Preparation method and application of two-dimensional photonic crystal DNA hydrogel biosensor

Abstract

The invention belongs to the technical field of biological detection sensors, and particularly relates to a preparation method and application of a two-dimensional photonic crystal DNA hydrogel biosensor. The preparation method comprises the following steps: preparing a two-dimensional photonic crystal, preparing hydrogel, and functionalizing the hydrogel. The magnetic particles, the two-dimensional photonic crystals, the silver nanowires and other materials are introduced to prepare the biosensor, shrinkage and expansion generated by hydrogel are sensitively converted into optical signals and electric signals, and deformation of a hydrogel film can be verified through an optical method and resistance measurement; the carboxyl group in the hydrogel can be coupled with the amino group of the complementary chain to capture the target DNA molecule, and the high-sensitivity and specific detection of the P72 protein gene segment can be realized based on the base complementary pairing principle.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensing sensors, and specifically relates to a preparation method and application of a two-dimensional photonic crystal DNA hydrogel biosensor. Background Art

[0002] African swine fever (ASF) is a swine disease caused by an acute, highly contagious filterable virus. It is characterized by a short course of disease but a mortality rate as high as 100%. Clinically, affected pigs show fever, cyanosis of the skin, and obvious bleeding in lymph nodes, kidneys, and gastrointestinal mucosa. African swine fever virus (ASFV) is an important member of the genus Asfivirus in the family Asfarviridae. Some of its characteristics are similar to those of the families Iridoviridae and Poxviridae. The diameter of the virus particles is 175 - 215 nanometers, showing icosahedral symmetry and having an envelope. The genome is double-stranded linear DNA, with a size of 170 - 190 kb.

[0003] African swine fever virus P72 (p72) is an important capsid protein encoded by the B646L gene and is also one of the important antigenic proteins of the ASFV virion. It exists in the form of a trimer in the virion and is abundant, with 8280 copies. It is an important target for the serological and molecular biological detection of ASFV. The p72 protein plays a key role in ASFV infection, and the antigenic epitopes on its surface are considered to be protective antigens. Recombinant p72 protein and p72 baculovirus have been successfully constructed, and specific monoclonal antibodies against the ASFV p72 protein have been prepared. These antibodies show strong reactivity against ASFV-infected cells. In addition, the p72 protein is also the target gene of the African swine fever virus fluorescence PCR detection kit. This 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 applicable to the detection of all genotypes of African swine fever virus.

[0004] The existing patent CN 117571987 A discloses a PDMS / FeSiB / QDs magnetoelastic flexible immunosensing test strip and its preparation method and application. The magnetoelastic flexible immunosensing test strip includes a flexible substrate, and quantum dot CsPbBr 3 is conjugated to the surface of the flexible substrate, and quantum dot CsPbBr 3 is conjugated with a biological antibody; a silver nanolayer is attached to the back of the flexible substrate. However, such biosensors using flexible substrates have great limitations, resulting in a relatively complex sensor manufacturing process and very time-consuming operation, thus restricting their application. For example, PDMS has problems such as complex operations for modifying antibodies with a gold layer and easy detachment of the gold layer. Summary of the Invention

[0005] The present invention overcomes the deficiencies of the prior art and provides a preparation method and application of a two-dimensional photonic crystal DNA hydrogel biosensor; the present invention is realized through the following technical solutions: A preparation method of a two-dimensional photonic crystal DNA hydrogel biosensor, comprising the following steps: Step S1, preparation of two-dimensional photonic crystal: uniformly mix a polystyrene microsphere-coupled iron tetroxide solution and n-propanol to obtain a mixed solution; and spread the mixed solution on a glass substrate to form a 2DPC array; Step S2, preparation of hydrogel: 1) Dissolve acrylic acid, cross-linking agent Bis and acrylamide in ultrapure water and then add glycerol to obtain solution A; 2) Dissolve photoinitiator LAP in dimethyl sulfoxide to obtain solution B; 3) Mix solution B and solution A evenly to serve as a polymerization precursor solution for preparing a hydrogel film; 4) Drop the polymerization precursor solution on the surface of the 2DPC array, irradiate it with 365 nm violet light at room temperature for 3 - 6 min to prepare a two-dimensional photonic crystal hydrogel film; peel the two-dimensional photonic crystal hydrogel film from the glass substrate and wash it; 5) Immerse the two-dimensional photonic crystal hydrogel film in a buffer solution to make the film reach swelling equilibrium; Step S3, functionalize the hydrogel: 1) Dissolve N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in a buffer solution, immerse the two-dimensional photonic crystal hydrogel film, activate the carboxyl groups on the surface of the 2DPCH, and then wash it; 2) Mix cp1 and cp2 evenly, drop them on the surface of the two-dimensional photonic crystal hydrogel film, place it at room temperature for 1 - 1.5 h and then store it overnight at 3 - 5 °C, and then wash it.

[0006] 3) Spin-coat AgNWs on the other side of the hydrogel film treated in 2) to form a conductive layer, and finally prepare an AgNWs / Hydrogel / PS / Fe 3 O 4 composite film.

[0007] Preferably, the volume ratio of the polystyrene microsphere-coupled iron tetroxide solution to n-propanol is 3:1.

[0008] Preferably, the method for forming the 2DPC array is to obliquely insert the glass substrate below the liquid surface, suck the mixed solution with a syringe, slowly inject it on the surface of deionized water, and then lift the glass substrate upward, and dry it to obtain the 2DPC array.

[0009] Preferably, the buffer solution is PBS buffer solution.

[0010] Preferably, the mass ratio of acrylic acid, crosslinking agent Bis and acrylamide is 12:1:32.

[0011] Preferably, after peeling the two-dimensional photonic crystal hydrogel film from the glass substrate, it is washed 3 times with water and PBS buffer solution successively.

[0012] Preferably, the volume ratio of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:5.

[0013] Preferably, the two-dimensional photonic crystal hydrogel film is immersed in PBS buffer solution overnight to make the film reach swelling equilibrium; the two-dimensional photonic crystal hydrogel film after swelling equilibrium is cut into small pieces of 1×1 cm 2 in size.

[0014] An application of a two-dimensional photonic crystal DNA hydrogel biosensor for detecting the P72 protein gene of African swine fever virus; the biosensor is prepared by the preparation method of a two-dimensional photonic crystal DNA hydrogel biosensor described above.

[0015] The beneficial effects of the present invention compared with the prior art are as follows: The present invention proposes an AgNWs / Hydrogel / PS / Fe 3 O 4 composite film. Using hydrogel as the substrate of the sensor, it has good biocompatibility and can be used for the detection of P72 protein gene fragments. Based on the bilayer composite structure, the flexible substrate hydrogel is combined with polystyrene microspheres coupled with Fe 3 O 4 nanoparticles and a magnetic field is applied. AgNWs are sprayed on the surface of the hydrogel film, effectively improving the sensitivity of the sensor.

[0016] Based on the principle of base complementary pairing, two complementary strands are complementary paired with the P72 protein gene fragment, the hydrogel shrinks and the spacing between the two-dimensional photonic crystal microspheres decreases. Under the action of an external magnetic field, the hydrogel generates a greater deformation. By monitoring the resistance change of the sensing film, the detection of the P72 protein gene fragment is realized.

[0017] Based on the characteristics of two-dimensional photonic crystals, the shrinkage and expansion of the film can change the spacing between the embedded photonic crystals. By measuring the Debye diffraction ring, the deformation of the film can be verified. After introducing AgNWs, the deformation of the film can be verified by measuring the resistance, thereby realizing two measurement methods.

[0018] In summary, based on the good stability and biocompatibility of hydrogel, the excellent magnetic properties of iron oxide, and the photon characteristics of two-dimensional photonic crystals, etc., the present invention prepares a flexible DNA biosensor. It has good sensing performance, as well as advantages such as low cost, simple preparation, short time consumption, and simple testing method. The present invention introduces materials such as magnetic particles, two-dimensional photonic crystals, and silver nanowires to prepare a biosensor, and sensitively converts the shrinkage and expansion generated by the hydrogel into optical signals and electrical signals, and the deformation of the hydrogel film can be verified by optical methods and resistance measurement.

[0019] The carboxyl groups inside the hydrogel can be coupled with the amino groups of the complementary strand to achieve the capture of target DNA molecules. Based on the principle of base complementary pairing, highly sensitive and specific detection of the P72 protein gene fragment can be achieved. At the same time, by utilizing the characteristics of two-dimensional photonic crystals, the Debye diffraction ring measurement method is introduced into the DNA biosensor, and it can be measured by two detection methods, effectively improving the functionality, and having advantages such as simple operation, good specificity, and good sensitivity, and having good application prospects in the fields of flexible biosensors and medical detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a flowchart of the preparation method of the two-dimensional photonic crystal DNA hydrogel biosensor of the present invention; wherein, tDNA is the P72 protein gene fragment.

[0021] Figure 2 is a linear relationship diagram of the P72 protein gene concentration and the resistance change of the two-dimensional photonic crystal DNA hydrogel biosensor. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the present invention will be further described in detail below in combination with embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. The technical solutions of the present invention will be described in detail below in combination with embodiments and drawings, but the protection scope is not limited by this.

[0023] See Figure 1 , this embodiment proposes a preparation method and application of a two-dimensional photonic crystal DNA hydrogel biosensor; specifically including the following steps: Materials: polystyrene microspheres, acrylic acid, acrylamide, n-propanol, TE buffer solution, dimethyl sulfoxide, cross-linking agent, photoinitiator Lap, glycerol; Step S1, preparation of two-dimensional photonic crystal: 1) Uniformly mix a solution of polystyrene microspheres coupled with iron oxide (existing, directly purchased solution) and n-propanol with a volume ratio of 3:1; 2) Insert the glass slide obliquely below the liquid surface, suck a small amount of the above solution with a syringe, slowly inject it on the surface of deionized water, then lift the glass slide upward, and then place it in the air to dry, obtaining a 2DPC (two-dimensional photonic crystal) array.

[0024] Step S2, Preparation of hydrogel: 1) Weigh 0.45 g of acrylic acid, 0.0375 g of crosslinking agent Bis (disuccinimidyl suberate sodium salt) and 1.2 g of acrylamide respectively, dissolve them in ultrapure water to obtain a 10 ml solution, and then add 5 mL of glycerol to obtain solution A; 2) Weigh 0.063 g of photoinitiator LAP and dissolve it in 0.25 mL of dimethyl sulfoxide to obtain solution B; 3) Take 20 μL of solution B and mix it evenly with 1 mL of solution A as the polymerization precursor solution for preparing the hydrogel film; 4) Take 200 μL of the polymerization precursor solution and drop it on the surface of the 2DPC array. Irradiate it with 365 nm ultraviolet light at room temperature for 5 min. Peel the prepared two-dimensional photonic crystal hydrogel film from the glass substrate, and wash it successively with water and PBS buffer solution 3 times; 5) Immerse the two-dimensional photonic crystal hydrogel film in PBS buffer solution overnight to make the film reach swelling equilibrium; 6) Cut the two-dimensional photonic crystal hydrogel film after swelling equilibrium into small pieces of 1×1 cm 2 for standby.

[0025] Step S3, Functionalization of hydrogel: 1) Weigh 3 mg of NHS (N-hydroxysuccinimide) and 15 mg of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) and dissolve them in 1 mL of PBS solution. Immerse the cut small pieces of two-dimensional photonic crystal hydrogel film in it for 1 h to activate the carboxyl groups on the surface of 2DPCH, and then wash it 3 times with PBS solution to remove the unreacted NHS and EDC; Since the dry DNA powder adheres to the tube wall in a very light dry film state, centrifugation before opening can prevent loss. Centrifuge the dry DNA powder at 4000 rpm for 30 - 60 s in a centrifuge. Then slowly open the tube cap, add an appropriate amount of TE buffer to the dry DNA powder (cp1, cp2, P72 protein gene fragment) to prepare different concentrations, cover the cap and shake well.

[0026] 10 μL of cp1 and cp2 with a certain concentration were respectively taken (cp1 and cp2 are capture probes, that is, DNA complementary strands for capturing the P72 protein gene fragment; cp1 and cp2 are respectively DNA strands semi-complementary to the P72 protein gene fragment; the concentration was selected as 90 μM after optimization), mixed evenly, dropped on the surface of the hydrogel, left at room temperature for 1 h and then overnight in a refrigerator at 4 °C. After taking out, it was rinsed 3 times with PBS solution, and finally the hydrogel sensing film was prepared.

[0027] cp1 and cp2 were dropped on one side of the hydrogel film, and silver nanowires were compounded on the other side. When the analyte was dropped on the cp side, it would cause the hydrogel to deform, and the conductive path of the silver nanowires would also change. At this time, we used KEITHLEY 2400 to measure the resistance value on the side containing silver nanowires; Detection results: As Figure 2 shown, as the concentration of the P72 protein gene increased, the relative change rate of the resistance also gradually increased, and a good linear relationship was presented.

[0028] The above content is a further detailed description of the present invention in combination with specific preferred embodiments. It cannot be determined that the specific embodiments of the present invention are limited thereto. For those of ordinary skill in the technical field to which the present invention pertains, without departing from the premise of the present invention, several simple deductions or substitutions can still be made, which should all be regarded as belonging to the patent protection scope determined by the claims submitted by the present invention.

Claims

1. A method for preparing a two-dimensional photonic crystal DNA hydrogel biosensor, characterized in that: The following steps are involved: Step S1, preparation of two-dimensional photonic crystals: uniformly mixing polystyrene microspheres coupled with ferroferric oxide solution and n-propanol to obtain a mixed solution; and spreading the mixed solution on a glass substrate to form a 2DPC array; Step S2, preparation of hydrogel: 1) Dissolve acrylic acid, crosslinking agent Bis and acrylamide in ultrapure water and add glycerol to obtain solution A; 2) dissolving the photoinitiator LAP in dimethyl sulfoxide to obtain solution B; 3) Mix solution B and solution A evenly to prepare a polymerization precursor solution for preparing a hydrogel film; 4) Dropping a polymerization precursor on the surface of the 2DPC array, irradiating it with 365nm violet light for 3-6 minutes at room temperature to obtain a two-dimensional photonic crystal hydrogel film; peeling the two-dimensional photonic crystal hydrogel film from the glass substrate and washing it; 5) Immersing the two-dimensional photonic crystal hydrogel film in a buffer solution to allow the film to reach swelling equilibrium; Step S3, functionalized hydrogel: 1) Dissolve N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride in a buffer solution, immerse the two-dimensional photonic crystal hydrogel film in it, activate the carboxyl groups on the surface of 2DPCH, and then wash it; 2) Mix cp1 and cp2 evenly, drop them on the surface of the two-dimensional photonic crystal hydrogel film, place them at room temperature for 1-1.5 hours, store them at 3-5°C overnight, and then wash them; 3) Spin-coating AgNWs on the other side of the hydrogel film treated in 2) to form a conductive layer, and finally preparing an AgNWs / Hydrogel / PS / Fe3O4 composite film.

2. The method for preparing a two-dimensional photonic crystal DNA hydrogel biosensor according to claim 1, characterized in that: The volume ratio of polystyrene microspheres coupled with ferroferric oxide solution and n-propanol is 3:

1.

3. The method for preparing a two-dimensional photonic crystal DNA hydrogel biosensor according to claim 1, characterized in that: The method for forming a 2DPC array is to insert a glass substrate obliquely below the liquid surface, absorb the mixed solution with a syringe, slowly inject it on the surface of deionized water, then pull up the glass substrate, and obtain the 2DPC array after drying.

4. The method for preparing a two-dimensional photonic crystal DNA hydrogel biosensor according to claim 1, characterized in that: The buffer is PBS buffer.

5. The method for preparing a two-dimensional photonic crystal DNA hydrogel biosensor according to claim 1, characterized in that: The mass ratio of acrylic acid, cross-linking agent Bis and acrylamide is 12:1:

32.

6. The method for preparing a two-dimensional photonic crystal DNA hydrogel biosensor according to claim 1, characterized in that: After the two-dimensional photonic crystal hydrogel film was peeled off from the glass substrate, it was washed three times with water and then with PBS buffer.

7. The method for preparing a two-dimensional photonic crystal DNA hydrogel biosensor according to claim 1, characterized in that: The volume ratio of N-hydroxysuccinimide to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:

5.

8. The method for preparing a two-dimensional photonic crystal DNA hydrogel biosensor according to claim 1, characterized in that: The two-dimensional photonic crystal hydrogel film was immersed in PBS buffer overnight to allow the film to reach swelling equilibrium; the two-dimensional photonic crystal hydrogel film after swelling equilibrium was cut into 1×1 cm 2 Small piece of.

9. An application of a two-dimensional photonic crystal DNA hydrogel biosensor, characterized in that: Used to detect the P72 protein gene of African swine fever virus; the biosensor is prepared by the preparation method of a two-dimensional photonic crystal DNA hydrogel biosensor as described in any one of claims 1-8.

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