A hydrogel confined photoexcitation ratio electrochemical sensing method for detecting aflatoxin b1

Abstract

The present application belongs to the technical field of biosensor, and particularly relates to a hydrogel confined light excitation ratio electrochemical sensing method for detecting AFB1; the present application introduces a hydrogel as a confined recognition site of a target AFB1, and based on base complementary pairing between an aptamer and a complementary strand, a double-stranded DNA structure is obtained to adsorb MB; when the target AFB1 exists, the MB is combined with the aptamer to be released, based on electrostatic repulsion, DNA chains are distributed on the surface of the hydrogel, and the MB is distributed in the pore diameter of the hydrogel, so that the MB signal is enhanced. With K3[Fe(CN)6] as a reference probe and the MB as a response probe, sensitive analysis of the AFB1 is realized, the detection range is 0.001-1000 ng / mL ‑1 , and the detection limit is 0.18 pg / mL.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biosensors, and particularly relates to a hydrogel confined light excitation ratio electrochemical sensing method for detecting AFB1. BACKGROUND

[0002] Hydrogel is a cross-linked 3D framework rich in water, which has good biocompatibility and can provide a stable reaction microenvironment for biomolecules. Based on the porous network structure of hydrogel, the analyte can penetrate into the matrix of hydrogel. By regulating the pore size and surface charge of hydrogel, the analyte is confined in the outer layer, interior and surface of hydrogel. Studies have shown that confining electrochemical reactions in small droplets can simplify the construction process of the sensor and improve the sensitivity of the sensor. However, the conductivity problem of hydrogel material hinders its wide application in electrochemical sensors.

[0003] By compounding metal nanoparticles or conductive polymers with good conductivity with hydrogel, the conductivity of hydrogel can be improved, and the electron transfer of hydrogel can be promoted. Light excitation is a controllable external stimulus that can enhance the signal of conductive hydrogel. For example, the local surface plasmon resonance (LSPR) based on gold nanoparticles (AuNPs) improves the conductivity of hydrogel.

[0004] However, so far, this functional hydrogel that combines all reactions in a 3D matrix has not been developed and applied to the construction of electrochemical sensors. SUMMARY

[0005] In view of the deficiencies of the prior art, the application provides a hydrogel confined light excitation ratio electrochemical sensing method for detecting AFB1, proposes a new construction strategy of electrochemical sensor, and realizes sensitive analysis of aflatoxin B1 (AFB1).

[0006] Hydrogel has good biocompatibility, the conductivity of hydrogel is improved by introducing AuNPs, and the electrochemical probe methylene blue (MB) adsorbed by DNA double strands is used as an electrochemical response probe, which is confined in the hydrogel. The electrochemical probe potassium ferricyanide (K3[Fe(CN)6]) in the electrolyte solution is used as a reference probe. Under light excitation, based on the LSPR effect of AuNPs, the MB signal is significantly enhanced.

[0007] The application constructs a hydrogel confined light excitation ratio electrochemical sensing method for detecting AFB1, takes AFB1 as a target molecule, obtains a double-stranded DNA structure based on base complementary pairing of an aptamer and a complementary chain, adsorbs MB through electrostatic action, releases MB when the target AFB1 exists, and due to electrostatic action, the negatively charged DNA chain is distributed on the surface of the hydrogel, and MB is distributed in the pore size of the hydrogel, so that the MB signal is enhanced. The K3[Fe(CN)6] is used as a reference probe, and the MB is used as a response probe, so that the sensitive analysis of AFB1 is realized.

[0008] In order to realize the technical purpose of the application, the specific scheme is as follows.

[0009] A hydrogel confined light excitation ratio electrochemical sensing method for detecting AFB1, the steps are as follows:

[0010] (1) Synthesis of hydrogel: uniformly mix polyethylene glycol (PEG), polyethylene glycol diacrylate (PEG-DA), 2-hydroxy-2-methyl propiophenone (2-H-2-M-Pro), acrylate polyethylene glycol carboxyl (Acryl-PEG-COOH) and ultrapure water in a certain proportion to form a hydrogel matrix;

[0011] (2) After the HAuCl4 solution is heated to boiling, Na3C6H5O7·2H2O is added, and after a period of reaction, the color of the solution changes to bright red, that is, a gold nanoparticle solution is obtained, which is recorded as AuNPs solution; then, the AuNPs solution is mixed with the hydrogel matrix synthesized in step (1) to obtain a hydrogel mixed matrix;

[0012] (3) After mixing the aptamer solution of the target AFB1 and the cDNA solution, the double-stranded DNA is obtained by PCR reaction; then the double-stranded DNA is mixed with the MB solution to obtain a mixed solution, and finally mixed with the hydrogel mixed matrix of step (2) to obtain a sensing matrix based on hydrogel;

[0013] (4) First, prepare an AFB1 standard solution, then mix it with the hydrogel-based sensing matrix prepared in step (3), and obtain a mixed solution which is incubated at room temperature, modified on the ITO surface after incubation, and obtained after ultraviolet polymerization based on the hydrogel space confinement ratio electrochemical sensor;

[0014] (5) Electrolyte solution: add potassium ferricyanide solution (K3[Fe(CN)6]) as a reference probe in the PBS buffer solution to form an electrolyte solution;

[0015] (6) Constructing a standard curve: Using the hydrogel-based ratiometric electrochemical sensor obtained in step (4) as the working electrode, saturated Ag / AgCl as the reference electrode, and a platinum wire electrode as the counter electrode, an external light source was introduced to excite the electrochemical sensing interface. During the excitation process, electrochemical tests were performed in the electrolyte solution. Two electrochemical signals were obtained for each concentration of AFB1 standard solution modified electrode, denoted as I0 and I1 respectively. MB and I K3[Fe(CN)6] A standard curve was constructed based on the ratio of the two obtained current values ​​and the logarithmic relationship of the AFB1 concentration.

[0016] (7) Detection of AFB1 in the sample: First, obtain the sample solution, and follow the steps (4)-(6), except that the AFB1 standard solution is replaced with the sample solution; finally, by electrochemical testing, I is obtained. MB and I K3[Fe(CN)6] Substituting the ratio of the two into the standard curve constructed in step (6), the concentration of AFB1 in the sample can be obtained; thus realizing the purpose of detecting AFB1 in unknown samples.

[0017] Preferably, in step (1), the amounts of polyethylene glycol (PEG), polyethylene glycol diacrylate (PEG-DA), 2-hydroxy-2-methylphenylacetone (2-H-2-M-Pro), acrylate polyethylene glycol carboxyl group (Acryl-PEG-COOH), and ultrapure water are in the following ratio: 2 mL: 2 mL: 0.5 mL: 0.5 mL: 1 mL; wherein the molecular weight of polyethylene glycol is 200 and the purity is greater than 90%; the molecular weight of polyethylene glycol diacrylate is 700 and the purity is greater than 90%; the molecular weight of 2-hydroxy-2-methylphenylacetone is 200 and the purity is 97%; and the concentration of acrylate polyethylene glycol carboxyl group is 20 mM and the purity is greater than 90%.

[0018] Preferably, in step (2), the concentration of the HAuCl4 solution is 0.4 mM, and the concentration of Na3C6H5O7·2H2O is 100 mg / mL. -1 The ratio of HAuCl4 solution to Na3C6H5O7·2H2O was 25.2 mL: 0.25 mL; the reaction time was 15-20 min.

[0019] Preferably, in step (2), the volume ratio of the hydrogel matrix to the AuNPs solution is 3:1; wherein the concentration of the AuNPs solution is 5.45 nM.

[0020] Preferably, in step (3), the volume ratio of the AFB1 aptamer solution to the cDNA solution is 1:1, the concentration of the double-stranded DNA is 4 μM, the concentration of the MB solution is 400 μM, and the volume ratio of the hydrogel, MB solution, and double-stranded DNA in the hydrogel-based sensing matrix is ​​4:1:2.

[0021] Preferably, in step (4), the concentration of the AFB1 standard solution is 0.001–1000 ng / mL. -1 The incubation time is 1.0 to 1.4 hours.

[0022] Preferably, in step (4), the amount of the mixture used for modification is 10-15 μL; the volume ratio of the AFB1 standard solution to the hydrogel-based sensing matrix is ​​1:7; and the ultraviolet light polymerization operation is as follows: the ultraviolet light polymerization time is 25 s at a wavelength of 365 nm.

[0023] Preferably, the concentration of K3[Fe(CN)6] in the electrolyte solution in step (5) is 0.01 mM; the PBS buffer solution is 0.1 M and pH = 7.4.

[0024] Preferably, the electrochemical detection in step (6) is recorded and detected by an electrochemical workstation of model CHI1040C; the scanning voltage range is -0.4 to 0.4V, the amplitude is 0.025V, and the frequency is 25Hz.

[0025] Preferably, the conditions for excitation by the external light source in step (6) are: power of 500mW / cm². 2 The vertical distance from the working electrode is 3 to 5 cm.

[0026] The beneficial effects of this invention are:

[0027] (1) The present invention introduces hydrogel to realize the spatial confinement of the target AFB1 recognition process, which restricts the recognition process of target AFB1 and its aptamer to a certain space, thereby improving the sensitivity of the photoexcitation ratio electrochemical sensor.

[0028] (2) The present invention introduces a photoexcitation strategy, based on the LSPR effect of AuNPs, which further improves the sensitivity of the photoexcitation ratio electrochemical sensor.

[0029] (3) The photoexcitation ratio electrochemical sensor based on hydrogel confinement reaction constructed in this invention has a detection range of 0.001 to 1000 ng / mL for AFB1 and a detection limit of 0.8 pg / mL. Attached Figure Description

[0030] Figure 1 The curve in the middle represents the relationship between the electrochemical signal under photoexcitation conditions and the electrochemical signal response under dark conditions.

[0031] Figure 2 (A) Figure IFigure 1 shows the recognition of the target analyte AFB1 with its aptamer in solution; Figure 2 shows the recognition of AFB1 with its aptamer in a hydrogel-based sensing matrix; Figure 3 shows the electrochemical detection signal after AFB1 is recognized in solution; Figure 4 shows the electrochemical detection signal after AFB1 is recognized in a hydrogel matrix.

[0032] Figure 3 (A) shows the response of the photoexcited ratio electrochemical sensor to different concentrations of AFB1; (B) shows the logarithm of AFB1 concentration versus I. MB / I K3[Fe(CN)6] (C) The figure shows the variable relationship; the figure shows the selectivity of the photoexcitation ratio electrochemical sensor for different interfering substances. Detailed Implementation

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terminology used herein is merely for describing particular embodiments and is not intended to limit the invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] Reagent instructions:

[0037] PEG and PEG-DA were purchased from Shanghai McLean Biotech Co., Ltd.; 2-H-2-M-Pro and Acryl-PEG-COOH were purchased from Aladdin Biotech Co., Ltd.

[0038] Among them, the aptamer of AFB1:

[0039] 5′-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3′;

[0040] Complementary DNA (cDNA):

[0041] 5'-TG TGG GCC TAG CGA AGG GCA CGA GAC ACA GAG AGA CAA CAC GTG CCCAAC-3'

[0042] The aptamer and cDNA of AFB1 were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0043] Electrolyte solution: composed of PBS buffer solution and potassium ferricyanide solution (K3[Fe(CN)6]), wherein K3[Fe(CN)6] serves as a reference probe; the concentration of K3[Fe(CN)6] in the electrolyte solution is 0.01 mM; the PBS buffer solution is 0.1 M with pH = 7.4.

[0044] Example 1:

[0045] (1) Mix 2 mL of PEG with a molecular weight of 200 and a purity greater than 90%, 2 mL of PEG-DA with a molecular weight of 700 and a purity greater than 90%, 0.5 mL of 2-H-2-M-Pro with a purity of 97%, 0.5 mL of Acryl-PEG-COOH with a concentration of 20 mM and a purity greater than 90% and 1 mL of ultrapure water evenly to obtain a hydrogel matrix;

[0046] (2) Heat 25.2 mL of 0.4 mM HAuCl4 solution to a complete boil, then add 0.25 mL of HAuCl4 solution and 100 mg of HAuCl4 solution. - 1 Na3C6H5O7·2H2O, the color of the solution gradually turned bright red as the heating time increased (20 min), proving that AuNPs were successfully synthesized and AuNPs solution was obtained; take 60 μL of the hydrogel matrix prepared in step (1) and mix it with 20 μL of AuNPs solution to obtain hydrogel mixed matrix.

[0047] (3) Mix 20 μL of 4 μM double-stranded DNA solution (the aptamer solution of the target AFB1 and the cDNA solution are mixed for PCR reaction, the conditions are: heat up to 95℃, react for 5 min, then cool down to 4℃ and hold for 10 min to obtain double-stranded DNA) with 10 μL of 400 μM MB solution, and adsorb MB into the double-stranded DNA structure by electrostatic interaction. Then mix the mixed solution of the two with 40 μL of hydrogel mixing matrix from step (2) to obtain a hydrogel-based sensing matrix.

[0048] (4) Take 15 μL of the hydrogel-based sensing matrix obtained in step (3) and modify it on the ITO surface. Then, use ultraviolet light with a wavelength of 365 nm to polymerize for 25 s to obtain a ratio electrochemical sensor based on the spatial confinement of hydrogel.

[0049] (5) To examine the response of the hydrogel-based ratiometric electrochemical sensor constructed in step (4) under photoexcitation conditions, an external light source with a power of 500 mW / cm² was introduced. 2 The vertical distance from the working electrode is 3–5 cm, and the signal response under photoexcitation conditions is as follows: Figure 1 As shown, compared with the signal under no light excitation, the response signal of MB is significantly enhanced;

[0050] (6) In order to verify the signal response of the photoexcited ratio electrochemical sensor constructed in step (5) in the hydrogel confinement space, the hydrogel-based sensing matrix obtained in step (3) was mixed with 10 μL of the target AFB1 standard solution to obtain a mixed hydrogel-based sensing matrix; then 15 μL of the mixed hydrogel-based sensing matrix was modified on the ITO surface and polymerized with ultraviolet light at a wavelength of 365 nm for 25 s. The signal response of MB was examined according to the operation in step (5).

[0051] like Figure 2 As shown in Figure A, I is a schematic diagram illustrating the recognition of the target analyte AFB1 with its aptamer in solution. Figure 2 B represents the electrochemical response of the corresponding MB, II is a schematic diagram of the recognition of the target analyte AFB1 with its aptamer in the hydrogel-based sensing matrix, and 2C represents the electrochemical response of the corresponding MB. Data analysis demonstrates that the spatial confinement function of the hydrogel can effectively improve the sensitivity of the photoexcitation ratio electrochemical sensor.

[0052] Further research and analysis on the hydrogel-confined photoexcitation ratio electrochemical sensing method:

[0053] (1) The analytical performance of the prepared photoexcitation ratio electrochemical sensor was studied:

[0054] First, prepare standard solutions of AFB1 at different concentrations. Then, dispense 10 μL of AFB1 solutions at concentrations of 0.001, 0.01, 0.1, 1, 10, 100, and 1000 ng / mL. -1 Mix with the hydrogel-based sensing matrix of step (3) in Example 1 (the two are in a one-to-one correspondence), incubate for 1.2 h, take 15 μL to modify the ITO surface, polymerize with ultraviolet light at a wavelength of 365 nm for 25 s and then perform electrochemical testing.

[0055] (2) Using the photoexcitation ratio electrochemical sensor modified in step (1) as the working electrode, saturated Ag / AgCl as the reference electrode, and a platinum wire electrode as the counter electrode, a power of 500 mW / cm² was utilized. 2 White light was used to excite the working interface at a distance of 3–5 cm from the working electrode. The electrochemical signals I between MB and K3[Fe(CN)6] were recorded and detected by an electrochemical workstation of model CHI1040C. MB I K3[Fe(CN)6] Electrochemical tests were performed in an electrolyte solution; the scanning voltage range was -0.4 to 0.4 V, the amplitude was 0.025 V, and the frequency was 25 Hz; according to I... MB / I K3[Fe(CN)6] A standard curve was constructed using the logarithm of AFB1 concentration; as AFB1 concentration increases, I MB / I K3[Fe(CN)6] Gradually decrease the concentration of AFB1, which is between 0.001 and 1000 ng / mL. -1 Within the range, it showed a linear relationship with the two obtained electrochemical signals. Figure 3 A); The specific linear relationship diagram is as follows: Figure 3 As shown in B, the logarithm of AFB1 concentration versus I MB / I K3[Fe(CN)6] It exhibits a good linear relationship, and its linear curve is I. MB / I K3[Fe(CN)6] =0.74lgC AFB1 +2.99, with a regression coefficient of 0.990, proves that the sensor performs well.

[0056] To evaluate the selectivity of the sensor, interference experiments were conducted using AFB2, OTA, a mixture of two toxins (AFB2+OTA), and a mixture of three toxins (AFB2+OTA+AFB1). Figure 3 As shown in C, the sensor's response to interference is almost identical to the background signal; only AFB1 can respond to Δ(I) MB / I K3[Fe(CN)6] The significant changes indicate that the sensor has good selectivity.

[0057] Based on the sensor's excellent analytical performance, AFB1 in actual peanut samples, peanut soil, and moldy peanut samples was further analyzed using the sensor:

[0058] (1) Grind 5g of peanut sample, soil sample and moldy peanut sample and soak them in a mixed solution containing 5mL methanol and 15mL ultrapure water respectively. Shake for 1.5 hours, then centrifuge at 8000 rpm for 10 minutes and take the supernatant as the sample solution.

[0059] (2) Take 10 μL of sample solution and mix it with the hydrogel sensing matrix from step (3) of Example 1 to obtain a mixed hydrogel-based sensing matrix. After incubation at room temperature for 1.2 h, take 15 μL of the mixed hydrogel-based sensing matrix and modify it on the ITO surface. After polymerization under ultraviolet light at a wavelength of 365 nm for 25 s, perform electrochemical testing to obtain the corresponding current value I. MB and I K3[Fe(CN)6] Then I MB / I K3[Fe(CN)6] Substituting the values ​​into the constructed standard curve, the concentration of AFB1 in the sample was detected. Specific analytical results are shown in Table 1.

[0060] Table 1. Detection of AFB1 in samples using the constructed electrochemical sensing method and the standard method UPLC-FL

[0061]

[0062]

[0063] The sensing method of this invention was used to analyze different concentrations of AFB1 in actual peanut samples, soil, and moldy peanuts, with recoveries ranging from 75.8% to 106%. Compared with the national standard method HPLC-FL (105%–129%), it can be seen that the photoexcitation ratio electrochemical sensor proposed in this invention has high reliability and accuracy for the analysis of AFB1 in actual peanut samples, soil, and moldy peanuts.

[0064] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. An electrochemical sensing method for detecting the hydrogel-confined photoexcitation ratio of AFB1, characterized in that, The steps are as follows: (1) Polyethylene glycol, polyethylene glycol diacrylate, 2-hydroxy-2-methylphenylacetone, acrylate polyethylene glycol carboxyl groups and ultrapure water are mixed evenly in a certain proportion to form a hydrogel matrix; (2) After heating the HAuCl4 solution to boiling, add Na3C6H5O7·2H2O. After reacting for a period of time, the solution turns bright red, which is the gold nanoparticle solution, denoted as AuNPs solution. Then, mix the AuNPs solution with the hydrogel matrix synthesized in step (1) to obtain the hydrogel mixed matrix. (3) After mixing the aptamer solution of the target AFB1 with the cDNA solution, PCR reaction was performed to obtain double-stranded DNA; then the double-stranded DNA was mixed with MB solution to obtain a mixed solution, and finally mixed with the hydrogel mixed matrix of step (2) to obtain a hydrogel-based sensing matrix. (4) First, prepare an AFB1 standard solution, and then mix it with the hydrogel-based sensing matrix prepared in step (3). The resulting mixture is incubated at room temperature, and after incubation, it is modified on the ITO surface. After UV photopolymerization, a ratio electrochemical sensor based on the spatial confinement of hydrogel is obtained. (5) Add potassium ferricyanide solution to PBS buffer solution as a reference probe to form an electrolyte solution; (6) Constructing a standard curve: Using the hydrogel-based ratiometric electrochemical sensor obtained in step (4) as the working electrode, saturated Ag / AgCl as the reference electrode, and a platinum wire electrode as the counter electrode, an external light source was introduced to excite the electrochemical sensing interface, and electrochemical tests were performed in the electrolyte solution during the excitation process. Two electrochemical signals were obtained for each concentration of AFB1 standard solution modified electrode, denoted as I. MB and I K3[Fe(CN)6] A standard curve was constructed based on the ratio of the two obtained current values ​​and the logarithmic relationship of AFB1 concentration. (7) Detection of AFB1 in the sample: First, obtain the sample solution, and follow the steps (4)-(6), except that the AFB1 standard solution is replaced with the sample solution; finally, I is obtained by electrochemical testing. MB and I K3[Fe(CN)6] Substituting the ratio of the two into the standard curve constructed in step (6), the concentration of AFB1 in the sample can be obtained, thus realizing the detection of AFB1 in unknown samples.

2. The electrochemical sensing method for detecting the hydrogel-confined photoexcitation ratio of AFB1 according to claim 1, characterized in that, In step (1), the amounts of polyethylene glycol, polyethylene glycol diacrylate, 2-hydroxy-2-methylphenylacetone, acrylate polyethylene glycol carboxyl groups, and ultrapure water are in the following ratio: 2 mL: 2 mL: 0.5 mL: 0.5 mL: 1 mL; wherein the molecular weight of polyethylene glycol is 200 and its purity is greater than 90%; the molecular weight of polyethylene glycol diacrylate is 700 and its purity is greater than 90%; the molecular weight of 2-hydroxy-2-methylphenylacetone is 200 and its purity is 97%; and the concentration of acrylate polyethylene glycol carboxyl groups is 20 mM and its purity is greater than 90%.

3. The electrochemical sensing method for detecting the hydrogel-confined photoexcitation ratio of AFB1 according to claim 1, characterized in that, In step (2), the concentration of HAuCl4 solution is 0.4 mM, and the concentration of Na3C6H5O7·2H2O is 100 mg / mL. -1 The ratio of HAuCl4 solution to Na3C6H5O7·2H2O is 25.2 mL: 0.25 mL.

4. The electrochemical sensing method for detecting the hydrogel-confined photoexcitation ratio of AFB1 according to claim 1, characterized in that, In step (2), the volume ratio of the hydrogel matrix to the AuNPs solution is 3:1; the concentration of the AuNPs solution is 5.45 nM.

5. The electrochemical sensing method for detecting the hydrogel-confined photoexcitation ratio of AFB1 according to claim 1, characterized in that, In step (3), the concentration of double-stranded DNA is 4 µM; the concentration of MB solution is 400 µM; and the volume ratio of hydrogel, MB solution and double-stranded DNA in the hydrogel-based sensing matrix is ​​4:1:

2.

6. The electrochemical sensing method for detecting the hydrogel-confined photoexcitation ratio of AFB1 according to claim 1, characterized in that, In step (4), the concentration of the AFB1 standard solution is 0.001~1000 ng / mL. -1 The incubation time is 1.0~1.4 h.

7. The electrochemical sensing method for detecting the hydrogel-confined photoexcitation ratio of AFB1 according to claim 1, characterized in that, In step (4), the amount of the mixed solution used for modification is 10-15 µL; the volume ratio of the AFB1 standard solution to the hydrogel-based sensing matrix is ​​1:7; the UV polymerization operation is as follows: the UV polymerization time is 25 s at a wavelength of 365 nm.

8. The electrochemical sensing method for detecting the hydrogel-confined photoexcitation ratio of AFB1 according to claim 1, characterized in that, The concentration of K3[Fe(CN)6] in the electrolyte solution in step (5) is 0.01 mM; the PBS buffer solution is 0.1 M with pH=7.

4.

9. The electrochemical sensing method for detecting the hydrogel-confined photoexcitation ratio of AFB1 according to claim 1, characterized in that, The electrochemical test described in step (6) was recorded and detected by an electrochemical workstation of model CHI1040C; the scanning voltage range was -0.4~0.4 V, the amplitude was 0.025 V, and the frequency was 25 Hz.

10. The electrochemical sensing method for detecting the hydrogel-confined photoexcitation ratio of AFB1 according to claim 1, characterized in that, The conditions for excitation by the external light source are: power of 500 mW / cm². 2 The vertical distance from the working electrode is 3~5 cm.

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