Preparation method and application of multi-field coupling biological detection test paper based on PDMS / Chitosan / MPMs film

Abstract

The invention belongs to the technical field of biological detection sensors, and particularly relates to a preparation method and application of multi-field coupling biological detection test paper based on a PDMS / Chitosan / MPMs film. The preparation method comprises the following steps: preparing a PDMS / Chitosan / MPMs composite film and functionalizing the surface of the composite film. The biological detection test paper is applied to detection of an African swine fever virus p72 protein gene. According to the invention, a sensing layer of the test paper is formed by doping chitosan particles in a flexible material PDMS to couple antibody recognition molecules, and a conversion layer of the test paper is prepared by introducing magnetic particles, a proper uniform magnetic field and silver nanowires; the flexible biological detection test paper sensitively converts a biological signal into an electromagnetic signal; the problem that active materials of traditional biological detection test paper are not uniformly dispersed is solved, and the consistency of the test paper is improved; by using the surface sacrificial layer technology, the film is ensured not to be damaged, and the product quality is effectively improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection sensors, and specifically relates to a preparation method and application of a multi-field coupling biological detection test paper based on PDMS / Chitosan / MPMs film. Background Art

[0002] African swine fever (ASF) is an acute, highly infectious, filterable virus-induced disease of pigs, characterized by a short course of disease, but a mortality rate of up to 100%. Clinical manifestations of sick pigs include fever, cyanosis of the skin, and significant bleeding in the lymph nodes, kidneys, and gastrointestinal mucosa. African swine fever virus (ASFV) is an important member of the genus ASFV of the family Asfrinidae. Some characteristics of the virus are similar to those of the iridoviridae and poxviridae. The diameter of the virus particle is 175-215 nanometers, with icosahedral symmetry and an envelope. The genome is double-stranded linear DNA, with a size of 170-190 kb.

[0003] p72 (African swine fever virus p72) is an important capsid protein encoded by the B646L gene and one of the important antigenic proteins of ASFV virus particles. It exists in the form of a trimer in the virus particles and is abundant with 8280 copies. It is an important target for ASFV serological and molecular biological detection. The p72 protein plays a key role in ASFV infection, and the antigenic epitopes on its surface are considered to be protective antigens. The study has successfully constructed recombinant p72 protein and p72 baculovirus, and prepared specific monoclonal antibodies against p72 protein, which show strong reactivity to ASFV infected cells. In addition, p72 protein is also the target gene of the African swine fever virus fluorescent PCR detection kit. The kit is based on the working principle of Taqman hydrolysis probe and is used to detect African swine fever nucleic acid in samples. It has high sensitivity and specificity and is suitable for the detection of all genotypes of African swine fever virus. However, methods such as PCR (polymerase chain reaction) and ELISA (enzyme-linked immunosorbent assay) for detecting African swine fever virus have disadvantages such as high detection cost, low sensitivity and accuracy, long detection time and the need for large-scale instruments and equipment.

[0004] At present, biosensors are also used to detect African swine fever virus p72, but the flexible substrates used in such biosensors have great limitations, such as the difficulty in restoring the gold layer on the surface of the test paper and the problem of easy detachment. In addition, traditional flexible biological test strips have many defects in preparation, such as uneven dispersion of active materials, reduced sensing performance due to excessive thickness of the test paper, and difficulty in separating the test paper from the glass substrate due to excessive thickness. Summary of the invention

[0005] The present invention overcomes the shortcomings of the prior art and proposes a preparation method and application of a multi-field coupled biological detection test paper based on PDMS / Chitosan / MPMs film; the present invention is achieved through the following technical solutions: The preparation method of the multi-field coupling biological detection test paper based on PDMS / Chitosan / MPMs film comprises the following steps: S1. Preparation of PDMS / Chitosan / MPMs composite film: S1.1, first prepare a polyacrylic acid film layer on a substrate; add 4-6wt% chitosan particles to a PDMS prepolymer to obtain a mixture, and apply the mixture on the polyacrylic acid film layer to form a PDMS / chitosan film; then add water to the PDMS / chitosan film to form a water film; S1.2, mixing 15-25wt% Fe3O4 coupled polystyrene microsphere latex with 1-propanol at a volume ratio of 2.5-3.5:1, and then injecting it onto the water film, so that the Fe3O4 coupled polystyrene microspheres self-assemble on the water film to form a tightly arranged array; heating the water film to evaporate the water film to form a PDMS / Chitosan / MPMs composite film on the substrate; peeling the PDMS / Chitosan / MPMs composite film from the substrate, and spraying AgNWs on the back of the PDMS / Chitosan / MPMs composite film to form a silver nanowire layer; S2. Surface functionalization of PDMS / Chitosan / MPMs composite films: S2.1, mixing the diluted antibody solution with the EDC / NHS solution to fully activate the antibody to obtain an activated antibody solution; S2.2, immerse the PDMS / Chitosan / MPMs composite film in the activated antibody solution and place it at 35-38°C for 1-1.5 hours; S2.3. Immerse the PDMS / Chitosan / MPMs composite film in a bovine serum albumin solution and incubate to block the physical adsorption sites.

[0006] Preferably, the polyacrylic acid film layer is prepared on the substrate by spin coating a polyacrylic acid aqueous solution on the glass substrate and heating and curing to form the polyacrylic acid film layer.

[0007] Preferably, after the PDMS prepolymer and the curing agent are mixed at a mass ratio of 8-12:1, 5 wt % of chitosan particles are added, and bubbles are removed to obtain a mixture.

[0008] Preferably, the prepared PDMS / chitosan membrane is subjected to hydrophilic modification, and then deionized water is added to the PDMS / chitosan membrane to form a water film.

[0009] Preferably, PET plastic is used to peel the PDMS / Chitosan / MPMs composite film from the glass substrate.

[0010] Preferably, the antibody solution is a classical swine fever virus p72 antibody solution, which is diluted to 50 μg / mL with PBS buffer, and the diluted classical swine fever virus p72 antibody solution is mixed with EDC / NHS solution in a volume ratio of 1:1 and placed in an environment of 36-38°C for 25-35 minutes to activate the carboxyl groups on the antibody.

[0011] Preferably, the concentration of the EDC / NHS solution is 4 mg / mL.

[0012] The application of multi-field coupled biological detection test paper based on PDMS / Chitosan / MPMs film is applied to the detection of African swine fever virus p72 protein gene; the biological detection test paper is prepared by the preparation method of multi-field coupled biological detection test paper based on PDMS / Chitosan / MPMs film.

[0013] Preferably, the ASFV solution is added dropwise to the PDMS / Chitosan / MPMs composite film and incubated in a shaker at 37° C. for 15-25 min.

[0014] The beneficial effects of the present invention compared with the prior art are as follows: The present invention proposes for the first time a method for preparing a PDMS / Chitosan / MPMs composite film, which is used for highly sensitive and specific detection of African swine fever virus; it optimizes the traditional needle tip flow method, so that the active material is more evenly dispersed on the surface of the test paper without affecting the flexibility of the test paper. The surface sacrificial layer technology is used to prevent the composite film from being damaged when it is separated from the glass substrate, effectively improving the quality of the test paper.

[0015] The present invention forms a sensing layer of the test paper by doping chitosan particles in the flexible material PDMS to couple antibody recognition molecules, and introduces magnetic particles, an appropriate uniform magnetic field and silver nanowires to prepare the conversion layer of the test paper. The flexible biological detection test paper sensitively converts biological signals into electromagnetic signals. The biological detection test paper prepared by the present invention can adopt a variety of detection methods, is suitable for a variety of occasions, and is more flexible and convenient. At the same time, by changing the type of antibody, this flexible biological detection test paper can be applied to other biomarker detections to expand its scope of application.

[0016] In summary, the present invention is based on the advantages of good flexibility of PDMS, excellent electromagnetic properties of Fe3O4-coupled polystyrene microspheres, and good conductivity of AgNWs. The multi-field coupled biological detection test paper based on PDMS / Chitosan / MPMs film has the advantages of short detection time, simple process, low manufacturing cost, high sensitivity, good specificity, and adaptability to complex detection environments. By optimizing the traditional needle tip flow technology, the problem of uneven dispersion of active materials in traditional biological detection test papers is solved, and the consistency of the test paper is improved. By utilizing the surface sacrificial layer technology, the film is ensured not to be damaged, and the product quality is effectively improved. Based on the covalent bonding mechanism of the amino group on the surface of chitosan and the carboxyl group on the surface of the antibody, different antibodies can be functionalized on the surface of PDMS / Chitosan / MPMs to specifically recognize different antigens, thereby realizing the detection of different biomarkers. The present invention expands the functions and application scope of traditional biological detection test papers, and the preparation method is simple and easy, and can be mass-produced. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Flow chart of the method for preparing multi-field coupled biological detection test paper based on PDMS / Chitosan / MPMs film described in the examples. DETAILED DESCRIPTION

[0018] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the present invention is further described in detail in conjunction with the embodiments and the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The technical solutions of the present invention are described in detail below in conjunction with the embodiments and the accompanying drawings, but the scope of protection is not limited thereto.

[0019] See also Figure 1 This embodiment proposes a preparation method and application of a multi-field coupled biological detection test paper based on PDMS / Chitosan / MPMs film; specifically comprising the following steps: The materials mainly include: Fe3O4-coupled polystyrene microsphere latex (MPMs), PDMS prepolymer, silver nanowires (AgNWs), chitosan, polyacrylic acid (PAA), classical swine fever virus p72 antibody, classical swine fever virus p72; S1. Preparation of PDMS / Chitosan / MPMs composite film S1.1. Use ethanol to clean the glass substrate in an ultrasonic cleaner for 15 minutes, and then use an oxygen plasma cleaner to modify the hydrophilicity of the glass substrate. Since the glass substrate is a hydrophobic material, a plasma cleaner is used to change the hydrophilicity of the glass substrate. Then, a PAA aqueous solution with a concentration of 40 wt% was fully rotated on the glass substrate at a speed of 1000 rpm for 1 min using a spin coater and heated and cured at 100°C to form a PAA film.

[0020] After mixing the PDMS prepolymer and the curing agent at a mass ratio of 10:1, 5wt% of chitosan particles are added, and a deaerator is used to remove bubbles to obtain a mixture. The mixture is coated on the PAA film and heated for 30 minutes to prepare a PDMS / chitosan film, and then the hydrophilicity is modified using an oxygen plasma cleaner, that is, the PDMS / chitosan film is placed in a plasma cleaner, the processing power is set to 100w, and the treatment is performed for 100s. Deionized water is slowly added to the PDMS / chitosan film to form a water film on the PDMS / chitosan film; The curing agent used in this embodiment includes glue A and glue B (existing materials); glue A (prepolymer) is the main component of PDMS material, which contains polymer segments of polydimethylsiloxane. These polymer segments play a role in building the material skeleton during the curing process. Glue A usually has a lower viscosity, which is convenient for mixing evenly with glue B curing agent. Glue B (curing agent) contains active groups that can undergo cross-linking reactions with the polymer segments in glue A, and the ingredients of glue B are: methyl vinyl cyclosiloxane. These active groups act as bridges during the curing process, connecting the polymer segments in glue A to form a stable network structure.

[0021] S1.2. Mix Fe3O4-coupled polystyrene microsphere latex (20wt%) and 1-propanol in a volume ratio of 3:1, vortex for 2 minutes, and then slowly and steadily inject into the air-water interface (the water-air interface refers to the interface where the water film contacts the air. The micro-injector that sucked the microspheres and n-propanol mixed solution is smoothly contacted with the water-air interface, and the polystyrene microspheres will spread out and spread evenly on the water film). Due to the different surface tensions between water and 1-propanol, PM (polystyrene microspheres) diffuse outward and self-assemble on the water surface to form a tightly packed array. After the water film evaporates, a PDMS / Chitosan / MPMs composite film is formed on the glass substrate; the water film evaporation is carried out by heating at a temperature of 110°C; Then, the glass substrate together with the PDMS / Chitosan / MPMs composite film thereon was soaked in deionized water overnight to dissolve the PAA film, and the PDMS / Chitosan / MPMs composite film was peeled off from the glass substrate using PET plastic, that is, annular PET was used to fix the PDMS / Chitosan / MPMs composite film on both sides. PET facilitates the separation of the composite film from the glass substrate and does not need to be removed in the end. There is a circular hole in the center of PET, and silver nanowires, a conductive material, are dripped into the circular hole of PET on one side, so that the dispersed silver nanowires are dripped and fixed on one side of the PDMS / Chitosan / MPMs composite film, and then heated to evaporate the ethanol solvent of the silver nanowires, and the silver nanowire layer was successfully prepared.

[0022] S2. Surface functionalization of PDMS / Chitosan / MPMs composite films S2.1. Dilute the classical swine fever virus p72 antibody solution to 50 μg / mL with PBS buffer, prepare a 4 mg / mL EDC / NHS solution, mix the diluted classical swine fever virus p72 antibody solution with the EDC / NHS solution in a volume ratio of 1:1 and place at 37°C for 30 minutes to fully activate the carboxyl groups on the antibody so that it can bind to chitosan more effectively.

[0023] Among them, the full Chinese name of EDC is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride; The full Chinese name of NHS is N-hydroxysuccinimide; EDC and NHS are usually used together, so the combination is called EDC / NHS solution.

[0024] S2.2. Immerse the PDMS / Chitosan / MPMs composite film in the solution prepared in step S2.1 and place it at 37°C for 1 hour. After taking it out, rinse it three times with PBS buffer (phosphate buffer solution) to remove the unreacted antibodies; the pH of the PBS buffer is 7.4 and the concentration is 0.01 mol / L.

[0025] S2.3. Immerse the PDMS / Chitosan / MPMs composite film in BSA solution (bovine serum albumin solution) and incubate it at 37°C for 30 minutes to block the physical adsorption sites. Take it out and dry it at room temperature and then store it at 4°C.

[0026] S3. ASFV detection ASFV solutions of different concentrations were obtained by diluting with PBS buffer, and then dropped onto the PDMS / Chitosan / MPMs composite film and incubated in a shaker at 37°C for 20 min.

[0027] Due to the specific recognition of classical swine fever virus p72 antibodies and ASFV antigens, the resistance of the silver nanowires changes accordingly due to the stress generated on the surface of the PDMS / Chitosan / MPMs composite film, and the magnetic properties of the film based on the magnetoelastic effect will also change. Therefore, ASFV can be detected by detecting the resistance changes and magnetic signal changes of the PDMS / Chitosan / MPMs composite film.

[0028] A magnetic field generator is used to generate a uniform magnetic field of appropriate strength, and the PDMS / Chitosan / MPMs composite film is placed in the magnetic field, so that the Fe3O4 particles in the PDMS / Chitosan / MPMs composite film move along the direction of the magnetic field, effectively amplifying the deformation of the PDMS / Chitosan / MPMs composite film. Therefore, a magnetic field of appropriate strength can be introduced during detection to improve the detection sensitivity of the PDMS / Chitosan / MPMs composite film.

[0029] The above content is a further detailed description of the present invention in combination with a specific preferred embodiment. It cannot be determined that the specific embodiments of the present invention are limited to this. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the present invention, which should be regarded as belonging to the present invention and the scope of patent protection determined by the submitted claims.

Claims

1. A method for preparing a multi-field coupled biological test strip based on PDMS / Chitosan / MPMs film, characterized in that: The following steps are involved: S1. Preparation of PDMS / Chitosan / MPMs composite film: S1.1, first prepare a polyacrylic acid film layer on a substrate; add 4-6wt% chitosan particles to a PDMS prepolymer to obtain a mixture, and apply the mixture on the polyacrylic acid film layer to form a PDMS / chitosan film; then add water to the PDMS / chitosan film to form a water film; S1.2, mixing 15-25wt% Fe3O4 coupled polystyrene microsphere latex with 1-propanol at a volume ratio of 2.5-3.5:1, and then injecting it onto the water film, so that the Fe3O4 coupled polystyrene microspheres self-assemble on the water film to form a tightly arranged array; heating the water film to evaporate the water film to form a PDMS / Chitosan / MPMs composite film on the substrate; peeling the PDMS / Chitosan / MPMs composite film from the substrate, and spraying AgNWs on the back of the PDMS / Chitosan / MPMs composite film to form a silver nanowire layer; S2. Surface functionalization of PDMS / Chitosan / MPMs composite films: S2.1, mixing the diluted antibody solution with the EDC / NHS solution to fully activate the antibody to obtain an activated antibody solution; S2.2, immerse the PDMS / Chitosan / MPMs composite film in the activated antibody solution and place it at 35-38°C for 1-1.5 hours; S2.

3. Take out the PDMS / Chitosan / MPMs composite film from the antibody solution and immerse it in a bovine serum albumin solution for incubation to block the physical adsorption sites.

2. The method for preparing a multi-field coupled biological test paper based on PDMS / Chitosan / MPMs film according to claim 1, characterized in that: The polyacrylic acid film layer is prepared on the substrate by spin coating a polyacrylic acid aqueous solution on the glass substrate and heating and curing to form the polyacrylic acid film layer.

3. The method for preparing a multi-field coupled biological test paper based on PDMS / Chitosan / MPMs film according to claim 1, characterized in that: After mixing the PDMS prepolymer and the curing agent at a mass ratio of 8-12:1, 5 wt % of chitosan particles were added, and bubbles were removed to obtain a mixture.

4. The method for preparing a multi-field coupled biological test paper based on PDMS / Chitosan / MPMs film according to claim 1, characterized in that: The prepared PDMS / chitosan membrane is hydrophilically modified, and then deionized water is added to the PDMS / chitosan membrane to form a water film.

5. The method for preparing a multi-field coupled biological test strip based on PDMS / Chitosan / MPMs film according to claim 1, characterized in that: The PDMS / Chitosan / MPMs composite film was peeled off from the substrate using PET plastic.

6. The method for preparing a multi-field coupled biological detection test paper based on PDMS / Chitosan / MPMs film according to claim 1, characterized in that: The antibody solution is a classical swine fever virus p72 antibody solution, which is diluted to 50 μg / mL with a PBS buffer solution, and the diluted classical swine fever virus p72 antibody solution is mixed with an EDC / NHS solution at a volume ratio of 1:1 and placed in an environment of 36-38°C for 25-35 minutes to activate the carboxyl group on the antibody.

7. The method for preparing a multi-field coupled biological test strip based on PDMS / Chitosan / MPMs film according to claim 6, characterized in that: The concentration of the EDC / NHS solution was 4 mg / mL.

8. Application of multi-field coupled biological detection test strips based on PDMS / Chitosan / MPMs film, characterized in that: Used for detecting the p72 protein gene of African swine fever virus; the biological detection test paper is prepared by the preparation method of the multi-field coupling biological detection test paper based on PDMS / Chitosan / MPMs film as described in claim 6.

9. The use of the multi-field coupled biological detection test paper based on PDMS / Chitosan / MPMs film according to claim 8, characterized in that: The African swine fever virus solution was dropped onto the PDMS / Chitosan / MPMs composite film and incubated in a shaker at 37°C for 15-25 min.

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