A polyacrylic acid polymer modified substrate and its preparation method and application in preparing biochip

Abstract

The present invention discloses a polyacrylic acid polymer modified substrate, a preparation method thereof and an application thereof in preparing a biochip. The polyacrylic acid polymer modified substrate is prepared by modifying a polyacrylic acid polymer layer on a substrate, and the specific steps are substrate cleaning and activation, aminosilanization of the substrate surface, substrate surface modification initiator and substrate surface modification polyacrylic acid polymer. The polyacrylic acid polymer modified substrate prepared by the above method has good stability and is universal in the preparation of biochips, and the preparation method has the characteristics of simple operation, flexible and simple control, no need for special equipment and low cost.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to a polyacrylic acid polymer modified substrate, a preparation method thereof, and an application of the substrate in preparing a biochip. Background Art

[0002] Microarray biochips, which are integrated with high-density biomolecules, have the characteristics of high throughput and miniaturization. They can complete the analysis of a large number of biomolecules in a short time and have a wide range of application needs in the field of biomedicine. The preparation of biochips usually uses covalent or non-covalent methods to firmly fix biomolecules on the substrate surface. How to efficiently fix multiple biomolecules on the substrate through special surface chemical treatment is an important link in the preparation and application of biochips.

[0003] Since the surface of ordinary substrates (such as flat glass) has limited immobilization capacity for biological molecules, the strategy of modifying the surface with nanoparticles and / or polymers can significantly increase the amount of biological molecules fixed on the surface by making full use of the three-dimensional space perpendicular to the substrate surface, thereby significantly improving the sensitivity of analytical detection based on microarray chip technology.

[0004] Polyacrylic acid polymers are common macromolecular compounds, which have the characteristics of multiple carboxyl groups and are widely used. However, there are currently no reports on the preparation and application of polyacrylic acid polymers in biochips. Summary of the invention

[0005] The purpose of the present invention is to provide a polyacrylic acid polymer modified substrate and a preparation method thereof and application thereof in the preparation of a biochip. The multifunctional substrate prepared by the present invention has good stability and is universal in the preparation of a biochip. The preparation method has the characteristics of simple operation, flexible and simple control, no need for special equipment, and low cost.

[0006] The present invention provides a method for preparing a polyacrylic acid polymer modified substrate, wherein the polyacrylic acid polymer modified substrate is prepared by modifying a polyacrylic acid polymer layer on a substrate, and the specific steps are as follows:

[0007] (1) Substrate cleaning and activation: After ultrasonic cleaning, the substrate is blown dry and heated to 60°C-80°C. 2 SO 4 / H 2 O 2 Soak in the cleaning solution for 30 min-60 min, and blow dry after cleaning;

[0008] (2) Aminosilanization of the substrate surface: soak the substrate obtained in step (1) in a 5%-10% (v / v) 3-aminopropyltrimethoxysilane ethanol solution for 2 h-5 h, wash and blow dry;

[0009] (3) Substrate surface modification initiator: soak the substrate obtained in step (2) in a mixed solution of 1%-3% (v / v) 1-bromoisobutyryl bromide and 1%-3% (v / v) dichloromethane, react for 30 min-60 min, and then wash and blow dry;

[0010] (4) Modifying the surface of the substrate with polyacrylic acid polymer: Mix water, methanol and polyacrylic acid polymer in a volume ratio of 1:1:1-3 and degas. Add 2,2'-bipyridine with a final concentration of 20 mM-50 mM, ascorbic acid with a final concentration of 120 mM-140 mM and CuBr with a final concentration of 10 mM-20 mM, and stir until fully dissolved. Immerse the substrate in step (3) in the mixed solution and react under the protection of an inert gas until the polyacrylic acid polymer is successfully modified on the substrate, thereby obtaining a polyacrylic acid polymer-modified substrate.

[0011] Furthermore, the thickness of the polyacrylic acid polymer layer ranges from 0.01 nm to 1000 nm.

[0012] Furthermore, the substrate includes an ordinary glass substrate, a quartz glass substrate, a silicon substrate, a gold / gold-plated substrate, and a silver / silver-plated substrate.

[0013] Further, the polyacrylic acid polymer is acrylic acid, methacrylic acid, ethyl acrylic acid, propyl acrylic acid, butyl acrylic acid, α-carboxymethyl acrylic acid, 2-isobutyl acrylic acid, 4-hydroxybutyl acrylic acid, trans-2,6-difluorocinnamic acid, 2,4-dihydroxycinnamic acid, 2-acetamido acrylic acid, β-phenyl acrylic acid, 4-methylcinnamic acid, 3,4-dimethoxycinnamic acid, 2-chlorocinnamic acid, 4-chloro-2-fluorocinnamic acid, trans-3,4-difluorocinnamic acid, 3,4-methylenedioxycinnamic acid, 2,4-dimethylcinnamic acid, 3-(trifluoromethoxy)cinnamic acid, 2,5-dimethoxycinnamic acid, α-cyano At least one of 3-hydroxycinnamic acid, α-cyano-4-hydroxycinnamic acid, 2-acetoxycinnamic acid, 4-chlorocinnamic acid, trans-3,5-dichlorocinnamic acid, 3,3-dimethoxyacrylic acid, 4-nitrocarboxylic acid, 3-(2-thienyl)acrylic acid, trans-3-(3-thienyl)acrylic acid, 2-thiophene-acrylic acid, 3-(4-pyridyl)acrylic acid, pyridine-2-acrylic acid, trans-3-(3-fluoro)acrylic acid, terephthalic acid, 2-phenylacrylic acid, 4-imidazoleacrylic acid, 3-indoleacrylic acid, trans-urocanic acid, 4-aminocinnamic acid, 2-furanacrylic acid, trans-2-butenoic acid, and sorbic acid.

[0014] Furthermore, the inert gas is nitrogen.

[0015] The invention also provides a polyacrylic acid polymer modified substrate prepared by the preparation method.

[0016] The present invention also provides the use of the polyacrylic acid polymer modified substrate in the preparation of a biochip.

[0017] Furthermore, the biochip comprises a polyacrylic acid polymer modified substrate, a biological molecule layer and a deactivated molecular layer; the polyacrylic acid polymer modified substrate is connected to the biological molecule layer or the deactivated molecular layer through an amide bond after being activated.

[0018] Furthermore, the preparation steps of the biochip are as follows:

[0019] (1) The polyacrylic acid polymer modified substrate is activated for 30 min to 60 min using a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide with a final concentration of 50 mM to 100 mM and N-hydroxysuccinimide with a final concentration of 20 mM to 50 mM, and then washed and blown dry;

[0020] (2) preparing biomolecular compounds into aqueous solutions;

[0021] (3) using a manual spotter, spot the biomolecule compound solution in step (2) onto the surface of the polyacrylic acid polymer modified substrate activated in step (1) to form a micro-dot array, placing it in a sealed container, incubating overnight with moisturizing, washing and then blowing dry;

[0022] (4) The polyacrylic acid polymer modified substrate with micro-dot array in step (3) is immersed in a deactivated molecular compound solution with a concentration of 1M-3M for reaction for 2 h-5 h, and then washed and blown dry to obtain a biochip.

[0023] Furthermore, the biomolecule compound includes amino acids, polypeptides, proteins, glycoproteins, amino sugars, amino-derived sugar compounds, DNA containing an amino terminus, RNA containing an amino terminus, and lipids containing an amino terminus.

[0024] Furthermore, the deactivated molecular compound solution includes ethanolamine, aminopolyethylene glycol, cyclobutylamine, heptylamine, hexadecylamine, octadecylamine, furfurylamine, 3-chlorobenzylamine, 4-chlorobenzylamine, 1,5-diaminopentane, bovine serum albumin, and skimmed milk powder.

[0025] Furthermore, the biochip includes a sugar chip, a glycoprotein chip, a protein chip, a polypeptide chip, a DNA chip, and a lipid chip.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0027] The present invention utilizes a polymethacrylic acid polymer to modify a glass substrate, the substrate has good stability, can be used to prepare a biochip, and has universal applicability in the preparation of a biochip, and the preparation method has the characteristics of simple operation, flexible and simple control, no need for special equipment, and low cost. The polymethacrylic acid polymer modified glass substrate of the present invention has a wide range of applications, and can be applied to the field of high-throughput biochip preparation, research on interactions between biomolecules, evaluation of biomolecule activity, disease diagnosis, high-throughput screening, drug screening, fluorescence array imaging, and surface plasmon resonance imaging, and therefore has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is an atomic force microscopy image of polymethacrylic acid modified on the surface of a glass substrate.

[0029] Figure 2 This is the fluorescence array imaging of the sugar chip.

[0030] Figure 3 This is the protein chip fluorescence array imaging image.

[0031] Figure 4 This is a picture of the DNA chip fluorescence array imaging.

[0032] Figure 5 It is the fluorescence signal value of the sample point before and after placement and ultrasonic treatment. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described in detail below in conjunction with specific examples. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are all commercially available unless otherwise specified.

[0034] Example 1: Preparation of polymethacrylic acid polymer glass substrate

[0035] Modify the polymethacrylic acid polymer layer on the glass substrate. The specific steps are as follows:

[0036] 1) Glass substrate cleaning and activation

[0037] The glass substrate was treated with acetone, H 2 O were ultrasonically cleaned 3 times, 5 min each time, N 2 Blow dry; at 70℃ H 2 SO 4 / H 2 O 2 Soak in washing solution (v / v=3 / 1) for 30 min, H 2 After washing 3 times, 2 Blow dry.

[0038] 2) Aminosilanization of glass substrate surface

[0039] The glass substrate treated in step 1) was immersed in a 10% (v / v) 3-aminopropyltrimethoxysilane ethanol solution for 3 h. After the reaction was completed, ethanol and H 2 After cleaning, 2 Blow dry.

[0040] 3) Glass substrate surface modification initiator

[0041] The aminosilanized glass substrate was immersed in a dichloromethane solution of 1-bromoisobutyryl bromide (1%, v / v) and triethylamine (1%, v / v) and reacted with slight shaking for 30 min. 2 O for 3 times and dried with nitrogen.

[0042] 4) Substrate surface modification with polymethacrylic acid polymer

[0043] H 2 O, methanol and methacrylic acid were mixed in a volume ratio of 1:1:1, degassed, and 2,2'-bipyridine (final concentration 30 mM), ascorbic acid (final concentration 121 mM) and CuBr (final concentration 11.5 mM) were added and stirred until fully dissolved. The glass substrate with the surface modified with the initiator was immersed in the above solution and heated under N 2 Reaction under protective conditions.

[0044] See also Figure 1 , polymethacrylic acid polymer was successfully modified on a glass substrate.

[0045] Example 2: Preparation of sugar chip based on glass substrate modified with polymethacrylic acid polymer and its application

[0046] 1) The glass substrate modified with polymethacrylic acid polymer in Example 1 was activated by immersion in a mixed aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (final concentration 70 mM) and N-hydroxysuccinimide (final concentration 40 mM) at room temperature for 30 min. 2 O for 3 times and blow dry with nitrogen.

[0047] 2) The sugar with amino terminal modification (4-aminophenyl-α-D-mannopyranoside) was prepared into aqueous solution with a series of concentrations (0, 0.01, 0.025, 0.05, 0.075, 0.1, 1, 10, 100 mM).

[0048] 3) Use a manual spotter to spot the sample onto the surface of the glass substrate modified with polymethacrylic acid activated in step 1) to form a micro-dot array. Place the substrate in a sealed container and incubate overnight to keep it moist. 2O for 3 times and blow dry with nitrogen.

[0049] 4) The substrate with micro-dot arrays was immersed in ethanolamine solution (1 M, pH = 8.6) for 3 h. 2 O for 3 times and dried with nitrogen to prepare the sugar chip.

[0050] 5) The sugar chip was incubated with FITC-labeled concanavalin A lectin solution at room temperature for 30 min (the concentration of FITC-labeled concanavalin A lectin solution was 100 μg / mL, which was dissolved in 25 mM pH 7.60 containing 1 mM MnCl). 2 , 1 mM CaCl 2 and 100 mM NaCl in Tris-HCl buffer). 2 The cells were washed three times with 4% O and dried with nitrogen. The images were imaged using a GenePix 4300 fluorescence chip scanner (Molecular Devices, Inc., USA) with an excitation light of 488 nm and an emission light of 513-555 nm.

[0051] Results Figure 2 The sugar chip can recognize the fluorescence signals of the sample points of 4-aminophenyl-α-D-mannopyranoside with a series of concentrations by using fluorescein isothiocyanate-labeled concanavalin A lectin.

[0052] Example 3: Preparation of protein chip based on glass substrate modified with polymethacrylic acid polymer and its application

[0053] 1) The glass substrate modified with polymethacrylic acid polymer in Example 1 was activated by immersion in a mixed aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (final concentration 70 mM) and N-hydroxysuccinimide (final concentration 40 mM) at room temperature for 30 min. 2 O for 3 times and blow dry with nitrogen.

[0054] 2) FITC-labeled concanavalin A was dissolved in PBS to prepare a series of solutions (0, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2 mM).

[0055] 3) Use a manual spotter to spot the sample onto the surface of the glass substrate modified with polymethacrylic acid activated in step 1) to form a micro-dot array. Place the substrate in a sealed container and incubate overnight to keep it moist. 2 O for 3 times and blow dry with nitrogen.

[0056] 4) The substrate with micro-dot arrays was immersed in ethanolamine solution (1 M, pH = 8.6) for 3 h. 2O for 3 times and dried with nitrogen to prepare a protein chip.

[0057] 5) Imaging was performed using a GenePix 4300 fluorescence microarray scanner (Molecular Devices, Inc., USA) with an excitation light of 488 nm and an emission light of 513-555 nm.

[0058] Results Figure 3 The sample spots of concanavalin A lectin labeled with fluorescein isothiocyanate fixed on the surface of the chip have fluorescent signals.

[0059] Example 4: Preparation of DNA chip based on glass substrate modified with polymethacrylic acid polymer and its application

[0060] 1) The glass substrate modified with polymethacrylic acid polymer in Example 1 was activated by immersion in a mixed aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (final concentration 70 mM) and N-hydroxysuccinimide (final concentration 40 mM) at room temperature for 30 min. 2 O for 3 times and blow dry with nitrogen.

[0061] 2) The sequence is 5'-AGAGACTGGCGCTTTTT-NH 2 The ssDNA-1 was dissolved in 3× sodium citrate buffer containing 0.005% (w / v) sodium dodecyl sulfate to obtain a ssDNA-1 solution with a concentration of 1 μM.

[0062] 3) Use a manual spotter to spot the ssDNA-1 sample onto the surface of the glass substrate modified with polymethacrylic acid activated in step 1) to form a micro-dot array. Place the substrate in a sealed container and incubate overnight for 3 hours. 2 O for 3 times and blow dry with nitrogen.

[0063] 4) The substrate with micro-dot arrays was immersed in ethanolamine solution (1 M, pH = 8.6) for 3 h. 2 The sample was washed with O for 3 times and dried with nitrogen gas to prepare a DNA chip.

[0064] 5) The DNA chip was reacted with ssDNA-2 with the sequence of 5'-GCGCCAGTCTCTCCCAGGACAGGCA-3' in 4× sodium citrate buffer at a concentration of 0, 0.5, 1, 10, 50, 100 μM at 55°C for 1 h. 2 O for 3 times, dried under nitrogen, and mixed with 5'-Alexa Fluor 488-T in 4× sodium citrate buffer. 10The ssDNA of TGCCTGTCCTGGG-3' was incubated at 55℃ for 1 h. 2 The cells were washed three times with 4% O and dried with nitrogen. The images were imaged using a GenePix 4300 fluorescence chip scanner (Molecular Devices, Inc., USA) with an excitation light of 488 nm and an emission light of 513-555 nm.

[0065] Results Figure 4 , the ssDNA-1 sample spot has a fluorescent signal.

[0066] Example 5: Stability study of glass substrate modified with polymethacrylic acid polymer

[0067] 1) The glass substrate modified with polymethacrylic acid polymer in Example 1 was activated by immersion in a mixed aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (final concentration 70 mM) and N-hydroxysuccinimide (final concentration 40 mM) at room temperature for 30 min. 2 O for 3 times and blow dry with nitrogen.

[0068] 2) Spot the sample of 5-isothiocyanate fluorescein cadaverine solution (5 μM) onto the surface of the polymethacrylic acid-modified glass substrate activated in step 1) to form a micro-dot array. Place the substrate in a sealed container and incubate overnight under moisturizing conditions. 2 The samples were washed with 1% O for 3 times and dried with nitrogen. The fluorescence signal values ​​at the sample points were collected using a GenePix 4300 fluorescence chip scanner (Molecular Devices, Inc., USA) under the conditions of excitation light of 488 nm and emission light of 513-555 nm.

[0069] 3) The substrate is placed at room temperature for 24 hours and ultrasonically treated for 30 minutes using an ultrasonic cleaner (output power density: 0.3-0.6 W / cm2). The fluorescence chip scanner is then used again to collect the fluorescence signal value at the sample point according to the detection parameters of step (2).

[0070] Results Figure 5 There was no significant difference in the fluorescence signal values ​​obtained at the sample points after placement and ultrasonic treatment, indicating that the polymer-modified substrate was stable.

[0071] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A method for preparing a polyacrylic acid polymer modified substrate, characterized in that: The polyacrylic acid polymer modified substrate is prepared by modifying a polyacrylic acid polymer layer on a substrate, and the specific steps are as follows: (1) Substrate cleaning and activation: After ultrasonic cleaning, blow dry the substrate, soak it in H2SO4 / H2O2 washing solution at 60℃-80℃, clean it and blow dry it; (2) Aminosilanization of the substrate surface: soak the substrate obtained in step (1) in a 5%-10% (v / v) 3-aminopropyltrimethoxysilane ethanol solution for 2 h-5 h, wash and blow dry; (3) Substrate surface modification initiator: soak the substrate obtained in step (2) in a mixed solution of 1%-3% (v / v) 1-bromoisobutyryl bromide and 1%-3% (v / v) dichloromethane, react for 30 min-60 min, and then wash and blow dry; (4) Modifying the surface of the substrate with a polyacrylic acid polymer: water, methanol and polyacrylic acid polymer in a volume ratio of 1:1:1 are mixed and degassed, and 2,2'-bipyridine with a final concentration of 20 mM-50 mM, ascorbic acid with a final concentration of 120 mM-140 mM and CuBr with a final concentration of 10 mM-20 mM are added, and stirred until fully dissolved. The substrate in step (3) is immersed in the mixed solution and reacted under the protection of an inert gas until the polyacrylic acid polymer is successfully modified on the substrate, thereby obtaining a polyacrylic acid polymer modified substrate; The substrate includes a common glass substrate, a quartz glass substrate, a silicon substrate, a gold / gold-plated substrate, and a silver / silver-plated substrate; The polyacrylic acid polymer is at least one of methacrylic acid, ethylacrylic acid, propylacrylic acid, 2-isobutylacrylic acid, 4-hydroxybutylacrylic acid, and trans-2-butenoic acid.

2. The polyacrylic acid polymer modified substrate prepared by the preparation method according to claim 1.

3. Use of the polyacrylic acid polymer modified substrate according to claim 2 in the preparation of a biochip.

4. The use according to claim 3, characterized in that: The biochip comprises a polyacrylic acid polymer modified substrate, a biological molecule layer and a deactivated molecular layer; the polyacrylic acid polymer modified substrate is connected with the biological molecule layer or the deactivated molecular layer through an amide bond after being activated.

5. The use according to claim 4, characterized in that: The preparation steps of the biochip are as follows: (1) The polyacrylic acid polymer modified substrate is activated for 30 min to 60 min using a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide with a final concentration of 50 mM to 100 mM and N-hydroxysuccinimide with a final concentration of 20 mM to 50 mM, and then washed and blown dry; (2) preparing biomolecular compounds into aqueous solutions; (3) using a manual spotter, spot the biomolecule compound solution in step (2) onto the surface of the polyacrylic acid polymer modified substrate activated in step (1) to form a micro-dot array, placing it in a sealed container, incubating overnight with moisturizing, washing and then blowing dry; (4) The polyacrylic acid polymer modified substrate with micro-dot array in step (3) is immersed in a deactivated molecular compound solution with a concentration of 1M-3M for reaction for 2 h-5 h, and then washed and blown dry to obtain a biochip.

6. The use according to claim 5, characterized in that: The biomolecule compounds include amino acids, polypeptides, proteins, glycoproteins, amino-derived sugar compounds, DNA containing an amino terminal, RNA containing an amino terminal, and lipids containing an amino terminal.

7. The use according to claim 5, characterized in that: The deactivated molecular compound solution includes ethanolamine, aminopolyethylene glycol, cyclobutylamine, heptylamine, hexadecylamine, octadecylamine, furfurylamine, 3-chlorobenzylamine, 4-chlorobenzylamine, 1,5-diaminopentane, bovine serum albumin, and skimmed milk powder.

8. The use according to claim 3, characterized in that: The biochips include sugar chips, glycoprotein chips, protein chips, polypeptide chips, DNA chips, and lipid chips.

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