Photoelectrochemical biosensor based on bismuth vanadate photoelectrode, construction method and application

Abstract

The invention relates to the technical field of OTA detection, and discloses a photoelectrochemical biosensor based on a bismuth vanadate photoelectrode as well as a construction method and application of the photoelectrochemical biosensor. By integrating a BiVO4 nano-porous photoelectrode with OTA aptamer recognition, CHA / HCR double-chain amplification and ALP enzymatic BCP precipitation signal amplification mechanism, a multi-stage signal amplification PEC adaptive sensor is constructed; the system solves the problems of insufficient sensitivity, selectivity, stability and practicability, and provides a new thought and a new technical platform for OTA ultra-sensitive detection.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of OTA detection, in particular to a photoelectrochemical biosensor based on a bismuth vanadate photoelectrode, a construction method and application. BACKGROUND

[0002] Ochratoxin A (OTA) is a secondary metabolite produced by Aspergillus and Penicillium fungi, which widely pollutes agricultural products such as grains, coffee and wine. About 10% of the agricultural products in the world are threatened by it. It has nephrotoxicity, hepatotoxicity, immunotoxicity, teratogenicity and carcinogenicity, and is listed as a 2B potential carcinogen by IARC. The dihydroisocoumarin and L-phenylalanine residues in its structure make it easy to accumulate in the body. Long-term exposure can cause chronic kidney disease, Balkan endemic nephropathy and urinary system tumors, and may participate in the occurrence of neurodegenerative diseases such as Parkinson's disease by interfering with DNA replication, inducing oxidative stress and mitochondrial dysfunction. Therefore, it is urgent to develop a sensitive, rapid and low-cost detection technology.

[0003] Although traditional HPLC-FLD, LC-MS / MS and ELISA methods are sensitive, they have limitations such as expensive equipment, complex pretreatment and long detection period, and are difficult to meet the needs of on-site rapid screening. Photoelectrochemical (PEC) sensing technology has the advantages of low background interference, high sensitivity, simple equipment and easy integration due to the separation of excitation source (light) and detection signal (current). In recent years, it has attracted widespread attention in the fields of food safety and biological analysis. Among them, visible light responsive n-type semiconductor bismuth vanadate (BiVO4, band gap about 2.4 eV) has become an ideal material for PEC sensing platform due to its excellent light absorption performance, chemical stability, biocompatibility and environmental protection characteristics. However, its poor electrical conductivity and high carrier recombination rate, as well as the lack of signal amplification strategy in existing OTA detection based on BiVO4, limit its detection limit to pg~ng level, and the actual sample verification is insufficient, which makes the operation cumbersome and difficult to realize trace detection and on-site application. SUMMARY

[0004] The application provides a photoelectrochemical biosensor based on a bismuth vanadate photoelectrode, a construction method and application, which solves the problems of insufficient sensitivity, selectivity, stability and practicability by integrating BiVO4 nanoporous photoelectrode, OTA aptamer recognition, CHA / HCR double-stranded amplification and ALP enzyme-catalyzed BCP precipitation signal amplification mechanism, and provides a new idea and new technology platform for OTA super-sensitive detection.

[0005] Technical scheme: In a first aspect, the application provides a construction method of a photoelectrochemical biosensor based on a bismuth vanadate photoelectrode, which comprises the following steps: S1. Preparing a BiVO4 photoelectrode; S2. After the carboxylated magnetic beads are activated by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, the magnetic beads are connected with amino aDNA to obtain magnetic beads carrying amino aDNA; S3. Mixing ochratoxin A aptamer and trigger DNA with PBS buffer to obtain an aptamer-tDNA complex after annealing; S4. Mixing magnetic beads carrying amino aDNA, the aptamer-tDNA complex and ochratoxin A, releasing tDNA after the aptamer recognizes ochratoxin A, and pairing to obtain magnetic beads carrying aDNA-tDNA; then adding hairpin probe Bio-H1 and hairpin probe Bio-H2, mixing, initiating hairpin self-assembly and hybridization chain reaction to form long-chain DNA nanostructure on the surface of the magnetic beads; then adding streptavidin-alkaline phosphatase conjugate to fix alkaline phosphatase on the magnetic beads through biotin-streptavidin reaction; finally adding 5-bromo-4-chloro-3-indole phosphate / nitro blue tetrazolium substrate, incubating, and catalyzing by alkaline phosphatase to generate insoluble precipitate; S5. Dropping the insoluble precipitate on the surface of the BiVO4 photoelectrode to perform photoelectrochemical test; and obtaining the concentration of ochratoxin A in the to-be-tested liquid according to the corresponding relationship between the change of photocurrent signal and the concentration of ochratoxin A.

[0006] Further, in S1, the specific preparation method of the BiVO4 photoelectrode is as follows: taking FTO glass as a substrate, depositing BiOI on the surface of the substrate by electrodeposition to obtain a BiOI electrode; then adding vanoxyl acetylacetone solution on the surface of the BiOI electrode, performing annealing treatment, and cleaning to obtain the BiVO4 photoelectrode.

[0007] Further, the specific conditions of the annealing treatment are as follows: heating at a heating rate of 2-4 ℃ / min to 400-500 ℃ and maintaining for 1-3 hours.

[0008] Further, in S3, the use amount ratio of ochratoxin A aptamer to trigger DNA is 1:1; and the annealing conditions are as follows: annealing at 95-100 ℃ for 3-8 min.

[0009] Further, in S4, the use amount ratio of magnetic beads carrying amino aDNA, the aptamer-tDNA complex and ochratoxin A is 5:5:1.

[0010] Further, in S4, the molar ratio of hairpin probe Bio-H1 to hairpin probe Bio-H2 is 1:1, and the time of hairpin self-assembly and hybridization chain reaction is 1-3 hours.

[0011] Further, in S4, the streptavidin-alkaline phosphatase conjugate is added in an amount of 20-21 μL.

[0012] Preferably, in S4, the incubation is for a time of 20-40 minutes.

[0013] In a second aspect, the present application provides a photoelectrochemical biosensor based on a BiVO4 photoelectrode constructed by any of the above methods.

[0014] In a third aspect, the present application provides the use of a photoelectrochemical biosensor based on a BiVO4 photoelectrode constructed by any of the above methods in the detection of ochratoxin A.

[0015] The present application constructs a PEC adaptive sensor based on a BiVO4 photoelectrode, the core principle of which is to combine photoelectrochemical reaction with biomolecular recognition, to realize ultra-sensitive detection of OTA through a multi-stage signal amplification mechanism.

[0016] BiVO4 is a n-type semiconductor material with good visible light absorption performance and stable photoelectrochemical activity. Under light conditions, BiVO4 absorbs photon energy, exciting electrons to jump from the valence band to the conduction band, generating electron-hole pairs. These photo-generated carriers separate and migrate to the electrode surface under the action of an electric field, participate in redox reactions, and form a stable photocurrent signal. In the present application, the BiVO4 photoelectrode serves as a signal conversion platform, and its surface modification state directly affects the photocurrent intensity. When a biomolecular recognition event occurs on the electrode surface and forms an insoluble precipitate, it will hinder the electron transport path, resulting in a decrease in the photocurrent signal. This signal change is negatively correlated with the concentration of the target OTA, thereby enabling quantitative detection.

[0017] Specifically, the present application uses OTA aptamer as a recognition element, which specifically binds to the target toxin OTA and releases trigger DNA (tDNA), which further activates catalytic hairpin self-assembly (CHA) and hybridization chain reaction (HCR) to form long-chain DNA nanostructures. The structure introduces alkaline phosphatase (ALP) through the biotin-streptavidin system, and ALP catalyzes the substrate BCIP / NBT to form insoluble blue-purple precipitate, which deposits on the surface of the BiVO4 electrode, significantly increasing the interfacial charge transfer resistance and reducing the photocurrent response.

[0018] The detection method of the PEC biosensor based on the BiVO4 photoelectrode constructed by the present application realizes a complete detection chain from molecular recognition to signal conversion to signal amplification, with high sensitivity, high selectivity, and good stability. It realizes the detection of OTA at the fg level.

[0019] Beneficial Effects: This invention provides a PEC biosensor based on a BiVO4 photoelectrode, combining BCP and CHA / HCR signal amplification mechanisms to achieve high sensitivity, high selectivity, and rapid detection of OTA, possessing advantages such as speed, simplicity, and low cost. Compared with existing technologies, the specific beneficial effects are as follows: 1. High sensitivity: The detection limit is as low as 62.02 fg / mL, which is superior to traditional methods; 2. High selectivity: High aptamer recognition specificity and strong anti-interference ability; 3. Low cost: The materials used are inexpensive and the equipment requirements are low; 4. Wide range of applications: Applicable to fields such as food safety and agricultural product supervision. Attached image description:

[0020] Figure 1 This is a schematic diagram of the construction method of the photoelectrochemical biosensor based on bismuth vanadate photoelectrode in this invention; Figure 2 This is a scanning electron microscope (SEM) image of the BiVO4 photoelectrode prepared in this invention; Figure 3 The image shows the XRD pattern of the BiVO4 photoelectrode prepared in this invention. Figure 4 This is a performance analysis diagram of the photoelectrochemical biosensor based on bismuth vanadate photoelectrode constructed in this invention at different concentrations of target analytes. Detailed Implementation

[0021] The present invention will now be described in detail with reference to the embodiments. Implementation method 1:

[0022] This embodiment provides a method for constructing a photoelectrochemical biosensor based on a bismuth vanadate photoelectrode, as detailed below: (1) Preparation of BiVO4 photoelectrode: First, bismuth nitrate pentahydrate (Bi(NO3)3·5H2O) was dissolved in 10 mL of 0.4 M potassium iodide (KI) solution, and the pH was adjusted to 1.7 with nitric acid (HNO3) to obtain a 0.04 M Bi(NO3)3 solution. Then, this solution was mixed with 4 mL of anhydrous ethanol (containing 0.23 M p-benzoquinone) and stirred thoroughly for several minutes.

[0023] Electrodeposition was performed using a three-electrode system: the working electrode was fluorine-doped tin oxide (FTO), the reference electrode was an Ag / AgCl electrode, and the counter electrode was a platinum wire electrode. A constant potential of -0.1 V (vs. Ag / AgCl) was applied at room temperature, and the deposition time was 240 seconds. Under the influence of the cathode potential, p-benzoquinone was reduced to hydroquinone, causing an increase in pH near the working electrode, which promoted the deposition of BiOI crystals on the electrode surface. The electrode was then washed with deionized water and dried.

[0024] Subsequently, 20 μL of a dimethyl sulfoxide (DMSO) solution containing 0.2 M vanadium acetylacetonate (VO(acac)2) was drop-coated onto a BiOI electrode (2 cm × 0.5 cm). The electrode was then heated to 450 °C in a muffle furnace at a heating rate of 2 °C / min and held at that temperature for 2 hours to convert BiOI to BiVO4. Next, the electrode was immersed in a 1 M NaOH solution and stirred for 30 minutes to remove residual V2O5. Finally, it was rinsed with deionized water and dried at room temperature to obtain a pure BiVO4 photoelectrode.

[0025] (2) Biosensor construction: 200 μL of carboxylated magnetic beads (10 mg / mL) were mixed with 700 μL, 0.1 M PBS buffer containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (2.5 mg / mL) and N-hydroxysuccinimide (NHS) (2.5 mg / mL) and reacted at 37 °C for 15 min to activate the carboxyl groups on the magnetic beads. Aminated aDNA (100 μL, 100 μM) was added, and the mixture was shaken at 37 °C for 12 h. After magnetic separation, the resulting magnetic beads carrying aminated DNA (MBs-aDNA) were washed with phosphate-buffered saline (PBS, 0.01 M, pH 7.4), then redispersed in 1 mL PBS buffer and stored at 4 °C for later use.

[0026] 10 μL of OTA aptamer (100 μM) and 10 μL of trigger DNA (100 μM) were added to 80 μL of PBS buffer (0.01 M, pH=7.4). The synthesized solution was annealed at 95 °C for 5 min and then gradually cooled to room temperature, and this solution was denoted as Apt-tDNA.

[0027] 50 μL of MBs-aDNA and 50 μL of Apt-tDNA were injected into centrifuge tubes containing 10 μL of OTA standards at different concentrations, and the mixture was continuously shaken at 37°C for 1 h. After magnetic separation and washing, a mixture containing 50 μL of Bio-H1 (50 μM) and Bio-H1 (50 μM) was added, and the mixture was shaken at 37°C for 2 h for HCR reaction. After magnetic separation, 20 μL of streptavidin-alkaline phosphatase conjugate (SA-ALP) (0.1 mg / mL) was added, and the reaction was carried out for 2 h to immobilize ALP on magnetic beads (ALP-MBs) via the biotinylate reaction. The entire mixture was directly poured into (and the ALP-MBs were collected) and transferred to a solution of 2 mL PBS (0.01 M, pH=7.4) containing 1 mL of 5-bromo-4-chloro-3-indole phosphate / nitroblue tetrazolium (BCIP / NBT) and incubated for 30 min. 10 μL was dropped onto the BiVO4 photoelectrode for PEC testing. The concentration of OTA in the test solution was obtained based on the correlation between the change in photocurrent signal and the OTA concentration.

[0028] A schematic diagram of the construction method of the photoelectrochemical biosensor based on the bismuth vanadate photoelectrode in this embodiment is shown below. Figure 1 As shown, (a) is the BiVO4 photoelectrode preparation process; (b) is the principle of OTA toxin-induced CHA, HCR and subsequent ALP, which realizes the BCP reaction with the help of magnetic separation; (c) is the working principle of constructing a PEC adapter sensor based on the BCP regulation effect.

[0029] In this embodiment, a detection strategy based on enzymatic biocatalytic precipitation (BCP) is employed. High-sensitivity magnetic separation detection of the target anaerobic OTA is achieved through dual amplification via catalytic hairpin self-assembly (CHA) and hybridization chain reaction (HCR). First, MB is linked to amino-coated aDNA, and OTA and the prepared aptamer-tDNA are added. The aptamer specifically recognizes OTA and separates from the tDNA. Complementary pairing results in magnetic beads carrying aDNA-tDNA (MB-aDNA-tDNA). Then, through hybridization chain reaction (HCR), when the target molecule tDNA is present, it binds to the complementary sequence of hairpin H1, opening the hairpin structure of H1 and exposing a new single-stranded region. This newly exposed region then binds to hairpin H2, opening the hairpin structure of H2 and releasing the same sequence as the target molecule, thereby further opening more H1 hairpins. This process is continuously repeated, ultimately forming a long double-stranded DNA nanowire, achieving amplification and improved detection sensitivity. Finally, BCIP (5-bromo-4-chloro-3-indole phosphate) was hydrolyzed under the catalysis of ALP (alkaline phosphatase). The resulting product reacted with NBT (nitroblue tetrazolium) to form an insoluble dark blue to blue-violet BCP precipitate. This precipitate significantly increased the charge transfer resistance at the electrode interface and weakened the light absorption capacity of the material, leading to a significant decrease in the photocurrent signal. The change in photocurrent was negatively correlated with the concentration of OTA, thus achieving highly sensitive quantitative detection of the target analyte.

[0030] The scanning electron microscope (SEM) image of the BiVO4 photoelectrode prepared in this embodiment is shown below. Figure 2 As shown, the monoclinic bismuth vanadate (BVO) photoanode exhibits a clear and uniform nanoporous morphology. Figure 2 a) Its pore size distribution range is relatively narrow, mainly concentrated between 200 and 500 nanometers, showing good structural consistency. Furthermore, from the cross-sectional image ( Figure 2 b) It can be observed that the porous film has a uniform thickness, averaging about 500 nanometers.

[0031] The XRD pattern of the BiVO4 photoelectrode prepared in this embodiment is shown below. Figure 3 As shown in the figure, the 2θ diffraction peaks of bismuth vanadate at 26.8°, 37.8°, 51.7°, 54.4°, 61.7°, and 65.6° correspond to the characteristic diffraction peaks of the FTO substrate. Furthermore, the 2θ diffraction peaks at 19.1°, 29.1°, 30°, 34.7°, 40°, 42.5°, 47.4°, 53.5°, and 58.8° can all be attributed to the monoclinic BVO crystal system (JCPDS standard card number 14-0688).

[0032] Performance analysis of the photoelectrochemical biosensor based on bismuth vanadate photoelectrode constructed in this embodiment at different concentrations of target analytes: Figure 4 (a) The photocurrent variation of the photoelectrochemical biosensor based on bismuth vanadate photoelectrode constructed in this embodiment under different concentrations of OTA can be seen. It can be seen that the current (I) gradually decreases with the increase of the target OTA concentration (from 100 fg / mL to 200 ng / mL). Figure 4 (b) shows the calibration curve between the logarithm of OTA concentration and the photocurrent signal. It can be seen that the photocurrent exhibits a linear relationship with the logarithm of OTA concentration (logc), with the regression equation I = -0.75lgC + 3.55 and R² = 0.9927. The limit of detection (LOD) is 62.02 fg / mL, and S / N = 3. This indicates that the photoelectrochemical biosensor based on a bismuth vanadate photoelectrode constructed in this embodiment has a wide detection range and a low limit of detection, achieving sensitive detection of OTA and demonstrating good PEC analytical performance. Implementation Method 2:

[0033] The photoelectrochemical biosensor based on bismuth vanadate photoelectrode constructed in Implementation Method 1 was used for OTA detection in peanut butter: Considering the extremely low background OTA content in the actual sample matrix, this study used the standard spiking method for spiked recovery experiments to verify the reliability of the method: After extraction, centrifugation, and filtration, peanut butter samples were spiked with different concentrations of OTA standards, and the method in Implementation Method 1 was used for detection; the results are shown in Table 1. Under different spiking levels, the recovery rate of OTA ranged from 98.2% to 105%, and the relative standard deviation (RSD) was between 3.092% and 6.245%.

[0034] The results show that the photoelectrochemical biosensor based on bismuth vanadate photoelectrode constructed in this invention has good accuracy and repeatability in actual samples.

[0035]

[0036] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent transformations or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for constructing a photoelectrochemical biosensor based on a bismuth vanadate photoelectrode, characterized in that, Includes the following steps: S1. Fabrication of BiVO4 photoelectrode; S2. Carboxylated magnetic beads are activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide and then linked with aminoated aDNA to obtain magnetic beads carrying aminoated aDNA. S3. Mix the ochratoxin A aptamer and trigger DNA with PBS buffer and anneal to obtain the aptamer-tDNA complex. S4. Magnetic beads carrying amino-aDNA, aptamer-tDNA complexes, and ochratoxin A are mixed. After the aptamer recognizes ochratoxin A, it releases tDNA, which pairs complementaryly to obtain magnetic beads carrying aDNA-tDNA. Then, hairpin probes Bio-H1 and Bio-H2 are added and mixed to initiate hairpin self-assembly and hybridization chain reaction, forming long-chain DNA nanostructures on the surface of the magnetic beads. Then, streptavidin-alkaline phosphatase conjugate is added, and alkaline phosphatase is immobilized on the magnetic beads through the biotinylate reaction. Finally, 5-bromo-4-chloro-3-indole phosphate / nitroblue tetrazolium substrate is added and incubated. Alkaline phosphatase catalyzes the formation of an insoluble precipitate. S5. The insoluble precipitate is drop-coated onto the surface of the BiVO4 photoelectrode and photoelectrochemical testing is performed; the concentration of ochratoxin A in the test solution is obtained based on the correspondence between the change in photocurrent signal and the concentration of ochratoxin A.

2. The method for constructing a photoelectrochemical biosensor based on a bismuth vanadate photoelectrode according to claim 1, characterized in that: In S1, the specific preparation method of the BiVO4 photoelectrode is as follows: using FTO glass as a substrate, BiOI is deposited on its surface by electrodeposition to obtain a BiOI electrode; then, acetylacetone vanadium oxide solution is dropped onto the surface of the BiOI electrode, followed by annealing and cleaning to obtain the BiVO4 photoelectrode.

3. The method for constructing a photoelectrochemical biosensor based on a bismuth vanadate photoelectrode according to claim 2, characterized in that: The specific conditions for the annealing treatment are: heating to 400-500℃ at a heating rate of 2-4℃ / min and holding at that temperature for 1-3 hours.

4. The method for constructing a photoelectrochemical biosensor based on a bismuth vanadate photoelectrode according to claim 1, characterized in that: In S3, the ratio of ochratoxin A aptamer to trigger DNA is 1:1; the annealing conditions are: annealing at 95-100℃ for 3-8 min.

5. The method for constructing a photoelectrochemical biosensor based on a bismuth vanadate photoelectrode according to claim 1, characterized in that: In S4, the ratio of the magnetic beads carrying amino-aDNA, the aptamer-tDNA complex, and ochratoxin A is 5:5:

1.

6. The method for constructing a photoelectrochemical biosensor based on a bismuth vanadate photoelectrode according to claim 1, characterized in that: In S4, the molar ratio of hairpin probe Bio-H1 to hairpin probe Bio-H2 is 1:1, and the time for hairpin self-assembly and hybridization chain reaction is 1-3 hours.

7. The method for constructing a photoelectrochemical biosensor based on a bismuth vanadate photoelectrode according to claim 1, characterized in that: In S4, the amount of the streptavidin-alkaline phosphatase conjugate added is 20-21 μL.

8. The method for constructing a photoelectrochemical biosensor based on a bismuth vanadate photoelectrode according to claim 1, characterized in that: In S4, the incubation time is 20-40 minutes.

9. A photoelectrochemical biosensor based on a bismuth vanadate photoelectrode, constructed by the method described in any one of claims 1-8.

10. The application of a photoelectrochemical biosensor based on a bismuth vanadate photoelectrode constructed by any one of claims 1-8 in the detection of ochratoxin A.

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