Polyactivated ester microarray chip as well as preparation method and application thereof

Abstract

The invention discloses a polymer activated ester microarray chip as well as a preparation method and application thereof, and relates to the technical field of microarray chip preparation. The invention provides a poly (activated ester) microarray chip and a preparation method and application thereof, a poly (lysine) glass slide is modified by dicarboxylic acid activated ester, the poly (activated ester) microarray chip with a three-dimensional substrate is constructed, the process is simplified through a one-step coupling reaction, the technical bottleneck of a traditional microarray chip is broken through, and the production cost is reduced. The method has the advantages of simplicity and convenience in operation, high fixing efficiency and ultra-sensitive detection capability, provides an efficient tool for high-throughput analysis in the field of biomedicine, and has remarkable scientific value and market application prospect.

Description

Technical Field

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

[0002] Microarray is a cutting-edge high-throughput biotechnology that can achieve parallel detection of massive biological molecules such as DNA, RNA, proteins, and sugar molecules in biological samples. It can simultaneously analyze the expression abundance and existence status of these molecules, providing key data support for life science research and clinical diagnosis.

[0003] Microarray technology relies on flat substrates such as silicon wafers, glass slides or nylon membranes, and uses micromachining technology to arrange tiny probe sites in an orderly manner on the surface. Each site is loaded with specific molecular probes, such as oligonucleotides, antibodies, etc., which can accurately combine with target molecules in the sample through specific recognition mechanisms, thereby quickly obtaining the expression characteristics or existence information of biological molecules.

[0004] Microarrays, as a highly efficient analytical model, play an irreplaceable role in biomedical research, early disease diagnosis, and drug target screening. However, the efficiency and stability of biomolecule immobilization on the substrate directly determine the sensitivity and reliability of chip detection.

[0005] Traditional surface chemical treatment methods are cumbersome and involve multiple steps such as substrate hydroxyl activation, amino modification, and carboxyl functionalization. Not only are the operations complex and costly, but the way the molecular probes are fixed on a two-dimensional plane limits space utilization, making it difficult to meet the needs of trace biomarker detection.

[0006] In view of this, how to develop a microarray chip substrate with a simple preparation process, high-density molecular probe fixation capability and high curing efficiency, and which can significantly improve detection sensitivity has become a key technical problem that needs to be overcome in this field.

[0007] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the Invention

[0008] In response to the above technical problems, the embodiments of the present invention provide a polymer activated ester microarray chip and a preparation method and application thereof to solve the problems raised in the above background technology.

[0009] A polymer activated ester microarray chip comprises a polylysine glass slide and polycarboxylic acid activated esters modified on the polylysine glass slide through covalent bonds.

[0010] Preferably, the polycarboxylic acid activated ester refers to a compound containing at least two carboxylic acid activated ester groups. In this patent, the compound containing at least two carboxylic acid activated ester groups is also a polycarboxylic acid activated ester.

[0011] A method for preparing the above-mentioned polymer activated ester microarray chip comprises the following steps:

[0012] Adding a polycarboxylic acid activated ester and a base to a solvent respectively, and stirring until dissolved to obtain a first mixed solution;

[0013] The poly-lysine substrate is immersed in the first mixed solution and reacted under the protection of an inert gas to obtain a poly-activated ester microarray chip.

[0014] Preferably, the volume ratio of the base to the solvent is 1:1-1:1000; and the concentration of the polycarboxylic acid activated ester is 0.1-200 mg / mL.

[0015] Preferably, the volume ratio of the base to the solvent is 1:100; and the concentration of the polycarboxylic acid activated ester is 5 mg / mL.

[0016] Preferably, the solvent includes one of N,N-dimethylformamide, ethyl acetate, dichloromethane, tetrahydrofuran, dimethyl sulfoxide, acetone, chloroform, methanol, ethanol, acetonitrile, toluene, pyridine, and petroleum ether.

[0017] Preferably, the base includes: N, N-diisopropylethylamine, triethylamine, imidazole, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), pyrrole, quinoline, 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), quinuclidine, tetramethylguanidine, 2,6-lutidine, 4-dimethylaminopyridine (DMAP), sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, lithium carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide.

[0018] Preferably, the polycarboxylic acid activated ester includes one of: succinimide activated ester, sulfated succinimide activated ester, carbonyl imidazole activated ester, pentafluorophenol activated ester, p-nitrophenyl ester, activated sulfonate, HOBt / HOAt ester, fluoroalkyl ester, enol ester, trichloroethyl ester, and mixed anhydride;

[0019] The carboxylic acids in the polycarboxylic acid activated esters include: oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, other straight-chain alkane diacids, ethylene glycol-disuccinic acid, phthalic acid, terephthalic acid, isophthalic acid, maleic acid, fumaric acid, tartaric acid, muconic acid, malic acid, nitrilotriacetic acid, triacrylic acid, 1,3,5-cyclohexanetricarboxylic acid, 1,3,5-pyromellitic acid, 1,2,4-benzenetricarboxylic acid, 1,2,3-benzenetricarboxylic acid, pyridine-2,4,6-tricarboxylic acid, butane-1,2,4-tricarboxylic acid triethyl ester 2,3,5- One of tricarboxylic acid pyrrole, 2,4,6-trimethyl-1,3,5-triazine, hydrolyzed limonene, 1,3,5-tricarboxypentane, 2-methylpropanetricarboxylic acid, 3-pentylbenzene-1,3,5-tricarboxylic acid, 2'-nitro-[1,1'-biphenyl]-3,4',5-tricarboxylic acid, 2-hydroxy-1,2,3-propanetricarboxylic acid, 5-nitro-1,2,3-benzenetricarboxylic acid, citric acid, isocitric acid, aconitic acid, 1,2,4-butanetricarboxylic acid, pyrazinetetracarboxylic acid, cyclobutane-1,1,3,3-tetracarboxylic acid, butanetetracarboxylic acid, cyclopentanetetracarboxylic acid, biphenyl-tetracarboxylic acid, ethylenediaminetetraacetic acid, polygalactonic acid, hyaluronic acid, alginic acid, polyacrylic acid, etc.

[0020] An application of the above-mentioned polyactivated ester microarray chip in the detection of target biomolecules.

[0021] Preferably, the target biomolecule includes one of: a small molecule active compound, an amino acid, a polypeptide, a protein, a glycoprotein, a sugar, a derived sugar compound, DNA, RNA, and a lipid.

[0022] A multi-activated ester microarray chip, a preparation method thereof, and an application thereof provided in an embodiment of the present invention have the following beneficial effects: The present invention provides a multi-activated ester microarray chip, a preparation method thereof, and an application thereof, wherein a poly-lysine glass slide is modified with a dicarboxylic acid activated ester to construct a multi-activated ester microarray chip with a three-dimensional substrate. The process is simplified through a one-step coupling reaction, breaking through the technical bottleneck of traditional microarray chips. The chip has both simple operation, high fixation efficiency, and ultra-sensitive detection capabilities, providing an efficient tool for high-throughput analysis in the biomedical field, and having significant scientific value and market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a comparison chart of the screening effects of modifying different carboxylic acid activated esters on a poly-lysine substrate and commercially available activated ester chips;

[0024] Figure 2 The results of the lower limit of detection study of the polyactivated ester microarray chip prepared by the present invention are as follows;

[0025] Figure 3 This is a schematic diagram of the effect of the polyactivated ester microarray chip constructed in the present invention. DETAILED DESCRIPTION

[0026] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.

[0027] In response to the above technical problems, the embodiments of the present invention provide a polymer activated ester microarray chip and a preparation method and application thereof to solve the problems raised in the above background technology.

[0028] 1. The design concept of the present invention of a polymer activated ester microarray chip and its preparation method and application is as follows:

[0029] (1) The role of polylysine substrate: Polylysine is a polymer with a large number of amino groups, which is fixed on the surface of the glass substrate by physical adsorption or chemical coupling; its molecular chain extends upward perpendicular to the substrate surface to form a three-dimensional spatial structure, providing high-density amino sites for subsequent modification.

[0030] (2) Polycarboxylic acid activated esters, such as succinimide activated ester (NHS), contain activated ester groups (-COO-NHS), whose carbonyl carbons are highly reactive and can undergo nucleophilic acyl substitution reactions with amino groups on the surface of polylysine to form amide bonds, thereby fixing the activated esters on the substrate surface to generate polyactivated ester microarray chips.

[0031] (3) If the target biological molecules (such as proteins, DNA, peptides, etc.) contain amino groups, they can also react with activated ester groups through the amino groups to form covalent bonds for coupling; the coupling reaction between the target biological molecules and the polyactivated ester microarray chip does not require an additional activation step, and the molecules can be directly fixed through a one-step coupling, which simplifies the multi-step process of traditional chip preparation (such as silanization, carboxyl activation, etc.).

[0032] (4) The long-chain structure of polylysine enables the activated ester groups to form a three-dimensional array perpendicular to the plane on the substrate surface. Compared with traditional two-dimensional planar modifications (such as silanized substrates), it significantly increases the density of activated esters per unit area, thereby increasing the amount of molecular probes fixed.

[0033] (5) It is explained that a polycarboxylic acid activated ester contains at least two carboxylic acid activated ester groups, such as a dicarboxylic acid activated ester; a dicarboxylic acid activated ester has two reaction sites, namely the carbonyl carbons of the two ester groups; one of the reaction sites can react with the amino group on the polylysine chip to form an activated ester chip; the activated ester on the chip can undergo a substitution reaction with the amino group on the target biological molecule with the help of another reaction site, so that it can be fixed on the microarray for activity screening.

[0034] 2. Experimental part

[0035] The materials used in the present invention are as follows:

[0036] The polylysine substrate was purchased from Thermo Fisher Scientific. Capsular polysaccharide was purchased from the Serum Institute of India. Amino-modified 200 kD dextran and polyclonal antibodies were prepared in our laboratory. 200 kD dextran was purchased from the National Standard Material Platform.

[0037] Commercially available NHS-activated ester chip substrates were purchased from Qiyue Biotechnology. The novel coronavirus protein receptor binding domain (RBD) was donated by Chengdu Maikekang Biotechnology Co., Ltd. All other materials used in this invention, unless otherwise stated, are commercially available.

[0038] Example 1 - Comparative experiment on the screening effect of modified different carboxylic acid activated esters on polylysine substrate and commercial activated ester chip

[0039] The first step is chip preparation:

[0040] (1) Three activated succinimide esters of succinic acid, suberic acid, and suberic acid (1 mg / mL), as well as triethylamine and N, N-dimethylformamide, were prepared into a 40 ml solution (volume ratio of 1%) in a glass slide jar; then five polylysine substrates were immersed in the above mixed solution and reacted under the protection of inert gas for 4 hours. After the reaction, the glass slides were removed and washed three times with N, N-dimethylformamide. The obtained chips were dried under vacuum and stored at -20°C under argon. Chips 2, 3, and 4 were obtained. Chips 2, 3, and 4 are three different poly-activated ester microarray chips prepared by the present invention.

[0041] (2) Chip 1 is a lysine activated ester chip made in the previous laboratory. The preparation steps are as follows: the glass slide is treated with piranha wash solution, 3-aminopropyltrimethoxysilane reacts with the glass slide, succinate glycoside reacts with the amino group on the chip, and carboxylic acid is condensed with succinimide to obtain chip 1.

[0042] It is noted that chip 1 is prepared using a traditional substrate, which requires multiple steps of reaction and a complex preparation process. It relies on silanization and multi-step coupling to form a two-dimensional planar modification with a low density of activated ester groups.

[0043] (3) Chip 5 is an activated ester chip purchased from Qiyue Biotechnology.

[0044] Step 2: Sample fixation and detection:

[0045] (1) Sample configuration: Figure 1 Compound 1 is shown as a 1 mg / mL aqueous solution in 150 mM PBS pH=8.5; Compound 1 is an oligosaccharide.

[0046] (2) Spotting: Spot the compound 1 solution from step (1) onto five chips (6 replicates, 330 pL per spot) and react in a wet box for 12 hours.

[0047] (3) Blocking: Block the residual activated ester groups with 50 mM sodium phosphate buffer (pH 9.0) containing 100 mM ethanolamine for 1 h.

[0048] (4) Cleaning: Wash twice with 1×PBST, 2 minutes each time. After the time is up, pour out the cleaning solution and pat dry on dust-free paper.

[0049] (5) BSA blocking: Incubate with 1×TBS / 1% BSA for 1 hour and repeat step (4).

[0050] (6) Take the monoclonal antibody of compound 1 and dilute it at 1:100 (final concentration is 1 μg / mL) and incubate for 3 h, then repeat step (4).

[0051] (7) Secondary antibody incubation: Goat Anti-mouse IgG (Cy5) was diluted 1:1000 and incubated at 500 μL / well for 2 h. After the reaction, the chip and chip fence were completely immersed in 1× PBST and the chip fence was removed. Minimize the contact between the glass slide and air. Soak the slide in 1× PBST three times for 2 min each time. After the time is up, discard the cleaning solution and pat dry on a dust-free paper.

[0052] (8) Wash and centrifuge the plate, drain, and scan.

[0053] Filter results such as Figure 1 As shown: Figure 1 A shows the prepared chips 1-4 and the commercially available control chip 5. Figure 1 B is the comparison of chip fluorescence values. Figure 1 C is a schematic structural diagram of compound 1.

[0054] Figure 1The results showed that chips 3 and 4 had the best effects, indicating that the polylysine substrate prepared by the present invention performed optimally in terms of molecular immobilization efficiency, detection sensitivity, and background control.

[0055] This indicates that Compound 1 contains an amino group in its structure, which can undergo a nucleophilic acyl substitution reaction with activated ester groups on the chip surface, thereby covalently immobilizing it on the chip surface. By spotting Compound 1 on the chip and observing the fluorescence signal intensity and detection limit after immobilization, the chip's ability to immobilize biomolecules and its detection sensitivity can be intuitively assessed.

[0056] Example 2 - Detection limit comparison experiment

[0057] (1) Compound 1 (20 μg / mL, pH=8.5 PBS solution) and cell polysaccharide (CWPS, 1 μg / mL, pH=8.5 PBS solution) were diluted fivefold and spotted onto Chip 3 and Chip 5 in Example 1.

[0058] Repeat steps 3-8 in Example 1.

[0059] The test results are as follows Figure 2 As shown: For compound 1, the detection limit of the chip prepared by the present invention is 10 pg / mL, while the detection limit of the commercially available NHS chip is 32 ng / mL ( Figure 2 A). For cell wall polysaccharide CWPS, the detection limit of the chip prepared by the present invention is 0.32 ng / mL, while the detection limit of the commercially available NHS chip is 8 ng / mL ( Figure 2 B).

[0060] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A polymer activated ester microarray chip, characterized in that: include: Polylysine glass slide, and polycarboxylic acid activated ester modified on the polylysine glass slide by covalent bond.

2. Polymeric activated ester microarray chip according to claim 1, is characterized in that, The polycarboxylic acid activated ester refers to a compound containing at least two carboxylic acid activated ester groups.

3. A method for preparing a polymer activated ester microarray chip as claimed in claim 1, characterized in that: The following steps are involved: Adding a polycarboxylic acid activated ester and a base to a solvent respectively, and stirring until dissolved to obtain a first mixed solution; The poly-lysine substrate is immersed in the first mixed solution and reacted under the protection of an inert gas to obtain a poly-activated ester microarray chip.

4. The method for preparing a polymeric activated ester microarray chip according to claim 2, wherein The volume ratio of the base to the solvent is 1:1-1:1000; the concentration of the polycarboxylic acid activated ester is 0.1-200 mg / mL.

5. The method for preparing a polymeric activated ester microarray chip according to claim 2, wherein The volume ratio of base to solvent was 1:100; the concentration of polycarboxylic acid activated ester was 5 mg / mL.

6. The method for preparing a polymeric activated ester microarray chip according to claim 2, wherein The solvent includes one of N, N-dimethylformamide, ethyl acetate, dichloromethane, tetrahydrofuran, dimethyl sulfoxide, acetone, chloroform, methanol, ethanol, acetonitrile, toluene, pyridine and petroleum ether.

7. the preparation method of poly-activated ester microarray chip according to claim 2 is characterized in that, alkali include: N,N-diisopropylethylamine, triethylamine, imidazole, 1,8-diazabicyclo[5.4.0]undec-7-ene, pyrrole, quinoline, 1,5-diazabicyclo[4.3.0]non-5-ene, quinuclidine, tetramethylguanidine, 2,6-lutidine, 4-dimethylaminopyridine, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, lithium carbonate, sodium hydroxide, potassium hydroxide, one of lithium hydroxide.

8. The method for preparing a polymeric activated ester microarray chip according to claim 2, wherein The polycarboxylic acid activated ester includes one of: succinimide activated ester, sulfated succinimide activated ester, carbonyl imidazole activated ester, pentafluorophenol activated ester, p-nitrophenyl ester, activated sulfonate, HOBt / HOAt ester, fluoroalkyl ester, enol ester, trichloroethyl ester, and mixed anhydride; The carboxylic acids in the polycarboxylic acid activated esters include: oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, other straight-chain alkane diacids, ethylene glycol-disuccinic acid, phthalic acid, terephthalic acid, isophthalic acid, maleic acid, fumaric acid, tartaric acid, muconic acid, malic acid, nitrilotriacetic acid, triacrylic acid, 1,3,5-cyclohexanetricarboxylic acid, 1,3,5-pyromellitic acid, 1,2,4-benzenetricarboxylic acid, 1,2,3-benzenetricarboxylic acid, pyridine-2,4,6-tricarboxylic acid, butane-1,2,4-tricarboxylic acid triethyl ester 2,3,5- One of tricarboxylic acid pyrrole, 2,4,6-trimethyl-1,3,5-triazine, hydrolyzed limonene, 1,3,5-tricarboxypentane, 2-methylpropanetricarboxylic acid, 3-pentylbenzene-1,3,5-tricarboxylic acid, 2'-nitro-[1,1'-biphenyl]-3,4',5-tricarboxylic acid, 2-hydroxy-1,2,3-propanetricarboxylic acid, 5-nitro-1,2,3-benzenetricarboxylic acid, citric acid, isocitric acid, aconitic acid, 1,2,4-butanetricarboxylic acid, pyrazinetetracarboxylic acid, cyclobutane-1,1,3,3-tetracarboxylic acid, butanetetracarboxylic acid, cyclopentanetetracarboxylic acid, biphenyl-tetracarboxylic acid, ethylenediaminetetraacetic acid, polygalactonic acid, hyaluronic acid, alginic acid, polyacrylic acid, etc.

9. Use of the polymer activated ester microarray chip as claimed in claim 1 in detecting target biomolecules.

10. Use of the polyactivated ester microarray chip according to claim 9 in detecting target biomolecules, characterized in that: The target biological molecule includes: one of: small molecule active compounds, amino acids, polypeptides, proteins, glycoproteins, sugars, derived sugar compounds, DNA, RNA, and lipids.

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