A protein chip with high-density pore-like microstructure and its preparation method

Abstract

The present invention relates to the technical field of protein chips, and discloses a protein chip with a high-density porous microstructure and a preparation method thereof, comprising a polymer substrate, a porous microstructure layer fixed on the polymer substrate by photocrosslinking, and protein antigen molecules fixed on the activated porous microstructure layer; the polymer substrate refers to a polymer containing C-H bonds. Photocrosslinking is a preparation method with low energy consumption and low cost. It adopts a polymer containing C-H bonds as a substrate material, and crosslinks the porous microstructure layer and the polymer substrate together by photocrosslinking. The process is simple and fast, and the porous structure of the porous microstructure layer greatly increases the contact area with the protein antigen molecules, which is conducive to the high-density aggregation of protein antigen molecules, and effectively reduces the lowest limit of antibody concentration detected by the protein chip. In addition, the preparation process of the protein chip is simple and fast, with low energy consumption and low cost.

Description

Technical Field

[0001] The present invention relates to the technical field of protein chips, and in particular to a protein chip with a high-density pore-shaped microstructure and a preparation method thereof. Background Art

[0002] Biochips are widely used in biological detection and disease diagnosis due to their miniaturization, high throughput and low cost. Glass, an inorganic material, has the advantages of high transparency, resistance to organic solvents and mature surface modification methods. As a substrate material for biochips, it is used for the detection and analysis of different biologically active molecules (DNA, proteins, cells and tissues, etc.). However, due to its rigidity and brittleness, glass is difficult and expensive to process. As an alternative to glass, polymers have the advantages of low manufacturing and post-processing costs, flexibility and a wide variety of options. Polymer substrates have been used in the preparation of biochips.

[0003] Compared with glass, polymers have relatively "inert" surfaces. Therefore, inert surfaces must be converted into bioanalytical surfaces with active sites capable of binding active molecules. A common strategy is to introduce reactive functional groups on the surface, such as epoxy, carboxyl or aldehyde groups for covalent coupling of proteins; however, the modification of inert polymers requires a tedious and time-consuming multi-step chemical and physical process, which increases costs and makes product quality difficult to control. In addition, traditional two-dimensional surfaces have limited active sites and a low density of immobilized active molecules, which reduces the sensitivity of the biochip.

[0004] Chinese patent CN102565415A discloses a protein in situ expression chip, its construction method, and application. The chip includes a substrate, a surface modification layer, and a protein fixed on the surface modification layer. The surface modification layer includes a second chain molecule and a polyrotaxane composed of a first chain molecule and cyclodextrin. The second chain molecule and the polyrotaxane are fixed to the substrate via fixing groups at one end of the second chain molecule and the first chain molecule. However, the chip still tends to use traditional inorganic materials as the substrate contact surface in contact with the surface modification layer, and still cannot fully utilize the advantages of the polymer substrate. Summary of the Invention

[0005] In order to solve the problem that the existing technology has not yet developed a protein chip with a polymer as a substrate with a simple preparation process and high sensitivity, the present invention provides a protein chip with a high-density porous microstructure, which uses a polymer as a substrate and has the advantages of simple preparation, low cost, and high detection sensitivity; the present invention also provides a method for preparing a protein chip with a high-density porous microstructure, which has a simple preparation process and low cost, and the prepared protein chip uses a polymer as a substrate and has high detection sensitivity.

[0006] The present invention is achieved by the following technical solutions:

[0007] A protein chip with a high-density porous microstructure comprises a polymer substrate, a porous microstructure layer fixed on the polymer substrate by photocrosslinking, and protein antigen molecules fixed on the activated porous microstructure layer; the polymer substrate is a polymer containing CH bonds.

[0008] Preferably, the polymer substrate is one of polycarbonate PC, cycloolefin copolymer COC, polymethyl methacrylate PMMA, polystyrene PS, polypropylene PP, and polyvinyl chloride PVC.

[0009] Photocrosslinking is a surface modification method with low energy consumption and low cost. It uses a polymer containing C-H bonds as the base material. The porous microstructure layer is crosslinked with the polymer base through photocrosslinking. The process is simple and fast. The porous structure of the porous microstructure layer greatly increases the contact area with the protein antigen molecules, which is conducive to the high-density aggregation of protein antigen molecules and effectively reduces the minimum limit of antibody concentration detected by the protein chip.

[0010] Preferably, the porous microstructure is formed by dissolving monomers 4-benzoylphenyl methacrylate, N,N-dimethylacrylamide, sodium p-styrenesulfonate, acrylic acid and butyl methacrylate in an organic solvent, and initiating a reaction with azobisisobutyronitrile to obtain a copolymer. The copolymer is printed onto the polymer substrate using a biomolecular printer and cross-linked by light.

[0011] Preferably, the activated porous microstructure layer refers to a porous microstructure layer activated by EDC / NHS.

[0012] A method for preparing the above protein chip is characterized by comprising the following steps:

[0013] Step 1) dissolving monomers 4-benzoylphenyl methacrylate, N,N-dimethylacrylamide, sodium p-styrenesulfonate, acrylic acid, and butyl methacrylate in a solvent of N,N-dimethylformamide (DMF), adding azobisisobutyronitrile as a catalyst, and conducting a reaction under an inert gas atmosphere. The reaction is cooled to room temperature, and ether is added to precipitate a solid, which is dried to obtain a copolymer. The copolymer is then dissolved in a phosphate buffer solution (PBSI) to obtain a PBS solution of the copolymer.

[0014] Step 2) The polymer substrate is placed in water and cleaned with ultrasound, the copolymer PBS solution of step 1) is printed on the polymer substrate, dried at room temperature, irradiated with ultraviolet light, cleaned, and blown dry to obtain a porous microstructure surface;

[0015] Step 3) The surface of the porous microstructure of step 2) is activated by infiltration with an aqueous solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and an aqueous solution of N-hydroxysuccinimide (NHS) at room temperature, rinsed with water, and blown dry; the protein antigen solution is spotted on the surface of the porous microstructure after EDC / NHS activation for reaction II, rinsed with PBSII, blocked with an aqueous solution of bovine serum albumin (BSA), rinsed with PBSII, and blown dry to obtain a protein chip.

[0016] EDC / NHS activation activates the COOH on the surface of the porous microstructure so that it can react with NH2 in the protein to form an amide bond, which is beneficial for the fixation of IgG on the surface of the porous microstructure; BSA is used to block the nonspecific adsorption points on the porous microstructure to prevent nonspecific adsorption of Cy3-anti-IgG to sites other than IgG.

[0017] Preferably, in step 1), the mixing molar ratio of the monomers 4-benzoylphenyl methacrylate, N,N-dimethylacrylamide, sodium p-styrenesulfonate, acrylic acid, butyl methacrylate, azobisisobutyronitrile, and N,N-dimethylformamide is 0.3-1:2-6:0.5-1:1-5:1.2-2.5:0.005-0.1:10-200.

[0018] Preferably, in step 1), the molar ratio of the monomers 4-benzoylphenyl methacrylate, N,N-dimethylacrylamide, sodium p-styrenesulfonate, acrylic acid, butyl methacrylate, azobisisobutyronitrile, and N,N-dimethylformamide is 0.5:4:0.8:3:2:0.01:150.

[0019] Preferably, in step 1), the reaction temperature is 60-80° C., the reaction time is 12-24 h, the inert gas is nitrogen or argon, and the PBSI concentration is 1-50 mmol / L.

[0020] Preferably, the ultraviolet light in step 2) is 365 nm, 80 mW / cm 2 , the illumination time is 6 to 8 minutes.

[0021] Preferably, in step 3), the concentration of the EDC solution is 10-40 mmol / L, the concentration of the NHS solution is 5-20 mmol / L, the mixing volume ratio of the EDC solution and the NHS solution is 1:1, the activation time is 28-35 min, the reaction II time is 50 min-1.2 h, the concentration of the BSA aqueous solution is 10 mg / mL, the concentration of the PBSII is 8-15 mmol / mL, and the blocking time is 50 min-1.2 h.

[0022] Beneficial effects of the present invention:

[0023] (1) Organic polymer substrates have the advantages of low manufacturing and post-processing costs and a wide variety of options to meet the needs of biological detection and diagnosis in different scenarios;

[0024] (2) The porous microstructure of the obtained protein chip has an extremely high protein contact area, and the density of protein antigen molecules is greatly improved, which effectively reduces the detection limit concentration of antibody molecules.

[0025] (3) The preparation process is simple and fast, with low energy consumption and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the copolymer in Example 1.

[0027] Figure 2 These are electron microscope images of the porous microstructure surface of Example 3 and the non-porous microstructure surface of Comparative Example 1.

[0028] Figure 3 The protein immobilization density of the protein chip under different FITC-IgG concentrations in Example 1.

[0029] Figure 4 The protein fixation efficiency of the protein chip under different FITC-IgG concentrations in Example 1 is shown.

[0030] Figure 5 These are fluorescence intensity graphs of FITC-IgG proteins with different concentrations fixed on the porous microstructure protein chip of Example 4 or the non-porous microstructure protein chip of Comparative Example 1.

[0031] Figure 6 The immunofluorescence intensity of different concentrations of Cy3-anti-IgG in Example 2. DETAILED DESCRIPTION

[0032] Example 1

[0033] A method for preparing a protein chip with a high-density porous microstructure comprises the following steps:

[0034] Step 1) dissolving monomers 4-benzoylphenyl methacrylate, N,N-dimethylacrylamide, sodium p-styrenesulfonate, acrylic acid and butyl methacrylate in a solvent of N,N-dimethylformamide (DMF), adding an initiator azobisisobutyronitrile, and reacting under argon protection at a temperature of 80° C. for 12 hours, cooling to room temperature, adding ether to precipitate a solid, and drying to obtain a copolymer, which is dissolved in a phosphate buffer solution (PBS) with a concentration of 50 mmol / L to obtain a PBS solution of the copolymer; the mixing molar ratio of the monomers 4-benzoylphenyl methacrylate, N,N-dimethylacrylamide, sodium p-styrenesulfonate, acrylic acid, butyl methacrylate, azobisisobutyronitrile and N,N-dimethylformamide is 0.5:4:0.8:3:2:0.01:150.

[0035] Step 2) The COC polymer substrate was placed in water and ultrasonically cleaned for 4 minutes. The copolymer PBS solution of step 1) was printed on the polymer substrate using a biomolecular printer, and dried at room temperature. The wavelength was 365 nm and the power was 80 mW / cm 2 The product was irradiated with ultraviolet light for 8 minutes, cleaned, and dried to obtain a porous microstructure surface;

[0036] Step 3) A 40 mmol / L aqueous solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was mixed with a 20 mmol / L aqueous solution of N-hydroxysuccinimide (NHS) in a volume ratio of 1:1. The porous microstructure surface prepared in step 2) was activated at room temperature for 30 minutes, rinsed with water, and dried. A 2 mg / mL IgG protein antigen solution was spotted on the EDC / NHS-activated porous microstructure surface for reaction II for 1 hour. The reaction was then rinsed with 10 mmol / mL PBSII and blocked with a 10 mg / mL aqueous solution of bovine serum albumin (BSA) for 1 hour. The protein chip was then rinsed with PBSII and dried.

[0037] Example 2

[0038] A method for preparing a protein chip with a high-density porous microstructure comprises the following steps:

[0039] Step 1) dissolving monomers 4-benzoylphenyl methacrylate, N,N-dimethylacrylamide, sodium p-styrenesulfonate, acrylic acid, and butyl methacrylate in a solvent of N,N-dimethylformamide (DMF), adding an initiator azobisisobutyronitrile, and reacting under nitrogen protection at a temperature of 60° C. for 24 hours, cooling to room temperature, adding ether to precipitate a solid, and drying to obtain a copolymer, which is then dissolved in a phosphate buffer solution (PBS) with a concentration of 10 mmol / L to obtain a PBS solution of the copolymer; the mixing molar ratio of the monomers 4-benzoylphenyl methacrylate, N,N-dimethylacrylamide, sodium p-styrenesulfonate, acrylic acid, butyl methacrylate, azobisisobutyronitrile, and N,N-dimethylformamide is 1.2:6:0.5:5:1.2:0.1:200.

[0040] Step 2) The PC polymer substrate was placed in water and ultrasonically cleaned for 5 minutes. The copolymer PBS solution of step 1) was printed on the polymer substrate using a biomolecular printer and dried at room temperature. The wavelength was 365 nm and the power was 80 mW / cm 2 The product was irradiated with ultraviolet light for 8 minutes, cleaned, and dried to obtain a porous microstructure surface;

[0041] Step 3) A 40 mmol / L aqueous solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was mixed with a 20 mmol / L aqueous solution of N-hydroxysuccinimide (NHS) in a volume ratio of 1:1. The porous microstructure surface prepared in step 2) was activated at room temperature for 30 minutes, rinsed with water, and dried. A 2 mg / mL IgG protein antigen solution was spotted on the EDC / NHS-activated porous microstructure surface for reaction II for 1 hour. The reaction was then rinsed with 10 mmol / mL PBSII and blocked with a 10 mg / mL aqueous solution of bovine serum albumin (BSA) for 1 hour. The protein chip was then rinsed with PBSII and dried.

[0042] Example 3

[0043] The difference from Example 1 is that in step 1), the mixing molar ratio of monomers 4-benzoylphenyl methacrylate, N,N-dimethylacrylamide, sodium p-styrenesulfonate, acrylic acid, butyl methacrylate, azobisisobutyronitrile, and N,N-dimethylformamide is 1:6:1:5:2.5:0.1:200; and in step 2), the polymer base material is PP.

[0044] Example 4

[0045] The difference from Example 2 is that the reaction temperature in step 1) is 70° C. and the reaction time is 18 h; and the polymer base material in step 2) is PMMA.

[0046] Comparative Example 1

[0047] The difference from Example 1 is that the PBSI and PBSII solutions are replaced with deionized water.

[0048] Comparative Example 2

[0049] The difference from Example 1 is that the mixing molar ratio of the monomers 4-benzoylphenyl methacrylate, N,N-dimethylacrylamide, sodium p-styrenesulfonate, acrylic acid, butyl methacrylate, azobisisobutyronitrile, and N,N-dimethylformamide is 1.5:4:1.5:4:2:0.003:250.

[0050] Comparative Example 3

[0051] The difference from Example 1 is that no butyl methacrylate is used in the reaction of step 1).

[0052] Detection method

[0053] The copolymer prepared in Example 1 was dissolved in D2O, and the spectrum was detected by NMR. The protein chip prepared in Example 1 was sprayed with gold and the porous microstructure was detected by electron microscopy. IgG labeled with fluorescent molecules was used as an antibody on the protein chips prepared in each example and comparative example. Different concentrations of IgG labeled with fluorescent molecules were detected by fluorescence spectrophotometer to obtain a standard curve. The fluorescence detection results of each example and comparative example were then compared to obtain the fixed protein density. The protein fixation efficiency of Example 2 was then tested, i.e., the ratio of the fluorescence intensity after BSA blocking to that before blocking. The test results are shown in FIG. Figures 1 to 6 and as shown in Table 1.

[0054] Table 1 Protein immobilization density and minimum Cy3-anti-IgG detection limit of each embodiment and comparative example

[0055]

[0056] As shown in Table 1, the protein immobilization density of Examples 1 to 4 was maintained at 50 μg / cm 2 The lowest Cy3-anti-IgG detection limit is about 6-8 ng / mL. Compared with the comparative example 1, after the PBSI and PBSII solvents were replaced with deionized water, the chip produced had no porous microstructure and the protein fixation density also dropped to 1.1 μg / cm 2 , the lowest Cy3-anti-IgG detection limit increased to 500 ng / mL. It can be seen that the porous microstructure provides a large contact area with the protein, greatly increasing the protein immobilization density, thereby reducing the lowest Cy3-anti-IgG detection limit; the raw material ratio in the copolymer preparation process of Comparative Example 2 is incorrect, which also reduces the formation of the porous microstructure to a certain extent, reduces the protein immobilization area, and the protein immobilization density also drops to 3 μg / cm2 , the lowest Cy3-anti-IgG detection limit increased to 200 ng / mL; in the preparation process of the copolymer of Comparative Example 3, butyl methacrylate was not added. Not only could the porous microstructure not be formed, but the specific surface area was significantly reduced, and the protein immobilization amount decreased. Its protein immobilization density was only 0.8 μg / cm 2 , while the lowest detection limit of Cy3-anti-IgG increased to 610 ng / mL.

[0057] like Figure 1 As shown in FIG. 1 , the NMR spectrum of the copolymer generated by the reaction raw materials initiated by azobisisobutyronitrile; Figure 2 As shown in Example 3 with a porous microstructure and Comparative Example 1 without a porous microstructure, it can be seen that the porous microstructure provides a large surface area for protein contact. The copolymer is dissolved in PBS solution, which plays an important role in the formation of the porous microstructure. However, when the copolymer is dissolved in deionized water to prepare the smooth and non-porous Comparative Example 1, the surface area for protein contact is greatly reduced, and the protein immobilization density is also significantly reduced. Figure 3 The density of FITC-IgG immobilized on the chip increased with the increase of the initial FITC-IgG concentration, and the maximum immobilization density capacity reached nearly 60 μg / cm 2 ; Figure 4 The fixation efficiency of FITC-IgG is the ratio of the fluorescence intensity after BSA blocking to that before blocking. It can be seen that the fixation efficiency increases slowly with the concentration of FITC-IgG. When the FITC-IgG concentration increases 500 times, the fixation efficiency only increases by 20%, indicating that the protein fixation efficiency is very stable under different FITC-IgG concentrations. Figure 5 It shows that Example 4 immobilized a significantly larger amount of FITC-IgG at different FITC-IgG concentrations, generating a stronger fluorescence intensity, which increased significantly with the FITC-IgG concentration. However, Comparative Example 1, due to the lack of a porous microstructure, had a small specific surface area and a small amount of protein immobilization. Even with the increase in FITC-IgG concentration, it was unable to immobilize more FITC-IgG, and the fluorescence intensity was significantly lower than that of Example 4. Moreover, the fluorescence intensity did not change significantly with the increase in FITC-IgG concentration. Figure 6 It shows that Cy3-anti-IgG specifically binds to FITC-IgG on the protein chip and emits fluorescence. When the Cy3-anti-IgG concentration is close to zero, the fluorescence intensity still remains greater than 50, indicating that the lowest detection limit of Cy3-anti-IgG is already in the single digit ng / mL.

[0058] The technical features of the above-described embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification. The above-described embodiments only express several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several variations and improvements can be made, which all fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent of this invention shall be based on the attached claims.

Claims

1. A protein chip with a high-density pore microstructure, characterized in that: The invention comprises a polymer substrate, a porous microstructure layer fixed on the polymer substrate by photocrosslinking, and protein molecules fixed on the activated porous microstructure layer; the polymer substrate is a polymer containing C—H bonds; the porous microstructure is prepared by dissolving monomers 4-benzoylphenyl methacrylate, N,N-dimethylacrylamide, sodium p-styrenesulfonate, acrylic acid, and butyl methacrylate in an organic solvent, and initiating a reaction with azobisisobutyronitrile to obtain a copolymer; the copolymer is printed onto the polymer substrate using a biomolecular printer and crosslinked by photoirradiation; the mixing molar ratio of the monomers 4-benzoylphenyl methacrylate, N,N-dimethylacrylamide, sodium p-styrenesulfonate, acrylic acid, butyl methacrylate, azobisisobutyronitrile, and N,N-dimethylformamide is 0.3-1:2-6:0.5-1:1-5:1.2-2.5:0.005-0.1:10-200; The preparation method comprises the following steps: Step 1) dissolving monomers 4-benzoylphenyl methacrylate, N,N-dimethylacrylamide, sodium p-styrenesulfonate, acrylic acid, and butyl methacrylate in N,N-dimethylformamide (DMF) solvent, adding an initiator azobisisobutyronitrile, and conducting a reaction under inert gas protection. Ether is added at room temperature to precipitate a solid, which is dried to obtain a copolymer. The copolymer is then dissolved in phosphate buffer (PBSI) to obtain a PBS solution of the copolymer. Step 2) The polymer substrate is placed in water and ultrasonically cleaned, the copolymer PBS solution of step 1) is printed on the polymer substrate, dried at room temperature, irradiated with ultraviolet light, cleaned, and blown dry to obtain a porous microstructure surface; Step 3) The porous microstructure surface of step 2) is activated by infiltration with an aqueous solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and an aqueous solution of N-hydroxysuccinimide (NHS) at room temperature, rinsed with water, and air-dried; a protein antigen solution is spotted on the porous microstructure surface after EDC / NHS activation for reaction II, rinsed with PBS II, blocked with an aqueous solution of bovine serum albumin (BSA), rinsed with PBS II, and air-dried to obtain a protein chip.

2. A protein chip with a high-density porous microstructure according to claim 1, characterized in that: The polymer substrate is one of polycarbonate PC, cycloolefin copolymer COC, polymethyl methacrylate PMMA, polystyrene PS, polypropylene PP, and polyvinyl chloride PVC.

3. A protein chip with a high-density porous microstructure according to claim 1 or 2, characterized in that: The activated porous microstructure layer refers to a porous microstructure layer activated by EDC / NHS.

4. The protein chip with high-density pore microstructure according to claim 1, characterized in that: In step 1), the molar ratio of the monomers 4-benzoylphenyl methacrylate, N,N-dimethylacrylamide, sodium p-styrenesulfonate, acrylic acid, butyl methacrylate, azobisisobutyronitrile, and N,N-dimethylformamide is 0.5:4:0.8:3:2:0.01:

150.

5. The protein chip with high-density pore microstructure according to claim 1, characterized in that: Step 1) The reaction temperature is 60-80°C, the reaction time is 12-24 hours, the inert gas is nitrogen or argon, and the PBSI concentration is 1-50 mmol / L.

6. The protein chip with high-density porous microstructure according to claim 1, characterized in that: Step 2) The ultraviolet light is 365nm, 40~120mW / cm 2 , the lighting time is 6~8min.

7. The protein chip with high-density porous microstructure according to claim 1, characterized in that: In step 3), the concentration of the EDC solution is 10-40 mmol / L, the concentration of the NHS solution is 5-20 mmol / L, the mixing volume ratio of the EDC solution and the NHS solution is 1:1, the activation time is 28-35 min, the reaction II time is 50 min-1.2 h, the concentration of the BSA aqueous solution is 10 mg / mL, the concentration of the PBSII is 8-15 mmol / mL, and the blocking time is 50 min-1.2 h.

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