A method for simultaneous detection of AFB1 and OTA using a homogeneous electrochemical-colorimetric dual-mode sensor based on a dual-functional probe.

Abstract

This invention belongs to the field of biosensor technology, specifically relating to a method for simultaneously detecting AFB1 and OTA using a homogeneous electrochemical-colorimetric dual-mode sensor based on bifunctional probes. This invention prepares Hemin@BSA@ZIF-90 nanozymes as bifunctional probes by encapsulating heme chloride and bovine serum albumin in a zeolite imidazole ester backbone-90 using a stirring method. The detection of AFB1 and OTA is designed as two independent homogeneous systems. The target analyte activates the CRISPR / Cas12a system, cleaving the single-stranded DNA connecting the magnetic bead and the probe, releasing the probe into the supernatant. The probe is integrated into a dual-channel electrochemical platform to generate a strong electrical signal. Its peroxidase-like catalytic activity is analyzed using RGB colorimetric analysis via a smartphone. This dual-mode biosensor exhibits high sensitivity and a low detection limit, providing an efficient and universal strategy for the simultaneous detection of fungal toxins.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology, specifically relating to a method for simultaneously detecting AFB1 and OTA using a homogeneous electrochemical-colorimetric dual-mode sensor based on a dual-functional probe. Background Technology

[0002] Agricultural products are susceptible to fungal contamination and the production of mycotoxins during growth, harvesting, storage, and processing. These toxins can be transmitted through the food chain, posing potential harm to human and animal health. In actual contamination scenarios, agricultural products may contain multiple mycotoxins simultaneously, with aflatoxin B1 (AFB1) and ochratoxin A (OTA) being the most common co-contamination combination. Studies have shown that these two mycotoxins have synergistic toxic effects, and even low-dose exposure may increase the risk of cancer. Therefore, developing high-performance sensors for the accurate and simultaneous detection of AFB1 and OTA is crucial.

[0003] Homogeneous electrochemical sensors have received widespread attention in recent years due to their advantages of not requiring complicated electrode modification and signal generation not being limited by the surface space of the electrode. However, in practical applications, achieving simultaneous monitoring of AFB1 and OTA still faces challenges: (1) Current strategies for achieving simultaneous detection still rely on multiple signal probes to generate distinguishable signals, which increases the complexity and cost of sensor fabrication and poses a challenge to selective identification; (2) Single-modal quantitative detection using only electrochemical methods is susceptible to operator differences and non-standard testing procedures, which may reduce the accuracy of the results.

[0004] Therefore, there is an urgent need to simplify the sensor fabrication process through rational design and develop dual-mode biosensors to achieve efficient simultaneous detection. Summary of the Invention

[0005] To address the shortcomings of existing detection methods, this invention provides a method for preparing a homogeneous electrochemical-colorimetric dual-mode sensor based on a bifunctional probe, for the simultaneous detection of aflatoxin B1 and ochratoxin A. Specifically, this invention relates to a homogeneous reaction system that integrates the bifunctional characteristics of ZIF-based nanozymes with the specific recognition and non-specific cleavage properties of CRISPR-Cas12a. The dual-channel homogeneous electrochemical-colorimetric dual-mode sensor constructed based on this system enables accurate and simultaneous quantitative detection of AFB1 and OTA in wheat.

[0006] This invention utilizes the zeolite imidazole ester backbone-90 as a framework to encapsulate heme chloride and bovine serum albumin (Hemin@BSA@ZIF-90) to prepare nanozymes as bifunctional signal probes. Then, Hemin@BSA@ZIF-90 and magnetic beads (MBs) are linked via single-stranded DNA (ssDNA) to form a homogeneous sensing system. In the presence of AFB1 / OTA, the aptamer recognizes and releases activating DNA (acDNA) from the double-stranded DNA (apt-acDNA). This acDNA binds to CRISPR / Cas12a-crRNA to form a Cas12a-crRNA-acDNA ternary complex, activating trans-cleavage capability and cleaving nearby ssDNA, leading to nanozyme release. The probes obtained from the two homogeneous systems are used for dual-channel electrochemical detection, based on the Fe(III) / Fe(II) redox reaction, enabling simultaneous detection of two electrical signals. Simultaneously, Hemin@BSA@ZIF-90, acting as a nanozyme, accelerates the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to oxTMB. Introducing this into a paper-based biochip integrated with a smartphone enables sensitive visual detection. This invention provides a simple, efficient, and universal strategy for the simultaneous detection of multiple mycotoxins.

[0007] The objective of this invention is achieved through the following technical solutions: This invention provides a method for simultaneously detecting AFB1 and OTA using a homogeneous electrochemical-colorimetric dual-mode sensor based on a dual-functional probe, comprising the following steps: (1) Preparation of Hemin@BSA@ZIF-90: First, hemin chloride (Hemin) and bovine serum albumin (BSA) were added to N,N-dimethylformamide (DMF) and stirred for the first time. Then, Zn(CH3COO)2∙2H2O was added and stirring continued. After stirring, a DMF solution containing imidazole-2-carboxaldehyde (2-ICA) was added and stirred for the third time to obtain a mixed solution. The precipitate was collected by centrifugation and washed sequentially with DMF, ethanol, and sodium dodecyl sulfate (SDS) (using SDS to wash away excess BSA). Finally, the precipitate was collected and dried to obtain the dried product, which is Hemin@BSA@ZIF-90. Preferably, in step (1), the amounts of heme chloride, bovine serum albumin, N,N-dimethylformamide and Zn(CH3COO)2∙2H2O, imidazole-2-carboxaldehyde and N,N-dimethylformamide are in the following ratio: 4 mg: 2 mg: 18 mL: 1 mL: 76 mg: 18 mL; the concentration of SDS solution is 1 wt%; the first stirring time and the continued stirring time are both 5 min, and the third stirring time is 15 min; the centrifugation conditions are: 10000 rpm, 10 min; the washing is performed three times each with DMF, ethanol and sodium dodecyl sulfate; the drying temperature is 60℃ and the time is 6~8 h. (2) Preparation of ssDNA-Hemin@BSA@ZIF-90-MBs: (a) Hemin@BSA@ZIF-90 was coupled with thiol-modified single-stranded DNA (ssDNA) to prepare an ssDNA-Hemin@BSA@ZIF-90 mixture, as follows: S1. Add tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) solution to ssDNA solution for reaction (react for a period of time to reduce thiol groups, the purpose of which is to eliminate disulfide bonds between DNA strands), and obtain TCEP-ssDNA solution after the reaction. S2. Simultaneously, the sulfosuccinimide-4-(N-maleimidemethyl)cyclohexane-2-carboxylate (sulfo-SMCC) solution was mixed with PBS buffer containing Hemin@BSA@ZIF-90 for the first incubation. After incubation, the precipitate was collected by centrifugation, washed with PBS buffer, and then resuspended in PBS buffer to obtain a sulfo-SMCC activated Hemin@BSA@ZIF-90 suspension. S3. Subsequently, the TCEP-ssDNA solution and the sulfo-SMCC activated Hemin@BSA@ZIF-90 suspension were mixed and incubated for a second time. After incubation, the conjugates were collected by centrifugation and washed with PBS buffer. Finally, the washed product was resuspended in ultrapure water to obtain the ssDNA-Hemin@BSA@ZIF-90 mixture. Preferably, in step (2) (a), the ratio of the amounts of TCEP solution and ssDNA solution is 10 μL: 150 μL, the concentration of TCEP solution is 30 mM, and the concentration of ssDNA solution is 10 μM. The concentration of the sulfo-SMCC solution was 0.1 mg / mL, and the final concentration of Hemin@BSA@ZIF-90 in PBS buffer was 0.5 mg / mL, where the PBS buffer contained 100 mM sodium chloride and had a pH of 7.4. The volume ratio of the sulfo-SMCC solution to the PBS buffer containing Hemin@BSA@ZIF-90 was 20 μL:200 μL. The first incubation reaction lasted for 1 h at room temperature, with a rotation speed of 10,000 rpm for 5 min. Washing was performed three times. The second incubation lasted for 24 h at room temperature. The concentration of the ssDNA-Hemin@BSA@ZIF-90 mixture obtained after resuspending was 1 mg / mL. (b) The ssDNA-Hemin@BSA@ZIF-90 biotin-streptavidin interaction prepared in (a) was immobilized on magnetic beads (MBs) in the following specific steps; First, MBs were washed with PBS buffer (PBST) containing Tween 20 and free of ribonuclease, and the washed MBs were redispersed in PBST to obtain an MBs solution. Then, the ssDNA-Hemin@BSA@ZIF-90 mixture obtained in (a) was added to the MBs solution, and after shaking for a period of time, magnetic separation was performed. The solid obtained by magnetic separation was collected and washed with PBS buffer containing BSA. Finally, the washed product was resuspended in reaction buffer to obtain the ssDNA-Hemin@BSA@ZIF-90-MBs mixture. Preferably, in step (2) (b), the concentration of the MBs solution is 10 mg / mL, the final concentration of Tween 20 in the PBS buffer is 0.1%; the concentration of the PBST buffer is 10 mM, and the pH is 7.4; the volume ratio of the MBs solution to the PBST buffer is 1:1; the shaking time is 24 h; the mass concentration of BSA in the PBS buffer containing BSA is 0.1%; and the reaction buffer is a solution composed of 20 mM of 4-hydroxyethylpiperazine ethanesulfonic acid, 50 mM of KCl, and 5 mM of MgCl2. (3) Preparation of activated CRISPR-Cas12a solution: S1. The aptamer solution of AFB1 (Apt1) and the activated DNA solution (acDNA1) are mixed and hybridized to generate double-stranded DNA, denoted as dsDNA1. Then, AFB1 standard solution is added to dsDNA1, and the resulting mixture is denoted as mixture A. Then, Cas12a, crRNA1 and 1×buffer are mixed and incubated to obtain unactivated CRISPR-Cas12a, denoted as mixture B. Finally, mixture A is added to mixture B and activated at a certain temperature to obtain activated CRISPR-Cas12a solution, denoted as mixture C. Preferably, in step (3), the concentrations of the AFB1 aptamer solution and the activated DNA solution in S1 are both 0.1 μM; the volume ratio of the AFB1 aptamer solution to the activated DNA solution is 1:1; AFB1 standard solution is added to dsDNA1, and the volume ratio of the two is 2:1; the final concentration of Cas12a in the mixture B is 100 nM, and the final concentration of crRNA1 is 120 nM; the incubation conditions are 37°C for 10 min; the volume ratio of the mixture A to the unmixed solution B is 1:3; and the activation conditions at a certain temperature are 37°C for 20 min. S2. Hybridize the OTA aptamer solution (Apt2) with the activated DNA solution (acDNA2) to generate double-stranded DNA, denoted as dsDNA2; add OTA standard solution to dsDNA2, and the resulting mixture is denoted as mixture D; mix Cas12a, crRNA2 and 1×buffer and incubate to obtain inactive CRISPR-Cas12a, denoted as mixture E; add mixture D to mixture E to obtain an activated CRISPR-Cas12a solution, denoted as mixture F; Preferably, in step (3), the concentrations of the OTA aptamer solution and the activated DNA solution in S2 are both 0.1 μM; the volume ratio of the OTA aptamer solution to the activated DNA solution is 1:1; OTA standard solution is added to dsDNA2, and the volume ratio of the two is 2:1; the final concentration of Cas12a in the mixture B is 100 nM, and the final concentration of crRNA2 is 120 nM; the incubation conditions are 37°C for 10 min; the volume ratio of the mixture E to the unmixed solution B is 1:3; and the activation conditions at a certain temperature are 37°C for 20 min. (4) The ssDNA-Hemin@BSA@ZIF-90-MBs mixture obtained in step (2) is mixed with mixture C and mixture F obtained in step (3) and incubated. After incubation, the supernatant is obtained by magnetic separation, and supernatant C and supernatant F are obtained accordingly. Preferably, the volume ratio of the mixture C, the mixture F and the ssDNA-Hemin@BSA@ZIF-90-MBs mixture in step (4) is 1:1; the incubation conditions are 37°C for 1 h.

[0008] (5) The supernatant C and supernatant F obtained in step (4) are respectively modified onto the surfaces of working electrode 1 and working electrode 2; a four-electrode system is formed by working electrode 1, working electrode 2, saturated Ag / AgCl electrode and platinum wire electrode, and electrochemical tests are performed based on the four-electrode system connected to a dual potentiostat to obtain electrochemical signals; and a standard curve is established with the concentration of AFB1 / OTA standard solution as the abscissa and the electrochemical signal as the ordinate. Preferably, the working electrode in step (5) uses pretreated GCE; the pretreatment step is as follows: take a glassy carbon electrode with a diameter of 3 mm and polish the electrode surface to a mirror finish using aluminum oxide powder with a particle size of 0.05 μm, then ultrasonically treat it in ethanol and ultrapure water for 30 s in sequence, and dry it after treatment to obtain pretreated GCE; the electrochemical signal is acquired by a CHI750E dual potentiostat; the supernatant modified on the electrode surface is 6 μL; using PBS solution (pH 7.4) as electrolyte, the differential pulse voltammetry (DPV) curve is scanned in the potential range of 0.25V to -0.65V, and the electrochemical signal is recorded; (6) Soak the circular filter paper in the supernatant obtained in step (4), and dry it to obtain a paper-based biochip; then add the reaction solution containing H2O2 and TMB to the paper-based biochip. After reacting for a period of time, use a smartphone to collect the colorimetric signal of the paper-based biochip, and use the concentration of AFB1 / OTA standard solution as the abscissa and the colorimetric signal as the ordinate to establish a standard curve. Preferably, the diameter of the paper-based biochip in step (6) is 1 cm; the reaction solution is an acetate buffer containing 2.5 mM TMB and 2 mM H2O2, with a pH of 4.5; the amount of reaction solution added is 10 μL, and the reaction time is 10 min.

[0009] (7) Detection of AFB1 and OTA in actual samples: First, obtain the test solution, and then follow the steps (3)-(6), except that the standard solutions of AFB1 and OTA of different concentrations are replaced with the test solution. Finally, the electrochemical signal and colorimetric signal are obtained at the same time, and the corresponding standard curve is substituted to realize the detection of AFB1 and OTA in unknown samples. Beneficial effects:

[0010] This invention focuses on improving the construction process of a dual-target, dual-mode sensor. Two independent homogeneous sensing systems are constructed using a single dual-function signal probe, allowing signals from different targets to be spatially separated and synchronously acquired via independent detection channels. This method eliminates the reliance on multiple probes for multi-target detection in traditional methods and significantly simplifies the construction process of sensors for simultaneous multi-target detection.

[0011] (1) For the first time, a four-electrode system and paper-based biochip are used to achieve sensitive electrochemical detection and intuitive colorimetric analysis of dual targets using only one signal probe; compared with other multi-target simultaneous detection strategies, it has the advantages of high efficiency and high accuracy in the detection process.

[0012] (2) This invention is the first to synthesize a nanozyme that encapsulates heme chloride and BSA using ZIF-90. BSA helps to highly disperse heme chloride and provides abundant active sites. As a bifunctional probe, it has excellent electrochemical and nanozyme catalytic properties.

[0013] (3) The present invention is based on the trans-cleavage of ssDNA by CRISPR-Cas12a with nuclease properties, which causes the probe to be released in the presence of the target. The unreleased signal probe is quickly removed by an external magnetic field, giving the homogeneous system the advantage of low background. At the same time, the signal amplification effect based on the dual enzyme cascade significantly improves the detection sensitivity.

[0014] (4) The homogeneous electrochemical-colorimetric dual-mode sensor constructed in this invention achieves accurate detection of AFB1 and OTA, with good selectivity and stability, wide linear range (electrochemical: 10 pg / mL~1000 ng / mL, colorimetric: 100 pg / mL~500 ng / mL), and low detection limit (electrochemical: 0.36 pg / mL (AFB1) and 0.29 pg / mL (OTA), colorimetric: 55 pg / mL (AFB1) and 48 pg / mL (OTA)). Attached Figure Description

[0015] Figure 1 The schematic diagram shows a dual-channel homogeneous electrochemical / colorimetric dual-mode biosensor for simultaneous detection of AFB1 and OTA: (A) The target triggers the trans-cutting process of the CRISPR / Cas12a system; (B) Dual-channel electrochemical signal output based on the homogeneous sensing system and colorimetric signal output based on the paper-based biochip.

[0016] Figure 2(A) is the SEM image of Hemin@BSA@ZIF-90; (B) is the TEM image of Hemin@BSA@ZIF-90; (C) is the XPS full spectrum of Hemin@BSA@ZIF-90; (D) is the XRD pattern of Hemin@BSA@ZIF-90.

[0017] Figure 3 In the middle (A) and (B), the electrochemical and UV-Vis spectra of the biosensor at different AFB1 and OTA concentrations (ad: 0, 1, 10, 100 ng / mL) are respectively; the inset shows photographs of the paper-based biochips of the four corresponding samples.

[0018] Figure 4 (A) is the DPV curve of the biosensor at different AFB1 and OTA concentrations; (B) is the linear regression curve of the DPV response with the logarithm of AFB1 and OTA concentrations; (C) is the colorimetric response of the biosensor at different AFB1 and OTA concentrations; (D) is the linear regression curve of ΔC with the logarithm of AFB1 and OTA concentrations. Detailed Implementation

[0019] The present invention will be further explained below with reference to specific embodiments and accompanying drawings.

[0020] Note on reagents involved in the sequence: The reagents used in this invention are all conventional primers and do not involve sequence listing inventions. They can all be obtained through commercial channels.

[0021] The aptamer, activating DNA, and crRNA used for AFB1 detection are denoted as Apt1, acDNA1, and crRNA1, respectively; the aptamer, activating DNA, and crRNA used for OTA detection are denoted as Apt2, acDNA2, and crRNA2, respectively. The primers and reagents used in this invention were all purchased from Sangon Biotech (Shanghai) Co., Ltd., and the corresponding sequences are shown in the table below:

[0022] Example 1: This embodiment provides a method for simultaneously detecting aflatoxin B1 and ochratoxin A using a homogeneous electrochemical-colorimetric dual-mode sensor based on a bifunctional probe, such as... Figure 1 As shown, the specific steps are as follows: (1) Preparation of Hemin@BSA@ZIF-90: 4 mg of heme chloride (Hemin) and 2 mg of bovine serum albumin (BSA) were dissolved in N,N-dimethylformamide (DMF) (8 mL) and stirred for 5 min. Then, 88 mg of Zn(CH3COO)2∙2H2O was added and stirred for another 5 min. After that, 76 mg of 2-ICA was dissolved in 12 mL of DMF and slowly added to the above mixture. After stirring for 15 min, the precipitate was collected by centrifugation at 10000 rpm for 10 min. The precipitate was washed with sodium dodecyl sulfate (SDS) to remove excess BSA, and then washed three times with DMF and ethanol, respectively. Finally, the collected precipitate was dried at 60°C for 8 h to obtain Hemin@BSA@ZIF-90; Figure 2 (A) and (B) are SEM and TEM images of Hemin@BSA@ZIF-90. It can be seen from the images that the Hemin@BSA@ZIF-90 particles have a uniform dodecahedral structure with an average size of about 90 nm.

[0023] Figure 2 The middle (C) image shows the XPS full spectrum of Hemin@BSA@ZIF-90. As can be seen from the image, Hemin@BSA@ZIF-90 is mainly composed of C, N, O and Zn elements, with a small amount of Fe element evenly distributed within the particles. Figure 2 The middle (D) is the XRD pattern of Hemin@BSA@ZIF-90. It can be seen from the figure that the XRD pattern of Hemin@BSA@ZIF-90 is basically the same as that of ZIF-90, except that the intensity is reduced. This indicates that the addition of Hemin and BSA did not destroy the crystal structure of ZIF-90.

[0024] (2) Preparation of ssDNA-Hemin@BSA@ZIF-90-MBs: (a) First, Hemin@BSA@ZIF-90 was coupled with thiol-modified ssDNA. To eliminate interstrand disulfide bonds in DNA, 10 μL of TCEP solution (30 mM) and 10 μL of PBS buffer (10 mM, pH 5.5) were added to 150 μL of thiol-modified ssDNA solution (10 μM) and incubated at room temperature for 1 h to reduce thiol groups. Simultaneously, 20 μL of sulfosuccinimide-4-(N-maleimidemethyl)cyclohexane-2-carboxylate (sulfo-SMCC) solution (0.1 mg / mL) was mixed with 200 μL of 100 mM PBS buffer (pH 7.4, 100 mM NaCl) containing Hemin@BSA@ZIF-90 (1 mg / mL) and incubated at room temperature for 1 h, followed by centrifugation at 10,000 rpm for 5 min. Then wash three times with 100 mM PBS buffer (pH 7.4, 100 mM NaCl); Subsequently, the above ssDNA solution and Hemin@BSA@ZIF-90 suspension were mixed. After incubation at 25°C for 24 h, the resulting conjugate was collected by centrifugation at 8000 rpm for 5 min and washed three times with 100 mM PBS buffer (pH 7.4, 100 mM NaCl). Finally, the washed product was resuspended in 200 μL PBS buffer (pH 7.4) to obtain the ssDNA-Hemin@BSA@ZIF-90-MBs mixture. (b) ssDNA-Hemin@BSA@ZIF-90 was immobilized on magnetic beads (MBs) via biotin-streptavidin interaction. First, 100 μL of MBs (10 mg / mL) was washed three times with 10 mM nuclease-free PBS buffer (PBST) containing 0.1% Tween 20, and then dispersed in 100 μL of PBST (10 mM). Next, ssDNA-Hemin@BSA@ZIF-90 was added to the MBs solution, and the mixture was gently shaken at 25°C for 24 h. After magnetic separation, the MBs were washed three times with 100 mM PBS buffer (pH 7.4) containing 0.1% BSA. Finally, ssDNA-Hemin@BSA@ZIF-90-MBs were resuspended in 100 μL of reaction buffer (20 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 50 mM KCl, 5 mM MgCl2) and stored at 4°C for later use. (3) Preparation of activated CRISPR-Cas12a solution, such as Figure 1 As shown in (A): S1. A double-stranded DNA, denoted as dsDNA1, was generated by hybridization of 0.1 μM MAFB1 aptamer solution (Apt1) and 0.1 μM activated DNA solution (acDNA1). Then, AFB1 standard solution was added to dsDNA1 at a volume ratio of 2:1, and the resulting mixture was denoted as mixture A. Cas12a, crRNA1, and 1×buffer were mixed and incubated (37°C, 10 min) to obtain unactivated CRISPR-Cas12a, denoted as mixture B. The final concentration of Cas12a in mixture B was 100 nM, and the final concentration of crRNA1 was 120 nM. Finally, mixture A was added to mixture B at a volume ratio of 1:3, and the mixture was activated at 37°C for 20 min to obtain activated CRISPR-Cas12a solution, denoted as mixture C. S2. The aptamer (Apt1) at a concentration of 0.1 μM OTA was mixed with 0.1 μM activated DNA (acDNA2) to generate double-stranded DNA, denoted as dsDNA2. OTA standard solution was added to dsDNA2 at a volume ratio of 2:1, and the resulting mixture was denoted as mixture D. Cas12a, crRNA2, and 1×buffer were mixed and incubated (37°C, 10 min) to obtain unactivated CRISPR-Cas12a, denoted as mixture E. The final concentration of Cas12a in mixture E was 100 nM, and the final concentration of crRNA2 was 120 nM. Finally, mixture D was added to mixture E at a volume ratio of 1:3, and the mixture was activated at 37°C for 20 min to obtain an activated CRISPR-Cas12a solution, denoted as mixture F. (4) Take 10 μL of the ssDNA-Hemin@BSA@ZIF-90-MBs mixture obtained in step (2) and incubate it with mixture C and mixture F (10 μL) obtained in step (3) for 1 h. After magnetic separation, take the supernatant. (5) such as Figure 1 (B) As shown in the left part, 6 μL of the supernatant obtained in step (4) was used to modify the surfaces of working electrode 1 and working electrode 2, respectively. Electrochemical tests were performed by connecting a four-electrode system to a dual potentiostat. Differential pulse voltammetry (DPV) curves were scanned in the potential range of 0.25V to -0.65V, and the electrochemical signals were recorded. A standard curve was established with the concentration of AFB1 / OTA standard solution as the abscissa and the electrochemical signal as the ordinate. (6) For example Figure 1(B) As shown on the right, a circular filter paper disc (1 cm in diameter) was immersed in 10 μL of the supernatant obtained in step (4), and dried to obtain a paper-based biochip. After drying, 10 μL of a mixed reaction solution (including TMB (2.5 mM) and H2O2 (2 mM)) was added to trigger a colorimetric reaction. The colorimetric signal of the paper-based biochip was collected using a smartphone, and a standard curve was established with the concentration of the AFB1 / OTA standard solution as the x-axis and the colorimetric signal as the y-axis. (7) Detection of AFB1 and OTA in actual samples: First, obtain the test solution, and then follow the steps (3)-(6), except that the standard solutions of AFB1 and OTA of different concentrations are replaced with the test solution. Finally, the electrochemical signal and colorimetric signal are obtained at the same time, and the corresponding standard curve is substituted to realize the detection of AFB1 and OTA in unknown samples. Example 2:

[0025] like Figure 3 As shown, the feasibility analysis of the method for simultaneously detecting AFB1 and OTA based on a homogeneous electrochemical-colorimetric dual-mode sensor with a dual-functional probe is as follows: A dual-mode biosensor for the simultaneous detection of AFB1 and OTA was successfully established using Hemin@BSA@ZIF-90 as a bifunctional probe. In the absence of AFB1 and OTA, almost no electrochemical or colorimetric signal response was observed in the supernatant. This is because, in the absence of a target, the MBs immobilized the probe, resulting in a low background signal in the homogeneous reaction system. Figure 3 As shown in (A), a distinct reduction peak of heme chloride can be observed in the presence of AFB1 / OTA. The electrical signal gradually increases with increasing concentrations of AFB1 and OTA from 1 to 10 and 100 ng / mL. Similarly, in the TMB−H2O2 system, the intensity of the UV absorption peak at 652 nm gradually increases due to the oxidation of TMB. Figure 3 As shown in the illustration in (B), the paper-based biochip changes color from colorless to blue-green, and the color gradually deepens with increasing target concentration. These experimental results demonstrate the successful construction of this homogeneous electrochemical-colorimetric dual-mode biosensor. The target concentration is positively correlated with the amount of acDNA released; the more acDNA released, the more thoroughly the CRISPR / Cas12a system cleaves the ssDNA, thus releasing more probes. Example 3:

[0026] The performance analysis of the method for simultaneous detection of AFB1 and OTA based on a homogeneous electrochemical-colorimetric dual-mode sensor with a dual-functional probe is as follows: For electrochemical detection, a standard curve was established with the logarithm of the AFB1 / OTA standard solution concentration on the x-axis and the obtained electrical signal on the y-axis. Figure 4As shown in (A), within the target concentration range of 10 pg / mL to 1000 ng / mL, the DPV response increases with increasing AFB1 and OTA concentrations. Figure 4 As shown in (B), the logarithmic values ​​of AFB1 and OTA concentrations exhibit a good linear relationship with the current response, with the linear regression equations being I = 1.174lgC. AFB1 +6.728 (R) 2 =0.998)(AFB1) and I=1.457lgC OTA +6.263 (R) 2 =0.996)(OTA), with detection limits of 0.36 pg / mL (AFB1) and 0.29 pg / mL (OTA), respectively.

[0027] For colorimetric detection, a smartphone is used to photograph the paper-based biochip, and its RGB values ​​are obtained through image analysis software. For example... Figure 4 As shown in (C), the color of the paper-based biochip gradually deepens within the target concentration range of 100 pg / mL to 500 ng / mL. Figure 4 In the figure (D), the linear relationship between the colorimetric signal response (ΔC) and the logarithm of the AFB1 / OTA standard solution concentration is expressed. The colorimetric signal response is determined by… We obtain ΔR, ΔG, and ΔB, which are the differences between the R, G, and B values ​​of the test sample and the R0, G0, and B0 of the blank sample, respectively. The linear regression equations are I = 17.85lgC. AFB1 +25.64 (R) 2 =0.996)(AFB1) and I=16.94lgC OTA +24.53 (R) 2 =0.993 (OTA), with detection limits of 55 pg / mL (AFB1) and 48 pg / mL (OTA), respectively. The dual-mode sensor developed in this invention has a wide detection range and a low detection limit. In the detection of dual targets, this method has outstanding advantages in terms of ease of development and versatility.

[0028] Example 4: The constructed homogeneous electrochemical-colorimetric biosensor was used to detect and analyze AFB1 and OTA in real samples.

[0029] The method for obtaining the test solution is as follows: The sample was thoroughly pulverized in a grinder to obtain particulate matter. Then, 5 g of the sample particulate matter and 1 g of sodium chloride were mixed and added to 20 mL of a mixed solution of methanol and water (4:1, v / v). The mixture was then extracted by shaking for 1 h, centrifuged at 8000 rpm for 10 min, and the supernatant was collected. 2 mL of the supernatant was added to water and brought to a final volume of 10 mL. Finally, the solution was filtered through a 0.22 µm filter until clear, yielding the test solution.

[0030] AFB1 and OTA were added to wheat samples at concentrations of 0.5, 5, and 50 ng / mL, respectively, using the standard addition method. The accuracy of the detection results was verified using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). The analytical results are shown in Tables 1 and 2. Table 1: Detection of AFB1 in wheat samples and validation of the national standard method (UPLC-MS / MS)

[0031] Table 2: Detection of OTA in wheat samples and validation of national standard method (UPLC-MS / MS)

[0032] Note: "ND": Not Detected Strong consistency was observed between the two detection modes and the UPLC-MS / MS method, with RSD values ​​≤ 5.1% and recoveries ranging from 96.70% to 102%. These results demonstrate the good reliability and accuracy of the developed biosensor in practical sample analysis.

[0033] 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. A method for simultaneous detection of AFB1 and OTA based on homogenous electrochemical-colorimetric dual-mode sensor using bifunctional probe, characterized in that, The steps are as follows: (1) Preparation of Hemin@BSA@ZIF-90: First, heme chloride and bovine serum albumin were added to N,N-dimethylformamide and stirred for the first time, then Zn(CH3COO)2∙2H2O was added and stirring continued; after stirring, DMF solution containing imidazole-2-carboxaldehyde was added and stirred for the third time to obtain a mixed solution; The precipitate was collected by centrifugation and washed sequentially with DMF, ethanol and sodium dodecyl sulfate. Finally, the precipitate was collected and dried to obtain the dried product, which is Hemin@BSA@ZIF-90. (2) Preparation of ssDNA-Hemin@BSA@ZIF-90-MBs: (a) Preparation of ssDNA-Hemin@BSA@ZIF-90 mixture: S1. Tris(2-carboxyethyl)phosphine hydrochloride solution was added to ssDNA solution for reaction, and TCEP-ssDNA solution was obtained after the reaction. S2. Simultaneously, the sulfosuccinimide-4-(N-maleimidemethyl)cyclohexane-2-carboxylate solution was mixed with PBS buffer containing Hemin@BSA@ZIF-90 for the first incubation. After incubation, the precipitate was collected by centrifugation, washed with PBS buffer, and then resuspended in PBS buffer to obtain a sulfo-SMCC activated Hemin@BSA@ZIF-90 suspension. S3. Subsequently, the TCEP-ssDNA solution and the sulfo-SMCC activated Hemin@BSA@ZIF-90 suspension were mixed and incubated for a second time. After incubation, the conjugates were collected by centrifugation and washed with PBS buffer. Finally, the washed product was resuspended in ultrapure water to obtain the ssDNA-Hemin@BSA@ZIF-90 mixture. (b) First, MBs were washed with PBS buffer containing Tween 20 and free of ribonuclease, and the washed MBs were redispersed in PBST to obtain an MBs solution; then, the ssDNA-Hemin@BSA@ZIF-90 mixture obtained in (a) was added to the MBs solution, and after shaking for a period of time, magnetic separation was performed. The solid obtained by magnetic separation was collected and washed with PBS buffer containing BSA; finally, the washed product was resuspended in reaction buffer to obtain the ssDNA-Hemin@BSA@ZIF-90-MBs mixture. (3) Preparation of activated CRISPR-Cas12a solution: S1. The aptamer solution of AFB1 is mixed with the activated DNA solution to generate double-stranded DNA, denoted as dsDNA1. Then, AFB1 standard solution is added to dsDNA1, and the resulting mixture is denoted as mixture A. Then, Cas12a, crRNA1, and 1×buffer are mixed and incubated to obtain unactivated CRISPR-Cas12a, denoted as mixture B. Finally, mixture A is added to mixture B and activated at a certain temperature to obtain activated CRISPR-Cas12a solution, denoted as mixture C. S2. Hybridize the OTA aptamer solution with the activated DNA solution to generate double-stranded DNA, denoted as dsDNA2; add OTA standard solution to dsDNA2 to obtain a mixture, denoted as mixture D; mix Cas12a, crRNA2 and 1×buffer and incubate to obtain unactivated CRISPR-Cas12a, denoted as mixture E; add mixture D to mixture E to obtain an activated CRISPR-Cas12a solution, denoted as mixture F; (4) The ssDNA-Hemin@BSA@ZIF-90-MBs mixture obtained in step (2) is mixed with mixture C and mixture F obtained in step (3) and incubated. After incubation, the supernatant is obtained by magnetic separation, and supernatant C and supernatant F are obtained accordingly. (5) The supernatant C and supernatant F obtained in step (4) are respectively applied to the surfaces of working electrode 1 and working electrode 2; A four-electrode system consisting of working electrode 1, working electrode 2, saturated Ag / AgCl electrode, and platinum wire electrode was used. Electrochemical tests were performed based on the four-electrode system connected to a dual potentiostat to obtain electrochemical signals. A standard curve was established with the concentration of AFB1 / OTA standard solution as the abscissa and the electrochemical signal as the ordinate. (6) Soak the circular filter paper in the supernatant obtained in step (4), and dry it to obtain a paper-based biochip; then add the reaction solution containing H2O2 and TMB to the paper-based biochip. After reacting for a period of time, use a smartphone to collect the colorimetric signal of the paper-based biochip, and use the concentration of AFB1 / OTA standard solution as the abscissa and the colorimetric signal as the ordinate to establish a standard curve. (7) Detection of AFB1 and OTA in actual samples: First, obtain the test solution, and then follow the steps (3)-(6), except that the standard solutions of AFB1 and OTA of different concentrations are replaced with the test solution. Finally, the electrochemical signal and colorimetric signal are obtained at the same time, and the corresponding standard curve is substituted to realize the detection of AFB1 and OTA in unknown samples.

2. The method for simultaneously detecting AFB1 and OTA using a homogeneous electrochemical-colorimetric dual-mode sensor based on a dual-functional probe according to claim 1, characterized in that, In step (1), the ratio of heme chloride, bovine serum albumin, N,N-dimethylformamide and Zn(CH3COO)2∙2H2O, imidazole-2-carboxaldehyde and N,N-dimethylformamide is 4 mg: 2 mg: 18 mL: 1 mL: 76 mg: 18 mL; the concentration of SDS solution is 1 wt%; the first stirring time and the continued stirring time are both 5 min, and the third stirring time is 15 min; the centrifugation conditions are: 10000 rpm, 10 min; the washing is performed three times each with DMF, ethanol and sodium dodecyl sulfate; the drying temperature is 60℃ and the time is 6~8 h.

3. The method for simultaneously detecting AFB1 and OTA using a homogeneous electrochemical-colorimetric dual-mode sensor based on a dual-functional probe according to claim 1, characterized in that, In step (2) (a), the ratio of the amounts of TCEP solution and ssDNA solution is 10 μL: 150 μL, the concentration of TCEP solution is 30 mM, and the concentration of ssDNA solution is 10 μM. The concentration of the sulfo-SMCC solution was 0.1 mg / mL, and the final concentration of Hemin@BSA@ZIF-90 in PBS buffer was 0.5 mg / mL, wherein the PBS buffer contained 100 mM sodium chloride and the pH was 7.4; the volume ratio of sulfo-SMCC solution to PBS buffer containing Hemin@BSA@ZIF-90 was 20 μL: 200 μL; the first incubation reaction time was 1 h at room temperature; the rotation speed was 10000 rpm for 5 min; the washing was performed three times; the second incubation time was 24 h at room temperature; the concentration of the ssDNA-Hemin@BSA@ZIF-90 mixture obtained after resuspension was 1 mg / mL.

4. The method for simultaneously detecting AFB1 and OTA using a homogeneous electrochemical-colorimetric dual-mode sensor based on a dual-functional probe according to claim 1, characterized in that, The concentration of the MBs solution in step (b) is 10 mg / mL, the final concentration of Tween 20 in the PBS buffer is 0.1%; the concentration of the PBST buffer is 10 mM, and the pH is 7.4; the volume ratio of the MBs solution to the PBST buffer is 1:1; the shaking time is 24 h; the mass concentration of BSA in the PBS buffer containing BSA is 0.1%; the reaction buffer is a solution composed of 20 mM of 4-hydroxyethylpiperazine ethanesulfonic acid, 50 mM of KCl and 5 mM of MgCl2.

5. The method for simultaneously detecting AFB1 and OTA using a homogeneous electrochemical-colorimetric dual-mode sensor based on a dual-functional probe according to claim 1, characterized in that, In step (3), the concentrations of the AFB1 aptamer solution and the activated DNA solution in S1 are both 0.1 μM; the volume ratio of the AFB1 aptamer solution to the activated DNA solution is 1:1; AFB1 standard solution is added to dsDNA1, and the volume ratio of the two is 2:1; the final concentration of Cas12a in the mixture B is 100 nM, and the final concentration of crRNA1 is 120 nM; the incubation conditions are 37°C for 10 min; the volume ratio of the mixture A to the unmixed solution B is 1:3; the activation conditions at a certain temperature are 37°C for 20 min.

6. The method for simultaneously detecting AFB1 and OTA using a homogeneous electrochemical-colorimetric dual-mode sensor based on a dual-functional probe according to claim 1, characterized in that, In step (3), the concentrations of the OTA aptamer solution and the activated DNA solution in S2 are both 0.1 μM; the volume ratio of the OTA aptamer solution to the activated DNA solution is 1:1; OTA standard solution is added to dsDNA2, and the volume ratio of the two is 2:1; the final concentration of Cas12a in the mixture B is 100 nM, and the final concentration of crRNA2 is 120 nM; the incubation conditions are 37°C for 10 min; the volume ratio of the mixture E to the unmixed solution B is 1:3; the activation conditions at a certain temperature are 37°C for 20 min.

7. The method for simultaneously detecting AFB1 and OTA using a homogeneous electrochemical-colorimetric dual-mode sensor based on a dual-functional probe according to claim 1, characterized in that, In step (4), the volume ratio of mixture C, mixture F and ssDNA-Hemin@BSA@ZIF-90-MBs mixture is 1:1; the incubation conditions are 37°C for 1 h.

8. The method for simultaneously detecting AFB1 and OTA using a homogeneous electrochemical-colorimetric dual-mode sensor based on a dual-functional probe according to claim 1, characterized in that, The working electrode mentioned in step (5) uses pretreated GCE; the pretreatment steps are as follows: take a glassy carbon electrode with a diameter of 3 mm and polish the electrode surface to a mirror finish using alumina powder with a particle size of 0.05 μm, then sonicate it in ethanol and ultrapure water for 30 s in sequence, and dry it after treatment to obtain pretreated GCE; the electrochemical signal is obtained by a CHI 750E dual potentiostat; the supernatant modified on the electrode surface is 6 μL; using PBS solution (pH 7.4) as electrolyte, the differential pulse voltammetry (DPV) curve is scanned in the potential range of 0.25V to -0.65V, and the electrochemical signal is recorded.

9. The method for simultaneously detecting AFB1 and OTA using a homogeneous electrochemical-colorimetric dual-mode sensor based on a dual-functional probe according to claim 1, characterized in that, In step (6), the diameter of the paper-based biochip is 1 cm; the reaction solution is an acetate buffer containing 2.5 mM TMB and 2 mM H2O2, with a pH of 4.5; the amount of reaction solution added is 10 μL, and the reaction time is 10 min.

Images