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

Abstract

The application 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 steps comprise preparation of a two-dimensional photonic crystal, preparation of a hydrogel, and functionalization of the hydrogel; the biosensor is prepared by introducing magnetic particles, two-dimensional photonic crystals, silver nanowires and other materials, the contraction and expansion sensitivity of the hydrogel is converted into optical signals and electrical signals, the deformation of the hydrogel film can be verified by optical methods and resistance measurement; the carboxyl groups in the hydrogel can be coupled with the amino groups of complementary chains to capture target DNA molecules, and based on the principle of base complementary pairing, high-sensitivity and specificity detection of P72 protein gene fragments can be realized.

Description

TECHNICAL FIELD

[0001] The application 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 biological sensor. BACKGROUND

[0002] African swine fever (ASF) is an acute, highly contagious fever caused by a filterable virus, characterized by a short course of disease but a mortality rate of up to 100%. The clinical manifestations of sick pigs are fever, cyanosis of the skin, lymph node, kidney, and gastrointestinal mucosa bleeding. African swine fever virus (ASFV) is an important member of the African swine fever virus family, and some of its characteristics are similar to those of the Choriovirus family and the Poxvirus family. The virus particle is 175-215 nanometers in diameter, has icosahedral symmetry, and has a capsule. The genome is a double-stranded linear DNA with a size of 170-190 kb.

[0003] ASFV p72 (p72) is an important capsid protein encoded by the B646L gene and 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, and is an important target for ASFV serological and molecular biology detection. The p72 protein plays a key role in ASFV infection, and the surface antigen epitopes are considered to be protective antigens. Studies have successfully constructed recombinant p72 protein and p72 baculovirus, and prepared specific monoclonal antibodies against ASFV p72 protein, which exhibit strong reactivity to ASFV-infected cells. In addition, the p72 protein is also the target gene of the ASFV fluorescent PCR detection kit. The kit is based on the working principle of Taqman hydrolysis probe and is used for detecting ASFV nucleic acid in samples, has high sensitivity and specificity, and is suitable for detecting all genotypes of ASFV.

[0004] The existing patent CN 117571987 A discloses a PDMS / FeSiB / QDs magnetoelastic flexible immunological detection test paper and a preparation method and application thereof. The magnetoelastic flexible immunological detection test paper comprises a flexible substrate, the surface of the flexible substrate is combined with quantum dots CsPbBr3, the quantum dots CsPbBr3 are coupled with biological antibodies, and the back of the flexible substrate is attached with a silver nano layer. However, the flexible substrate used by such a biological sensor has great limitations, resulting in a relatively complex sensor manufacturing process and a very time-consuming operation, thereby limiting the application. For example, the PDMS has complex operations of using a gold layer to modify antibodies and the gold layer is prone to falling off. SUMMARY

[0005] This invention overcomes the shortcomings of existing technologies and proposes a method for preparing and applying a two-dimensional photonic crystal DNA hydrogel biosensor. This invention is achieved through the following technical solution:

[0006] A method for fabricating a two-dimensional photonic crystal DNA hydrogel biosensor includes the following steps:

[0007] Step S1, Preparation of two-dimensional photonic crystal: Polystyrene microspheres are coupled with iron oxide solution and n-propanol and mixed uniformly to obtain a mixed solution; the mixed solution is then spread on a glass substrate to form a 2DPC array;

[0008] Step S2, Preparation of hydrogel:

[0009] 1) Dissolve acrylic acid, crosslinking agent Bis, and acrylamide in ultrapure water, then add glycerol to obtain solution A;

[0010] 2) Dissolve the photoinitiator LAP in dimethyl sulfoxide to obtain solution B;

[0011] 3) Mix solution B and solution A evenly to prepare the polymerization precursor solution for preparing the hydrogel film;

[0012] 4) Drop the polymerization precursor liquid onto the surface of the 2DPC array and irradiate it with 365nm ultraviolet light at room temperature for 3-6 min to obtain a two-dimensional photonic crystal hydrogel film; peel the two-dimensional photonic crystal hydrogel film off the glass substrate and clean it.

[0013] 5) Immerse the two-dimensional photonic crystal hydrogel film in a buffer solution to allow the film to reach swelling equilibrium;

[0014] Step S3, Functionalized Hydrogel:

[0015] 1) Dissolve N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in buffer solution, immerse the two-dimensional photonic crystal hydrogel film in the solution, activate the carboxyl groups on the 2DPCH surface, and then wash.

[0016] 2) Mix cp1 and cp2 evenly, drop them onto the surface of the two-dimensional photonic crystal hydrogel film, place at room temperature for 1-1.5 hours, then store overnight at 3-5°C, and then wash.

[0017] 3) Spin-coat AgNWs onto the other side of the hydrogel film treated in 2) to form a conductive layer, and finally prepare the AgNWs / Hydrogel / PS / Fe3O4 composite film.

[0018] Preferably, the volume ratio of polystyrene microspheres coupled with ferric oxide solution and n-propanol is 3:1.

[0019] Preferably, the method of forming the 2DPC array is to tilt the glass substrate into the liquid surface, to suck the mixed solution by a syringe, to slowly inject on the surface of deionized water, and then to pull up the glass substrate, and to obtain the 2DPC array after drying.

[0020] Preferably, the buffer solution is a PBS buffer solution.

[0021] Preferably, the mass ratio of the acrylic acid, the crosslinking agent Bis and the acrylamide is 12:1:32.

[0022] Preferably, after the two-dimensional photonic crystal hydrogel film is peeled from the glass substrate, the two-dimensional photonic crystal hydrogel film is cleaned with water and a PBS buffer solution for three times in sequence.

[0023] Preferably, the volume ratio of the N-hydroxysuccinimide and the 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is 1:5.

[0024] Preferably, the two-dimensional photonic crystal hydrogel film is immersed in a PBS buffer solution overnight to make the film reach a swelling equilibrium; and the two-dimensional photonic crystal hydrogel film after the swelling equilibrium is cut into pieces with a size of 1*1cm 2 .

[0025] The application of the two-dimensional photonic crystal DNA hydrogel biosensor is applied to detecting an African swine fever virus P72 protein gene; the biosensor is prepared by the preparation method of the two-dimensional photonic crystal DNA hydrogel biosensor.

[0026] The beneficial effects generated by the application relative to the prior art are as follows:

[0027] The application provides an AgNWs / Hydrogel / PS / Fe3O4 composite film, the hydrogel is used as a substrate of a sensor, has good biocompatibility, and can be used for detecting a P72 protein gene fragment. Based on the double-layer composite structure, the flexible substrate hydrogel is coupled with the Fe3O4 nanoparticles and the polystyrene microspheres, a magnetic field is applied, the AgNWs are sprayed on the surface of the hydrogel film, and the sensitivity of the sensor is effectively improved.

[0028] Based on the principle of base complementary pairing, two complementary chains are complementary paired with a P72 protein gene fragment, the hydrogel is shrunk, the spacing between the two-dimensional photonic crystal microspheres is reduced, the hydrogel is deformed more under the action of an external magnetic field, the resistance change of the sensing film is monitored, and the detection of the P72 protein gene fragment is realized.

[0029] The present application is 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, and the deformation of the film can be verified by measuring the Debye diffraction ring. After introducing AgNWs, the deformation of the film can be verified by measuring the resistance, thereby realizing two methods of measurement.

[0030] In summary, the present application is based on the advantages of good stability and biocompatibility of hydrogel, excellent magnetic properties of ferroferric oxide, and photonic properties of two-dimensional photonic crystals, etc. It has good sensing performance, low cost, simple preparation, short time consumption, simple testing method, etc. The present application introduces magnetic particles, two-dimensional photonic crystals and silver nanowires to prepare a biosensor, and the shrinkage and expansion of the hydrogel are sensitively converted into optical and electrical signals, which can be verified by optical method and resistance measurement.

[0031] The carboxyl groups in the hydrogel can be coupled with the amino groups of the complementary chain to capture the target DNA molecule. Based on the principle of base complementary pairing, high sensitivity and specificity detection of P72 protein gene fragments can be realized. At the same time, the characteristics of two-dimensional photonic crystals are used to introduce the Debye diffraction ring measurement method into the DNA biosensor, which can be measured by two detection methods, effectively improving the functionality, and has the advantages of simple operation, good specificity, good sensitivity, etc. It has good application prospect in the field of flexible biosensors and medical detection. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 The flow chart of the preparation method of the two-dimensional photonic crystal DNA hydrogel biosensor described in the present application; wherein tDNA is a P72 protein gene fragment.

[0033] Figure 2 The linear relationship between the P72 protein gene concentration and the resistance change of the two-dimensional photonic crystal DNA hydrogel biosensor. DETAILED DESCRIPTION

[0034] In order to make the technical problems, technical solutions and beneficial effects of the present application more clear and explicit, the present application is further described in detail in combination with the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present application, and are not used to limit the present application. The technical solutions of the present application are described in detail below in combination with the embodiments and drawings, but the protection scope is not limited thereto.

[0035] Reference Figure 1 The present embodiment proposes a preparation method and application of a two-dimensional photonic crystal DNA hydrogel biosensor; specifically including the following steps:

[0036] Materials: polystyrene microspheres, acrylic acid, acrylamide, n-propanol, TE buffer, dimethyl sulfoxide, crosslinking agent, photoinitiator LAP, glycerol;

[0037] Step S1, preparation of two-dimensional photonic crystal:

[0038] 1) Mix the polystyrene microspheres and the ferric oxide solution (commercially available) in a volume ratio of 3:1 with n-propanol uniformly;

[0039] 2) Insert the slide into the liquid surface obliquely, use a syringe to suck a small amount of the above solution, slowly inject it on the surface of deionized water, then pull the slide up, and place it in the air to dry, obtaining a 2DPC (two-dimensional photonic crystal) array.

[0040] Step S2, preparation of hydrogel:

[0041] 1) Weigh 0.45 g of acrylic acid, 0.0375 g of crosslinking agent Bis (sodium salt of succinimidyl succinate) and 1.2 g of acrylamide into ultrapure water to obtain 10 mL of solution, then add 5 mL of glycerol to obtain solution A;

[0042] 2) Weigh 0.063 g of photoinitiator LAP and dissolve it in 0.25 mL of dimethyl sulfoxide to obtain solution B;

[0043] 3) Mix 20 μL of solution B with 1 mL of solution A uniformly as the polymerization precursor solution for preparing the hydrogel film;

[0044] 4) Take 200 μL of the polymerization precursor solution and drop it on the surface of the 2DPC array, irradiate it with 365 nm purple light for 5 min, peel off the prepared two-dimensional photonic crystal hydrogel film from the glass substrate, and wash it with water and PBS buffer solution for 3 times respectively;

[0045] 5) Immerse the two-dimensional photonic crystal hydrogel film in PBS buffer solution overnight to make the film reach swelling equilibrium;

[0046] 6) Cut the two-dimensional photonic crystal hydrogel film after swelling equilibrium into small pieces of 1 × 1 cm 2 for standby.

[0047] Step S3, functionalization of hydrogel:

[0048] 1) Weigh 3 mg of NHS (N-hydroxysuccinimide) and 15 mg of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride) into 1 mL of PBS solution, immerse the cut small piece of two-dimensional photonic crystal hydrogel film into it for 1 h, activate the carboxyl group on the surface of 2DPC, then wash it with PBS solution for 3 times to remove unreacted NHS and EDC;

[0049] Because the DNA dry powder is very light and attached to the wall of the tube in the form of dry film, opening before centrifugation can prevent loss, and the dry powder DNA is centrifuged in the centrifuge at 4000 rpm for 30-60 s. Then slowly open the tube cap, add an appropriate amount of TE buffer to the DNA dry powder (cp1, cp2, P72 protein gene fragment) to configure different concentrations, and shake well after covering the cap.

[0050] 10 μL of cp1 and cp2 (cp1 and cp2 are capture probes, that is, DNA complementary chains of capturing P72 protein gene fragments; cp1 and cp2 are DNA chains that are semi-complementary to the P72 protein gene fragment; the concentration is optimized to be 90 μM) of a certain concentration are mixed and added dropwise on the surface of the hydrogel, and then placed at room temperature for 1 h and in a refrigerator at 4℃ overnight. After taking out, rinse with PBS solution for 3 times, and finally prepare the hydrogel sensing film.

[0051] The cp1 and cp2 are added dropwise on one side of the hydrogel film, and the silver nanowires are compounded on the other side. When the test substance is added to the cp side, the hydrogel will be deformed, and the conductive path of the silver nanowires will also change. At this time, we use KEITHLEY 2400 to measure the resistance value on the side containing the silver nanowires.

[0052] The detection results are shown in Table 1. Figure 2 As shown in Table 1, with the increase of the concentration of P72 protein gene, the relative change rate of resistance also gradually increases, and shows a good linear relationship.

[0053] The above is a further detailed description of the present application in combination with a specific preferred embodiment, and cannot be considered as limiting the specific embodiments of the present application. For ordinary skilled persons in the technical field to which the present application belongs, without departing from the present application, a number of simple deductions or substitutions can be made, and all should be considered as belonging to the scope of patent protection determined by the claims submitted.

Claims

1. A two-dimensional photonic crystal DNA hydrogel biosensor for detecting African swine fever virus P72 protein gene, characterized in that, The preparation method of the two-dimensional photonic crystal DNA hydrogel biosensor comprises the following steps: Step S1, preparation of a two-dimensional photonic crystal: uniformly mix a polystyrene microsphere coupled ferroferric oxide solution and n-propanol to obtain a mixed solution; and lay the mixed solution on a glass substrate to form a two-dimensional photonic crystal array; Step S2, preparation of a hydrogel: 1) dissolve acrylic acid, a crosslinking agent Bis, and acrylamide in ultrapure water, and then add glycerol to obtain solution A; 2) dissolve a photoinitiator LAP in dimethyl sulfoxide to obtain solution B; 3) uniformly mix solution B and solution A as a polymerization precursor solution for preparing a hydrogel film; 4) drop the polymerization precursor solution on the surface of the two-dimensional photonic crystal array, irradiate with 365 nm purple 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 clean it; 5) immerse the two-dimensional photonic crystal hydrogel film in a buffer solution to make the film reach a swelling equilibrium; Step S3, functionalization of 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 two-dimensional photonic crystal, and then clean it; 2) uniformly mix cp1 and cp2, drop them on the surface of the two-dimensional photonic crystal hydrogel film, and place them at room temperature for 1-1.5 h, and then store them at 3-5 °C overnight, and then clean them; cp1 and cp2 are capture probes, i.e., DNA complementary strands that capture P72 protein gene fragments; cp1 and cp2 are DNA strands that are semi-complementary to P72 protein gene fragments, respectively; 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 / Fe3O4 composite film.

2. The two-dimensional photonic crystal DNA hydrogel biosensor for detecting the P72 protein gene of African swine fever virus according to claim 1, characterized in that, The volume ratio of the polystyrene microsphere coupled ferroferric oxide solution and n-propanol is 3:

1. 3.The two-dimensional photonic crystal DNA hydrogel biosensor for detecting the P72 protein gene of African swine fever virus according to claim 1, wherein, The method for forming the two-dimensional photonic crystal array is to insert the glass substrate obliquely below the liquid surface, use a syringe to suck the mixed solution, slowly inject it on the surface of deionized water, then pull the glass substrate upward, and dry to obtain the two-dimensional photonic crystal array.

4. The two-dimensional photonic crystal DNA hydrogel biosensor for detecting the P72 protein gene of African swine fever virus according to claim 1, characterized in that, The buffer solution is a PBS buffer solution.

5. The two-dimensional photonic crystal DNA hydrogel biosensor for detecting African swine fever virus P72 protein gene according to claim 1, characterized in that, The mass ratio of the acrylic acid, the crosslinking agent Bis, and the acrylamide is 12:1:

32. 6.The two-dimensional photonic crystal DNA hydrogel biosensor for detecting the P72 protein gene of African swine fever virus according to claim 1, wherein, After peeling the two-dimensional photonic crystal hydrogel film from the glass substrate, wash it with water and a PBS buffer solution for three times, respectively.

7. The two-dimensional photonic crystal DNA hydrogel biosensor for detecting African swine fever virus P72 protein gene according to claim 1, characterized in that, The volume ratio of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is 1:

5. 8.The two-dimensional photonic crystal DNA hydrogel biosensor for detecting the P72 protein gene of African swine fever virus according to claim 1, wherein, The two-dimensional photonic crystal hydrogel film was immersed in PBS buffer overnight to make the film reach swelling equilibrium; the two-dimensional photonic crystal hydrogel film after swelling equilibrium was cut into 1x1 cm 2 pieces.

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