A kind of MOF-818 nanometer enzyme-based electrochemical biosensor, detection method and application thereof

Abstract

The present application relates to the technical field of analytical detection, and particularly relates to an electrochemical biosensor based on MOF-818 nanoscale enzyme, a detection method and application thereof.In the present application, glucose oxidase is introduced to the surface of a magnetic ball through catalytic hairpin self-assembly and hybridization chain reaction induced by target aflatoxin B1, H2O2 is generated in the presence of glucose, and the detection of AFB1 is realized by detecting the reduction current generated by MOF-818 modified screen-printed electrode on the surface of H2O2.Combination of catalytic hairpin self-assembly and hybridization chain reaction realizes double amplification of the signal, and the excellent electrocatalytic performance of MOF-818 to H2O2 further amplifies the detection signal.Meanwhile, the introduction of magnetic separation technology realizes efficient enrichment of the probe, and significantly reduces the background interference.Therefore, the constructed sensor has high sensitivity and wide linear range, and can realize the detection of aflatoxin B1 in actual samples.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, specifically to an electrochemical biosensor based on MOF-818 nanozymes, a detection method, and its applications. Background Technology

[0002] Aflatoxins are secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus, and are highly toxic and carcinogenic mycotoxins. Aflatoxins mainly include group B (B1 and B2), group G (G1 and G2), and group M (M1 and M2), among which aflatoxin B1 (AFB1) is the most toxic and is classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC). Aflatoxin B1 has an extremely stable chemical structure and is difficult to destroy by conventional high-temperature cooking. Even at low levels of exposure, aflatoxin B1 can cause serious liver toxicity, gene mutations, teratogenicity, and immunosuppression, posing a significant threat to human and public health.

[0003] Currently, the main methods for detecting aflatoxin B1 include instrumental methods such as liquid chromatography and liquid chromatography-tandem mass spectrometry, as well as immunoassay methods such as colloidal gold immunochromatography, fluorescence immunoassay, and enzyme-linked immunosorbent assay (ELISA). While instrumental methods are sensitive and accurate, the equipment is expensive, time-consuming, and requires highly skilled operators, making them unsuitable for screening large numbers of samples. Colloidal gold is low-cost and convenient to use, but it can only detect qualitatively, not quantitatively. Fluorescence immunoassay, while capable of quantitative detection, suffers from unresolved non-specific staining issues, resulting in insufficient objectivity in result interpretation and a relatively complex procedure. ELISA, on the other hand, has low precision and poor repeatability. Summary of the Invention

[0004] To overcome the above problems, the present invention provides an electrochemical biosensor based on MOF-818 nanozymes, a detection method, and its application.

[0005] To achieve the above technical objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides an electrochemical biosensor based on MOF-818 nanozymes, comprising: Arched probe Apt / S1, hairpin probe HP1, hairpin probe HP2, hairpin probe HP3, hairpin probe HP4, screen-printed electrode loaded with MOF-818 nanozyme, and glucose solution, which are composed of aptamer strand Apt and primer probe S1 hybridization combination. The hairpin probe HP1 is mounted on a magnetic ball. The 3' end of hairpin probe HP3 and the 5' end of hairpin probe HP4 are modified with glucose oxidase (GOx). The nucleic acid sequence of the aptamer strand Apt is shown in SEQ ID NO: 1; The nucleic acid sequence of primer probe S1 is shown in SEQ ID NO: 2; The nucleic acid sequence of the hairpin probe HP1 is shown in SEQ ID NO: 3; The nucleic acid sequence of the hairpin probe HP2 is shown in SEQ ID NO: 4; The nucleic acid sequence of the hairpin probe HP3 is shown in SEQ ID NO: 5; The nucleic acid sequence of the hairpin probe HP4 is shown in SEQ ID NO: 6.

[0006] In one or more embodiments, the electrochemical biosensor based on MOF-818 nanozymes also includes a Tris-HCl buffer solution.

[0007] In one or more embodiments, the molar ratio of the arched probe Apt / S1, which is a hybrid combination of aptamer strand Apt and primer probe S1, to hairpin probes HP1, HP2, HP3 and HP4 is (0.9~1.1):(0.75~0.85):(0.9~1.1):(1.8~2.2):(1.8~2.2), preferably (1∶0.8∶1∶2∶2).

[0008] In one or more embodiments, the concentration of the arched probe Apt / S1, which is a hybrid combination of the aptamer strand Apt and the primer probe S1, is 0.8~1.2 μM.

[0009] In one or more embodiments, the method for preparing a screen-printed electrode loaded with MOF-818 nanozymes includes: The MOF-818 nanoenzyme aqueous solution is dropped onto the surface of the screen-printed electrode and dried.

[0010] Preferably, the concentration of the MOF-818 nanozyme aqueous solution is 0.8~1.2 g / L.

[0011] In one or more embodiments, the solvent in the glucose solution is a phosphate buffer (PBS) solution, and the pH of the PBS buffer solution is 5-6, preferably 6; The concentration of the glucose solution is 8-12 mmol / L.

[0012] In a second aspect, the present invention provides an electrochemical biosensor based on MOF-818 nanozyme described in the first aspect for the detection of aflatoxin B1.

[0013] A third aspect of the present invention provides a method for detecting aflatoxin B1, comprising the following steps: The sample to be tested was mixed with the arched probe Apt / S1 solution and incubated for the first time. Then, hairpin probes HP1, HP2, HP3 and HP4 were added for the second incubation. Magnetic separation was performed, the precipitate was collected, and then the precipitate was dispersed in a glucose solution. After the reaction, the test solution was obtained. The test droplet was added to a screen-printed electrode loaded with MOF-818 nanozyme, and a current-time (it) curve test was performed. The concentration of aflatoxin B1 in the test sample was obtained based on the standard curve of aflatoxin B1 concentration versus current intensity.

[0014] In one or more embodiments, the solvent in the arched probe Apt / S1 solution is a Tris-HCl buffer solution.

[0015] In one or more embodiments, the temperature for the first incubation is 35-40°C and the time is 45-60 min, preferably 37°C and 50 min.

[0016] In one or more embodiments, the temperature for the second incubation is 35-40°C and the time is 45-60 min, preferably 37°C and 50 min.

[0017] In one or more embodiments, the reaction temperature is 33~37°C and the time is 20~45 min, preferably 35°C and 30 min.

[0018] In one or more embodiments, when performing a current-time curve (it) test, the potential is -0.5 to -0.7V and the time is 90 to 120 s.

[0019] The beneficial effects of this invention are as follows: In this invention, glucose oxidase is introduced onto the surface of a magnetic sphere via catalytic hairpin self-assembly (CHA) and hybridization chain reaction (HCR) induced by the target aflatoxin B1. In the presence of glucose, H2O2 is generated, and AFB1 is detected by detecting the reduction current generated by H2O2 on the surface of a screen-printed electrode modified with MOF-818. The nucleic acid amplification strategy combining catalytic hairpin self-assembly and hybridization chain reaction achieves dual signal amplification. The excellent electrocatalytic performance of MOF-818 for H2O2 further amplifies the detection signal. Simultaneously, the introduction of magnetic separation technology enables efficient probe enrichment, significantly reducing background interference. Therefore, the constructed sensor exhibits high sensitivity and a wide linear range, enabling the detection of aflatoxin B1 in real samples. Attached Figure Description

[0020] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1 The design principle of an electrochemical biosensor based on MOF-818 nanozymes is given, where A represents the design principle of the electrochemical biosensor based on MOF-818 nanozymes, B represents the synthesis process of MOF-818, and C represents the detection process of the electrochemical biosensor based on MOF-818 nanozymes. Figure 2 The following are characterizations of MOF-818, where A is the SEM image of MOF-818, B is the TEM image of MOF-818, C is the FT-IR spectrum of MOF-818, and D is the XRD spectrum of MOF-818. Figure 3 The above are XPS spectra of MOF-818, where A is the total spectrum, B is the C1s spectrum, C is the N1s spectrum, D is the O1s spectrum, E is the Cu2p spectrum, and F is the Zr3d spectrum. Figure 4 Cyclic voltammograms of H2O2 on bare screen-printed electrodes and MOF-818 / screen-printed electrodes are shown. In A, the cyclic voltammogram of H2O2 on the bare screen-printed electrode is shown, and in B, the cyclic voltammogram of H2O2 on the MOF-818 / screen-printed electrode is shown. In A and B, a represents PBS without H2O2 (pH=7.40); b represents PBS containing 5 mM H2O2 (pH=7.40). Figure 5 The cyclic voltammetry behavior of the MOF-818 / screen-printed electrode in the presence of glucose and glucose oxidase is shown. In this diagram, A is the CV curve of the MOF-818 / screen-printed electrode in different solutions, and B is the CV curve of the MOF-818 / screen-printed electrode in a buffer solution containing glucose and glucose oxidase at different pH values. Figure 6 The CV and it curves of the MOF-818 / screen-printed electrode in different solutions are shown, where A is the CV of the MOF-818 / screen-printed electrode in different solutions and B is the it curve of the MOF-818 / screen-printed electrode in different solutions. Figure 7The image shows the it response curves of MOF-818 in 0.1 PBS solution (pH=6.0). In this diagram, A represents the response to different glucose concentrations at a fixed GOx concentration (concentrations from top to bottom: 0, 2, 4, 6, 8, and 10 mM), B represents the response to different GOx concentrations at a fixed glucose concentration (concentrations from top to bottom: 0, 0.125, 0.250, and 0.500 mg / mL), and C represents the feasibility verification of the sensing technology (AFB1 concentrations from top to bottom: 0, 100 pg / mL, and 100 ng / mL). Figure 8 The effect of different signal amplification strategies on the measurement is shown in the figures: a) no CHA and HCR amplification strategies, b) only CHA amplification strategy, and c) both CHA and HCR strategies are present (target concentration is 100 pg / mL). Figure 9 To optimize the reaction conditions for detection using an electrochemical biosensor based on MOF-818 nanozymes, A is the optimization of the incubation time for the arched probes Apt / S1 and AFB1, and B is the optimization of the incubation time for HP1-HP4. Figure 10 The results of AFB1 detection by an electrochemical biosensor based on MOF-818 nanozyme are shown. In this figure, A is the it curve at different concentrations of AFB1 (0~100 ng / mL), and B is the linear curve between current intensity and lgC AFB1. Figure 11 The results show the selectivity, reproducibility, and stability of the electrochemical biosensor based on MOF-818 nanozymes. In this table, A represents selectivity, B represents reproducibility (CAFB1 = 1 ng / mL), and C represents long-term stability. Detailed Implementation

[0022] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0023] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0024] Currently, the main methods for detecting aflatoxin B1 include instrumental methods such as liquid chromatography and liquid chromatography-tandem mass spectrometry, as well as immunoassay methods such as colloidal gold immunochromatography, fluorescence immunoassay, and enzyme-linked immunosorbent assay (ELISA). While instrumental methods are sensitive and accurate, the equipment is expensive, time-consuming, and requires highly skilled operators, making them unsuitable for screening large numbers of samples. Colloidal gold is low-cost and convenient to use, but it can only detect qualitatively, not quantitatively. Fluorescence immunoassay, while capable of quantitative detection, suffers from unresolved non-specific staining issues, resulting in insufficient objectivity in result interpretation and a relatively complex procedure. ELISA, on the other hand, has low precision and poor repeatability.

[0025] To overcome the above problems, the present invention provides an electrochemical biosensor based on MOF-818 nanozymes.

[0026] Figure 1 The design principle of the electrochemical biosensor based on MOF-818 nanozymes is as follows: An arched probe Apt / S1, composed of an aptamer strand Apt and a primer probe S1, specifically binds to AFB1 in the presence of the target analyte AFB1, releasing primer S1. S1 triggers a hairpin self-assembly (CHA) reaction between HP1 and HP2 on the magnetic sphere surface, releasing S1 again. S1 then triggers another round of CHA, achieving the first round of signal amplification for the target analyte. Subsequently, the exposed ends of HP2 induce a hybridization chain reaction (HCR) between HP3 and HP4, generating a long double-stranded DNA structure on the magnetic sphere surface. The ends of HP3 and HP4 are modified with glucose oxidase (GOx), immobilizing a large amount of GOx on the magnetic sphere surface, achieving the second round of signal amplification for the target analyte. GOx catalyzes the oxidation of glucose to hydrogen peroxide (H2O2). AFB1 is detected by detecting the reduction current generated by H2O2 on the surface of a screen-printed electrode modified with MOF-818. This invention employs MOF-818, a metal-organic framework material with peroxidase-like properties. This material exhibits strong catalytic activity in the electrochemical reduction of hydrogen peroxide (H₂O₂), significantly improving detection sensitivity and achieving a third-round signal amplification of the target analyte. Based on aptamer-specific recognition, the constructed sensor demonstrates excellent selectivity for target analyte detection. Furthermore, the target-induced catalytic hairpin self-assembly and hybridization chain reaction, combined with the catalytic performance of MOF-818, constitute a triple signal amplification strategy that enables highly sensitive detection of the target analyte.

[0027] The electrochemical biosensor based on MOF-818 nanozyme provided by this invention exhibits a good linear relationship between the response current intensity and the logarithm (lgC) of AFB1 concentration within a concentration range of 10 pg / mL to 100 ng / mL. The standard curve based on aflatoxin B1 concentration and current intensity is y = 2.18x + 12.48 (where y is the current intensity in μA and x is lgC). AFB1 / ng•mL -1 The correlation coefficient (R) was 0.997, and the limit of detection (LOD) was 6.4 pg / mL.

[0028] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0029] Medicines and reagents Zirconium oxychloride octahydrate (ZrOCl2•8H2O), copper nitrate trihydrate (Cu(NO3)2•3H2O), tris(hydroxymethyl)aminomethane (Tris), β-d-glucose, glucose oxidase (GOx), tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP), and sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester (Sulfo-SMCC) were purchased from Maclean Biotechnology Co., Ltd. (Shanghai, China). Trifluoroacetic acid, zeaxanthin (derived from *Fusarium graminearum*), and Tween 20 were purchased from Aladdin Biotechnology Co., Ltd. (Shanghai, China). Ochratoxin A (OTA) was purchased from Tanmo Quality Inspection Technology Co., Ltd. (Changzhou, China). Aflatoxin B2 (AFB2) solution standard in acetonitrile and T-2 toxin solution standard in acetonitrile were purchased from Northern Weiye Metrology Group Co., Ltd. (Beijing, China). 1H-pyrazole-4-carboxylic acid (H2PyC) was purchased from Dipo Biotechnology Co., Ltd. (Shanghai, China). Streptavidin magnetic beads (MB) were purchased from Titan Technology Co., Ltd. (Shanghai, China). N,N-dimethylformamide (DMF), ethylenediaminetetraacetic acid (EDTA), acetone, disodium hydrogen phosphate (Na₂HPO₄), sodium dihydrogen phosphate (NaH₂PO₄), sodium chloride (NaCl), and magnesium chloride (MgCl₂) were purchased from Tianjin Chemical Reagent Co., Ltd. (Tianjin, China). All reagents were of analytical grade and required no further purification before use. All solutions were prepared using ultrapure water from a Milli-Q water purification system (resistivity 18.2 MΩ).

[0030] All oligonucleotide sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Fengzhifang cornmeal was purchased from Squirrel Convenience Store (Liaocheng, China).

[0031] All oligostranded DNAs were centrifuged at 4000 r / min for 1 min (this step is to centrifuge the powder on the test tube wall to the bottom), diluted to a specific concentration with 10 mM Tris-HCl buffer (containing 100 mM NaCl and 10 mM MgCl2, pH=7.4), and stored at 4°C. Thioylated HP3 and HP4 were reduced in Tris-HCl buffer containing 1 mM TCEP to cleave disulfide bonds.

[0032] Example 1 1.1 Synthesis of MOF-818 85.0 mg ZrOCl2·8H2O, 62.0 mg Cu(NO3)2·3H2O, and 65.0 mg H2PyC were dispersed in 20 mL DMF and ultrasonically dispersed for 5 min to ensure that all solid raw materials were fully dissolved and uniformly dispersed. 240 µL of trifluoroacetic acid was slowly added dropwise to the mixed solution, and the temperature was raised to 100°C. After reacting at this temperature for 10 h, 62.0 mg Cu(NO3)2·3H2O was added again, and the reaction was carried out at 100°C for another 10 h. The product was washed three times by centrifugation with DMF and acetone, and then dried in a vacuum drying oven at 60°C for 12 h to obtain the final product MOF-818.

[0033] The synthesized MOF-818 was dispersed in ultrapure water to prepare a solution with a concentration of 1 mg / mL, and then sonicated to ensure uniform dispersion before use.

[0034] 1.2 Preparation of the arched probe Apt / S1, which is composed of the aptamer strand Apt and the primer probe S1 hybridization combination: After mixing Apt and S1 in a molar ratio of 1.5:1, the mixture was heated at 95°C for 5 min and then slowly cooled at room temperature to obtain an arched probe Apt / S1.

[0035] 1.3 Fabrication of magnetic beads (HP1-MB) loaded with hairpin probe HP1: 500 μL of 1 μM HP1 was added to 100 μL of a solution containing streptavidin magnetic beads, and the mixture was rotated and mixed at room temperature for 30 min. After magnetic separation, the sediment was dissolved in 500 μL of 10 mM Tris-HCl buffer solution (containing 100 mM NaCl and 10 mM MgCl2, pH=7.4) to obtain magnetic beads loaded with hairpin probe HP1 (HP1-MB).

[0036] 1.4 Preparation of HP3-GOx and HP4-GOx: Mix 80 μL of 4 mg / mL sulfo-SMCC, 160 μL of 20 mg / mL GOx, and 760 μL of 0.1M PBS solution (pH=7.40) and incubate at room temperature for 1 h. Centrifuge for 10 min using a 30 kDA ultrafiltration tube to remove unreacted material. Transfer the activated GOx to a new tube and add 96 μL of 10 μM HP3, incubate at room temperature for 2 days. Centrifuge using a 30 kDA ultrafiltration tube to remove excess DNA. Dilute the separated solution to 480 μL to obtain a 2 μM HP3-GOx solution.

[0037] The preparation of HP4-GOx solution is the same as that of HP3-GOx, except that HP3 is replaced with HP4 of equal concentration and volume in the above steps.

[0038] 1.5 Construction of an electrochemical biosensor based on MOF-818 nanozymes: First, 20 μL of 1.0 μM arched probe Apt / S1 was incubated with 20 μL of AFB1 at different concentrations (0.01–100 ng / mL) at 37 °C for 50 min. Then, 20 μL of 0.8 μM HP1-MB, 20 μL of 1.0 μM HP2, 20 μL of 2.0 μM HP3-GOx, and 20 μL of 2.0 μM HP4-GOx were added, and the mixtures were incubated at 37 °C for 50 min. After magnetic separation and washing three times, the precipitate (HP3-GOx / HP4-GOx)n-HP1-MB was obtained. The precipitate was dispersed in 120 μL of 10 mM glucose solution and reacted at 35 °C for 0.5 h. The supernatant was then used for electrochemical detection.

[0039] 20 μL of a 1 mg / mL MOF-818 aqueous solution was added to a screen-printed electrode (SPCE). After natural drying, the supernatant from the previous incubation with added glucose was dropped onto the electrode for current-time (it) curve testing. The parameters were set as follows: potential -0.6 V, time 100 s.

[0040] Example 2 The synthesized MOF-818 material was characterized using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Figure 2 As shown in Figures A and B, MOF-818 exhibits an octahedral structure with a relatively uniform particle size distribution. Figure 2 The middle C is the Fourier transform infrared spectrum (FT-IR). 1659 cm⁻¹ -1 The strong absorption peaks at the left and right correspond to the carbonyl symmetric stretching vibration, 1556 cm⁻¹. -1 The absorption peak at 1294 cm⁻¹ corresponds to the C=N stretching vibration of the pyrazole ring.-1 The absorption peak at 661 cm⁻¹ is attributed to the pyrazole ring skeletal vibration and CN / CO stretching vibration. -1 The absorption peak at that location is attributed to the O-Zr-O bond. To investigate the structural characteristics of the prepared MOF-818 material, X-ray diffraction (XRD) was used to analyze its crystallinity. According to Bragg's law (2dsinθ=nλ), the long-range ordered arrangement of atoms or molecules within the material causes strong coherent scattering of incident X-rays. Figure 2 As shown in Figure D, the diffraction peaks of MOF-818 appear at 5.94° and 6.84°, corresponding to the (222) and (400) crystal planes, respectively, which is consistent with previous reports. The X-ray diffraction (XRD) pattern shows sharp and high-intensity characteristic diffraction peaks, indicating that the synthesized MOF-818 material has a highly ordered porous framework structure and good crystallinity.

[0041] X-ray photoelectron spectroscopy (XPS) is used to characterize the elemental composition and chemical state of materials. Figure 3 The XPS spectra indicate that the synthesized MOF-818 contains carbon (C), nitrogen (N), oxygen (O), fluorine (F), zirconium (Zr) and copper (Cu). Figure 3 The C 1s spectrum in B indicates that carbon exists primarily in three forms. The characteristic peak at 284.8 eV is attributed to C=C or C=C bonds, the peak at 289.1 eV is due to the presence of C=O bonds, and the peak at 292.6 eV corresponds to CN or C=N bonds on the pyrazole ring. Figure 3 In the N1s spectrum of C, the characteristic peak at a binding energy of 400.3 eV confirms the formation of N-Cu coordination bonds. In Zr... 3d In the spectrum, the characteristic peaks at 183.0 eV and 185.4 eV correspond to Zr 3d 5 / 2 and Zr 3d 3 / 2 The spin orbital splitting peak indicates that zirconium in MOF-818 is in the +4 valence (Zr). 4+ ).exist Figure 3 The Cu 2p spectrum in the middle E region shows that the fitted peaks at 934.8 eV and 954.8 eV belong to Cu. 2+ A characteristic peak was also observed at 933.5 eV, indicating the presence of Cu in the system. + In summary, the copper in the prepared MOF-818 material is simultaneously expressed as Cu. + and Cu 2+ The two valence states coexist.

[0042] Example 3 Characterization of the catalytic performance of MOF-818 for H2O2: Figure 4 Cyclic voltammograms of H2O2 on bare screen-printed electrodes and MOF-818 / screen-printed electrodes. Figure 4 As can be seen from Figure A, no obvious reduction peak of H2O2 appeared on the bare electrode. This is attributed to the low conductivity of the screen-printed electrode, which slows down the electron transfer kinetics of the H2O2 reduction reaction. When MOF-818 was modified onto the screen-printed electrode, a pair of obvious redox peaks appeared in a PBS solution at pH 7.40. This pair of redox peaks should be attributed to the Cu in MOF-818. 2+ / Cu + Redox reaction of the redox couple. After the addition of H2O2, a reduction peak of H2O2 appeared at around -0.24 V, and the reduction current was much larger than that at the bare electrode. The results indicate that MOF-818 has a strong catalytic performance for the electrochemical reduction reaction of H2O2, which is attributed to the good conductivity and large specific surface area of ​​MOF-818. The catalytic mechanism may be that Cu(II) in MOF-818 gains electrons from the electrode surface to generate Cu(I), and Cu(I) is then oxidized to Cu(II) by H2O2, while H2O2 gains electrons to generate H2O.

[0043] This invention modifies glucose oxidase onto the surface of magnetic spheres through target-induced catalytic hairpin self-assembly and a hybridization chain reaction. Based on the catalytic effect of glucose oxidase on the oxidation of glucose to H2O2, the detection of the target AFB1 is transformed into the detection of H2O2. To demonstrate the feasibility of this concept, the cyclic voltammetric behavior of glucose and glucose oxidase in coexistence was investigated. Figure 5 As shown in Figure A, the reduction current intensity in the buffer solution containing only glucose showed no significant change compared to the current measured in the blank buffer solution. However, the reduction current significantly increased after the addition of glucose oxidase. This indicates that no H₂O₂ is produced in the absence of glucose oxidase. Only in the presence of glucose oxidase can H₂O₂ be produced, resulting in a significant electrochemical response signal. Furthermore, the effect of solution pH on the catalytic performance of this system was investigated. Figure 5 As shown in Figure B, solution acidity has a significant impact on the electrochemical response of the system. When the solution pH increases from 4 to 6, the reduction current gradually increases, and then gradually decreases with further increases in pH. This may be because glucose oxidase is inactivated and H₂O₂ decomposes in neutral and weakly alkaline solutions. Therefore, in subsequent experiments, PBS with pH=6 was selected as the optimal acidity for the reaction of glucose with GOx.

[0044] In a solution at pH 6.0, the CV curves measured in PBS solution containing only glucose almost overlapped with those measured in blank PBS solution. However, in the presence of glucose oxidase, the reduction current was significantly enhanced. Furthermore, the current response of MOF / SPE in different solutions was measured using the current-time (it) method. Figure 6 As shown in Figure B, the current only changes significantly after the addition of glucose oxidase, a result consistent with cyclic voltammetry. However, cyclic voltammetry is not conducive to direct observation and analysis of the target analyte concentration. Therefore, the electrochemical characterization method of IT was used to replace cyclic voltammetry to demonstrate that the MOF-818 material only shows significant changes in the presence of both glucose oxidase and glucose solution, thus providing a basis for quantitative analysis.

[0045] To further investigate the catalytic performance of MOF-818 on the H2O2 generated from the reaction of glucose with glucose oxidase, its electrochemical characterization was performed using the IT method. Figure 7 As shown in Figure A, under the condition of a fixed GOx concentration, as the glucose concentration increases, the concentration of H2O2 generated in the reaction increases, and the it response current shows an increasing trend. Similarly, as... Figure 7 As shown in Figure B, when the glucose concentration is fixed, increasing the GOx concentration results in a positive correlation between the electrochemical response signal and the GOx concentration. These results demonstrate the catalytic performance of MOF-818 in the generation of H2O2 in this system, and the it electrochemical signal is correlated with glucose and glucose oxidase. Based on this, a sensor was constructed using a designed DNA sequence, and its feasibility was tested. Figure 7 The results of the C-test showed that the negative current value increased with the increase of AFB1 concentration. This indicates that AFB1 successfully triggered the expected DNA reaction and regulated the GOx loading at the electrode interface, proving the feasibility of the sensing.

[0046] Example 4 In this invention, besides MOF-818 modifying the electrode to improve detection sensitivity ( Figure 4 In addition to the above, catalytic hairpin self-assembly (CHA) and hybridization chain reaction (HCR) were introduced. The combination of these two signal amplification strategies significantly improved the detection sensitivity. To demonstrate the synergistic amplification effect, the electrochemical responses under different signal amplification strategies were measured. For comparison, the S1 chain and HP2 were modified with glucose oxidase, respectively, for verification. Figure 8As shown, the measured electrochemical signal is weak when CHA and HCR are absent (curve a). The current intensity increases when CHA is introduced (curve b). The current intensity increases significantly when both CHA and HCR are present (curve c). The experimental results indicate that the synergistic amplification strategy of CHA and CHR can significantly enhance the electrochemical response, thus improving the detection sensitivity.

[0047] Example 5 Optimization of reaction conditions for detection using an electrochemical biosensor based on MOF-818 nanozymes: To achieve better response results from the experimental sensor, the experimental conditions were optimized. Figure 9 Figure A shows the optimization of the incubation time for the arched probes Apt / S1 and AFB1. As can be seen from the figure, the incubation time has a significant impact on the electrochemical signal of the sensor. Short incubation times result in insufficient time for the AFB1 aptamer to react with AFB1, leading to less released S1 and thus less GOx binding, resulting in a lower signal. With increasing incubation time, the electrochemical signal gradually increases, but tends to decrease when the incubation time exceeds 50 min, possibly due to reduced DNA activity. Therefore, 50 min was chosen as the optimal condition for the reaction between the arched probes Apt / S1 and AFB1. Figure 9 Figure B represents the optimized incubation time for HP1-HP4. As shown in the figure, the reaction is not fully completed in the shortest time, resulting in a low electrochemical signal. The reaction efficiency improves over time, reflected in an increase in the it signal. However, once the reaction reaches a certain point, the electrochemical changes become less significant, almost reaching a plateau. This may be because the reaction has reached a relative upper limit, and further increasing the reaction time has no effect on increasing the electrochemical signal, but rather reduces efficiency. Therefore, 50 min is considered the optimal incubation time for HP1-HP4.

[0048] Example 5 Detection of AFB1 using an electrochemical biosensor based on MOF-818 nanozymes: Under the optimized experimental conditions described in Example 4, the it signals of different concentrations of AFB1 were detected. Figure 10 As shown in Figure A, the negative current in the it curve increases systematically with increasing AFB1 concentration. Experimental results indicate that within the concentration range of 10 pg / mL to 100 ng / mL, the response current intensity exhibits a good linear relationship with the logarithm (lgC) of AFB1 concentration. The standard curve (linear regression equation) based on aflatoxin B1 concentration and current intensity is y = 2.18x + 12.48 (where y is the current intensity in μA and x is lgC). AFB1 / ng•mL -1The correlation coefficient (R) was 0.997, and the limit of detection (LOD) was 6.4 pg / mL. These results indicate that the electrochemical biosensor based on MOF-818 nanozymes exhibits a wider linear range and a lower limit of detection.

[0049] Example 6 Selectivity, reproducibility, and stability of electrochemical biosensors based on MOF-818 nanozymes: When evaluating sensor performance, in addition to linear range, key indicators such as selectivity, repeatability, and anti-interference ability should be comprehensively considered. Figure 11 Figure A presents the evaluation results of the selectivity and anti-interference ability of the MOF-818 nanozyme-based electrochemical biosensor for AFB1. The interfering agents selected in the experiment were T-2 toxin (T-2), aflatoxin B2 (AFB2), zearalenone (ZEN), and ochratoxin A (OTA), each at a concentration 10 times that of AFB1 (i.e., 10 ng / mL). As shown in the figure, only the presence of AFB1 elicited a significant current response; the other interfering agents did not produce significant changes in current signal, and the electrochemical signal was essentially the same as the blank background signal. This indicates that the MOF-818 nanozyme-based electrochemical biosensor exhibits excellent selectivity for AFB1. Figure 11 Figure B presents the reproducibility evaluation results of the sensor. Six parallel determinations of 1 ng / mL AFB1 standard solution were performed using the sensor prepared in the same batch, and the relative standard deviation (RSD) was only 0.98%, indicating that the electrochemical biosensor based on MOF-818 nanozyme has excellent reproducibility. Figure 11 The temporal stability of the sensor was evaluated. The changes in the it signal were continuously monitored during storage at 4°C, with tests conducted on days 3, 6, 9, 12, and 15. The results showed that although the sensor's response current decreased slightly with prolonged storage, it remained generally stable. After 15 days of storage, the sensor still retained 92.30% of its initial signal, confirming that the MOF-818 nanozyme-based electrochemical biosensor exhibits good long-term stability and is suitable for detecting real-world samples.

[0050] Example 7 Testing of actual samples: Using cornmeal as the detection target, the feasibility of an electrochemical biosensor based on MOF-818 nanozyme was tested using a spiking method. The specific steps were as follows: Different concentrations of AFB1 standard solution were added to three 5 g portions of cornmeal, and the mixture was shaken thoroughly to allow the solvent to evaporate and bind to the matrix. 10.0 mL of acetonitrile was added to each centrifuge tube, and the mixture was shaken for 5 min. The tubes were then centrifuged at 5000 rpm for 5 min at room temperature. The supernatant was frozen at -20°C for 3 h, and then centrifuged at 10000 rpm for 5 min. The supernatant was filtered into vials using a 0.22 µm syringe filter, resulting in a final solution volume of 1 mL.

[0051] The detection results of AFB1 in corn flour samples are shown in Table 1.

[0052] Table 1. Detection results of AFB1 in corn flour samples

[0053] Table 1 shows the application performance of the MOF-818 nanozyme-based electrochemical biosensor in complex matrices. The data indicate that the MOF-818 nanozyme-based electrochemical biosensor can accurately quantify AFB1 in samples. Within the test range, the RSD of the test results was less than 5%, and the spiked recovery rate ranged from 93.54% to 108.06%, indicating that the sensor has good reliability and broad application prospects in the detection of complex real-world samples.

[0054] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An electrochemical biosensor based on MOF-818 nanozymes, characterized in that, include: Arched probe Apt / S1, hairpin probe HP1, hairpin probe HP2, hairpin probe HP3, hairpin probe HP4, screen-printed electrode loaded with MOF-818 nanozyme, and glucose solution, which are composed of aptamer strand Apt and primer probe S1 hybridization combination. The hairpin probe HP1 is mounted on a magnetic ball. The 3' end of hairpin probe HP3 and the 5' end of hairpin probe HP4 are modified with glucose oxidase (GOx). The nucleic acid sequence of the aptamer strand Apt is shown in SEQ ID NO: 1; The nucleic acid sequence of primer probe S1 is shown in SEQ ID NO: 2; The nucleic acid sequence of the hairpin probe HP1 is shown in SEQ ID NO: 3; The nucleic acid sequence of the hairpin probe HP2 is shown in SEQ ID NO: 4; The nucleic acid sequence of the hairpin probe HP3 is shown in SEQ ID NO: 5; The nucleic acid sequence of the hairpin probe HP4 is shown in SEQ ID NO:

6.

2. The electrochemical biosensor as described in claim 1, characterized in that, Electrochemical biosensors based on MOF-818 nanozymes also include Tris-HCl buffer solution.

3. The electrochemical biosensor as described in claim 1, characterized in that, The molar ratio of the arched probe Apt / S1, which is a hybridization combination of aptamer strand Apt and primer probe S1, to hairpin probes HP1, HP2, HP3 and HP4 is (0.9~1.1):(0.75~0.85):(0.9~1.1):(1.8~2.2):(1.8~2.2), preferably (1∶0.8∶1∶2∶2). The concentration of the arched probe Apt / S1, which is a hybridization combination of the aptamer strand Apt and the primer probe S1, is 0.8–1.2 μM.

4. The electrochemical biosensor as described in claim 1, characterized in that, The preparation method of screen-printed electrodes loaded with MOF-818 nanozymes includes: Add an aqueous solution of MOF-818 nanoenzyme to the surface of a screen-printed electrode and allow it to dry. Preferably, the concentration of the MOF-818 nanozyme aqueous solution is 0.8~1.2 g / L.

5. The electrochemical biosensor as described in claim 1, characterized in that, The glucose solution is in the form of a phosphate buffer (PBS) solution, and the pH of the PBS buffer solution is 5-6, preferably 6. The concentration of the glucose solution is 8-12 mmol / L.

6. The electrochemical biosensor based on MOF-818 nanozyme according to any one of claims 1 to 5 is applied to the detection of aflatoxin B1.

7. A method for detecting aflatoxin B1, characterized in that, Includes the following steps: The sample to be tested was mixed with the arched probe Apt / S1 solution and incubated for the first time. Then, hairpin probes HP1, HP2, HP3 and HP4 were added for the second incubation. Magnetic separation was performed, the precipitate was collected, and then the precipitate was dispersed in a glucose solution. After the reaction, the test solution was obtained. The test droplet was added to a screen-printed electrode loaded with MOF-818 nanozyme, and a current-time (it) curve test was performed. The concentration of aflatoxin B1 in the test sample was obtained based on the standard curve of aflatoxin B1 concentration versus current intensity.

8. The detection method as described in claim 7, characterized in that, The solvent in the Apt / S1 solution of the arched probe is Tris-HCl buffer solution.

9. The detection method as described in claim 7, characterized in that, The temperature for the first incubation is 35~40℃, and the time is 45~60 min, preferably 37℃ for 50 min; Alternatively, the temperature for the second incubation is 35-40℃ and the time is 45-60 min, preferably 37℃ for 50 min.

10. The detection method as described in claim 7, characterized in that, The reaction temperature is 33~37℃, and the time is 20~45min, preferably 35℃ and 30min; Alternatively, when performing a current-time curve (it) test, the potential is -0.5 to -0.7 V, and the time is 90 to 120 s.

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