detection signals

[0156] 1. Microarray fabrication: The glass is pre-cleaned according to standard procedures, a layer of SiO2 is deposited on the surface, and then SiNx is deposited on top of it. Periodic and regular dense array is etched by exposure. After the SiNx is etched at the intervals between the microarray units, the SiO2 film is fully exposed.

[0157] 2. Surface modification: The control group consisted of untreated chips, while the experimental group used compounds of formula (III) to modify the microarray:

[0158]

[0159] The compound of formula (III) was prepared as in Example 1 using the compound of formula (II), employing reactant monomers (COOHCH=CH2, CH2=CHC6H5, CH2=CH2, CHOH=CH2, NH2CH=CH2). The synthesized compound of formula (III) was allowed to self-assemble into a nanosheet structure, and the structure of the nanosheets was observed under an atomic force microscope (AFM). The structure is as follows: Figure 5 As shown.

[0160] Its negatively charged ends -COOH and -OH are arranged alternately, and the hydrophobic end group is benzene. The compound of formula (III) is uniformly coated on the chip surface and oscillated uniformly on a shaker at a period of -30° to +30° for 15 min for 5 h to form a self-assembled film layer.

[0161] 3. SNP detection: Single-stranded DNA probes (sequences shown in SEQ ID NO:2) are printed onto the corresponding microarray with a purity OD260 / 280 between 1.6 and 1.8. Under enzymatic catalysis, -OH groups bind to the 5' phosphate group, completing the chip fabrication. The analyte is broken into 150-800 bp fragments, and hybridization is performed at 42°C.

[0162] 4. Results: such as Figure 6 As shown, the chip modified with the compound of formula (III) (bottom figure) has a significantly higher fluorescence intensity compared to the chip not modified with the compound of this application (top figure).

[0163] Example 3. Improved antibody conjugation rate and detection limit of the immunoassay chip.

[0164] 1. Microarray fabrication: The glass is pre-cleaned according to standard procedures, a layer of SiO2 is deposited on the surface, and then SiNx is deposited on top of it. Periodic and regular dense array is etched by exposure. After the SiNx is etched at the intervals between the microarray units, the SiO2 film is fully exposed.

[0165] 2. Surface Modification: The control group consisted of unmodified chips, while the experimental group used compounds of formula (IV) to modify the microarray:

[0166]

[0167] The preparation method of the compound of formula (IV) is the same as that of the compound of formula (II) in Example 1, using reactant monomers (COOHCH=CH2, CH2=CH2, CH2O, CH2=CHC). 10H7, NH2CH=CH2). The synthesized compound of formula (IV) was allowed to self-assemble into a nanosheet structure, and the structure of the nanosheets was observed under an atomic force microscope (AFM). The structure is as follows. Figure 7 As shown.

[0168] Its negative terminal is -COOH, positive terminal is -NH2, and hydrophobic terminal group is biphenyl. The compound of formula (IV) is uniformly coated on the chip surface and oscillated uniformly on a shaker at a period of -30° to +30° for 15 min for 5 h to form a self-assembled film layer.

[0169] 3. Antibody conjugation: Anti-Aβ42 antibody was printed onto the corresponding microarray. The microarray was activated with n,n'-diisopropylcarbodiimide:n-hydroxysuccinimide (DIC:NHS) at a ratio of 1:1 and incubated overnight at 4°C and 80% humidity. The microarray was then washed sequentially with 10xPBS, 1xPBS, and ddH2O to remove any unconjugated antibody.

[0170] 4. Immunoassay: The blood sample is slowly passed through the chip or covered on the microarray surface, reacting for 15 minutes. The sample is then washed sequentially with 10xPBS, 1xPBS, and ddH2O to remove unbound or non-specifically bound antibodies. Next, Anti-Aβ42 antibodies with different epitopes and green fluorescent labels are slowly passed through the chip or covered on the microarray surface, reacting for 15 minutes. The sample is then washed sequentially with 10xPBS, 1xPBS, and ddH2O. The results are observed through fluorescence color development.

[0171] 5. Results: such as Figure 8 As shown, the left side represents the chip modified with the compound of formula (IV), while the right side represents the unmodified scheme. It is evident that the antibody conjugation efficiency is significantly improved.

[0172] Example 4. Reduction of non-specific adsorption on the chip

[0173] 1. Select the SNP detection microarray chip in Example 2, and set up an experimental group and a control group. The experimental group was modified with the compound of formula (III), and the control group was blocked with conventional BSA.

[0174] 2. After sealing, the SNP chip is tested using step 3 of Example 2, and the fluorescence intensity at the microarray intervals is compared.

[0175] 3. Results: such as Figure 9As shown, higher fluorescence intensity at the microarray spacers indicates greater non-specific binding during the experiment. The left side represents the control group blocked with conventional BSA, while the right side represents the experimental group modified with compounds of formula (III). In the experimental group on the right, the fluorescence intensity at the microarray spacers is significantly lower than that in the control group on the left, indicating that modification with compounds of formula (III) can significantly reduce non-specific binding in the microarray chip.

[0176] Example 5. Anti-crosstalk for dPCR detection unit

[0177] 1. This embodiment uses a dPCR chip, with a control group and an experimental group. The dPCR chip is prepared as in Example 1, wherein the experimental group is modified with the compound of formula (II), and the control group is tested in the original state of the chip.

[0178] 2. Prepare standards. Use nucleic acid standards telomerase reverse transcriptase C228T mutant nucleic acid DNA (TERT C228T) to set gradient copy numbers (5, 50, 500, 5000, 50000), and perform dPCR reaction according to the dPCR chip operation procedure;

[0179] 3. Based on the preset values, compare the experimental results of the modified and unmodified compounds according to formula (II), and analyze the deviation between the two. The results are as follows: Figure 10 As shown, the left side represents the control group, and the right side represents the experimental group modified with the compound of formula (II). This demonstrates that modification with the compound of formula (II) can significantly reduce crosstalk between detection units in the microarray chip.

Claims

1. The use of the compound represented by formula (I), or its geometrical isomers, tautomers, isotopic markers, hydrates, solvates, or salts thereof, in the modification of chip surfaces, wherein formula (I) is: in, X1 is CH or N, X2 is CH2, C=O or SS, and when X2 is the terminal group of the compound, it is CH3 or CH3C=O; R1 and R5 are each independently -COOH or -OH or other negatively charged groups in aqueous solution, R3 is -NH2 or -C(=O)OR, R6 is -NH2, -C(=O)OR or -OH, where R is a carbon chain with C≤5, R2 and R4 are each independently benzene, biphenyl, anthraquinone or other superhydrophobic conjugable groups, n≥1, n2≥3.

2. The use according to claim 1, wherein, n² is a multiple of 4.

3. The use according to claim 1, wherein n is 1; and / or R1 is -COOH, R5 is -OH, R3 is -NH2, and R6 is -OH; or, R1 is -COOH, R5 is -COOH, R3 is -NH2, and R6 is -NH2; and / or R2 and R4 are benzene, biphenyl, or anthraquinone.

4. The use according to claim 1, wherein, Equation (I) is: The definitions of each group in formulas (I-1), (I-2), and (I-3) are the same as those in formula (I).

5. The use according to claim 1, wherein, The compound represented by formula (I) is 6. A microarray chip substrate, said microarray chip substrate being modified with a compound of formula (I), or its geometric isomers, tautomers, isotope markers, hydrates, solvates, or salts thereof, said formula (I) being: in, X1 is CH or N, X2 is CH2, C=O or SS, and when X2 is the terminal group of the compound, it is CH3 or CH3C=O; R1 and R5 are each independently -COOH or -OH or other negatively charged groups in aqueous solution, R3 is -NH2 or -C(=O)OR, R6 is -NH2, -C(=O)OR or -OH, where R is a carbon chain with C≤5, R2 and R4 are each independently benzene, biphenyl, anthraquinone or other superhydrophobic conjugable groups, n≥1, n2≥3.

7. The microarray chip substrate according to claim 6, wherein, n² is a multiple of 4.

8. The microarray chip substrate according to claim 6, wherein n is 1; and / or R1 is -COOH, R5 is -OH, R3 is -NH2, and R6 is -OH; or, R1 is -COOH, R5 is -COOH, R3 is -NH2, and R6 is -NH2; and / or R2 and R4 are benzene, biphenyl, or anthraquinone.

9. The microarray chip substrate according to claim 6, wherein, Equation (I) is: The definitions of each group in formulas (I-1), (I-2), and (I-3) are the same as those in formula (I).

10. The microarray chip substrate according to claim 6, wherein, The compound represented by formula (I) is 11. The microarray chip substrate according to any one of claims 6-10, wherein, The material of the microarray chip substrate is selected from one or more of glass, metal, plasmonic materials, nitrocellulose, cellulose acetate, silicone wafers, nylon membranes, polypropylene membranes, micro-magnetic beads, latex microspheres, resin microspheres, and plastics.

12. A microarray chip, the microarray chip comprising the microarray chip substrate of any one of claims 6-11.

13. A kit comprising a microarray chip substrate according to any one of claims 6-11 or a microarray chip according to claim 12.

14. Use of the microarray chip substrate of any one of claims 6-11, the microarray chip of claim 12, or the kit of claim 13 in in situ nucleic acid synthesis, SNP detection, single-molecule immunoassay, or single-cell sequencing.

15. A method for modifying a microarray chip substrate, comprising: Hydrophilic materials are deposited or coated between microarrays on a microarray chip substrate, and hydrophobic materials are deposited or coated on the microarray. The compound of any one of claims 1-5 is uniformly coated on the chip surface, and then the chip is shaken uniformly on a shaker for 1-10 hours to form a self-assembled film layer.

16. The method according to claim 15, wherein, The hydrophilic material is SiO2 or polydopamine, and the hydrophobic material is SiNx or a resin-based hydrophobic material.

17. A method for in situ synthesis of nucleic acids, comprising: The nucleic acid monomer is linked to the microarray chip substrate of any one of claims 6-11 or the microarray chip of claim 12; Addressable microelectrode arrays are used to guide the molecular assembly of specific nucleic acid base sequences on the microarray chip substrate or on the microarray chip.

18. A method for SNP detection, comprising: The nucleic acid single-stranded probe is printed onto the microarray chip substrate of any one of claims 6-11 or the microarray chip of claim 12, and the -OH of the compound shown in formula (I) is bound to the 5' phosphate of the nucleic acid single-stranded probe under the catalysis of polymerase to complete the preparation of the SNP detection chip; The nucleic acid to be tested is broken into fragments of 150-800bp, and the broken nucleic acid fragments are hybridized with the single-stranded nucleic acid probe on the SNP detection chip.

19. A method for immunoassay, comprising: The probe molecules are printed onto the microarray chip substrate of any one of claims 6-11 or the microarray chip of claim 12 to complete the fabrication of the immune detection chip; The test sample containing the target molecule is slowly passed through or covered on the surface of the microarray. After the reaction, an antibody with a fluorescent label is slowly passed through or covered on the surface of the microarray. The reaction is then observed by fluorescence color development.

20. A compound represented by formula (I), or its geometric isomers, tautomers, isotopic labels, hydrates, solvates, or salts thereof, wherein formula (I) is: in, X1 is CH or N, X2 is CH2, C=O or SS, and when X2 is the terminal group of the compound, it is CH3 or CH3C=O; R1 and R5 are each independently -COOH or -OH or other negatively charged groups in aqueous solution, R3 is -NH2 or -C(=O)OR, R6 is -NH2, -C(=O)OR or -OH, where R is a carbon chain with C≤5, R2 and R4 are each independently benzene, biphenyl, anthraquinone or other superhydrophobic conjugable groups, n≥1, n2≥3.

21. The compound of claim 20, or its geometric isomers, tautomers, isotopic markers, hydrates, solvates, or salts thereof, wherein, n² is a multiple of 4.

22. The compound of claim 20, or its geometric isomers, tautomers, isotopic labels, hydrates, solvates, or salts thereof, wherein n is 1; and / or R1 is -COOH, R5 is -OH, R3 is -NH2, and R6 is -OH; or, R1 is -COOH, R5 is -COOH, R3 is -NH2, and R6 is -NH2; and / or R2 and R4 are benzene, biphenyl, or anthraquinone.

23. The compound of claim 20, or its geometric isomers, tautomers, isotopic markers, hydrates, solvates, or salts thereof, wherein, Equation (I) is: The definitions of each group in formulas (I-1), (I-2), and (I-3) are the same as those in formula (I).

24. The compound of claim 20, or its geometric isomers, tautomers, isotopic markers, hydrates, solvates, or salts thereof, wherein, The compound represented by formula (I) is

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