Electrochemical sensor based on FeCo-Anti-MCLR, preparation method of electrochemical sensor and application of electrochemical sensor in detection of microcystin-LR

Abstract

The invention relates to an electrochemical sensor based on FeCo-Anti-MCLR, a preparation method of the electrochemical sensor and application of the electrochemical sensor to detection of microcystic toxins-LR. FeCo nano-particles are constructed to be combined with a microcystic toxin antibody (Anti-MCLR), the surface of an electrode is coated with the combined electrode to construct a working electrode with electrochemical immunodetection capacity, namely, the FeCo-Anti-MCLR modified electrode, and the working electrode is used for detecting the microcystic toxins-LR. According to the present invention, the MC-LR can be rapidly detected on site, the sensitivity is high, the detection range is 0.02-0.09 nmol / L, the minimum detection limit is 0.02 nmol / L, the temporary guidance limit value of the MC-LR 1 [mu] g / L (1.01 nmol / L) in the drinking water of the World Health Organization (WHO) is met, and the technology provides the powerful tool for sensitively and rapidly monitoring the MC-LR in the environment.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical sensors, and in particular to an electrochemical sensor based on FeCo-Anti-MCLR, a preparation method thereof, and an application thereof in detecting microcystin-LR. Background Art

[0002] Microcystin-LR (MC-LR) is produced by a type of cyanobacteria (blue-green algae) when it overgrows (forming algal blooms) in eutrophic waters. It is a cyclic heptapeptide compound containing multiple non-protein amino acids and a unique Adda side chain. It is one of the most common and most toxic variants of microcystin. MC-LR has serious impacts on both the environment and humans, primarily affecting the liver. Acute exposure may cause hepatitis and liver failure, while chronic exposure may lead to diseases such as liver cancer.

[0003] Current methods for detecting MC-LR include liquid chromatography / mass spectrometry (HPLC-MS), thin-layer chromatography (TLC), enzyme-linked immunosorbent assay (ELISA), and protein phosphatase inhibition assay (PPIA). However, these methods generally have high analysis costs, expensive instruments, long detection times, susceptibility to interference, and low selectivity, making them unable to meet the requirements for rapid and sensitive on-site detection of MC-LR.

[0004] "Research on Microcystin Immunosensor Based on Screen-Printed Electrodes Modified with Fe3O4@Au Magnetic Nanoparticles" discloses a method of modifying the surface of a screen-printed working electrode with core-shell Fe3O4@Au magnetic nanoparticles. The antibody is then fixed to the electrode surface through the adsorption interaction between gold nanoparticles and microcystin-(leucine-arginine) antibodies (anti-MCLR). The nonspecific adsorption sites are blocked with bovine serum albumin (BSA). The resulting current-type immunosensor for detecting MCLR has a detection limit of 0.38μg / L. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problems of long detection time, interference from foreign substances, low selectivity and the like in the prior art and to provide an electrochemical sensor based on FeCo-Anti-MCLR and its preparation method and application in the detection of microcystin-LR.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] One of the technical solutions of the present invention is to provide a method for preparing an electrochemical sensor based on FeCo-Anti-MCLR, comprising the following steps:

[0008] S1. Mixing an iron salt, a cobalt salt, polyvinyl pyrrolidone (PVP) and water to obtain a mixed solution, adding a potassium cobalt cyanide solution, aging the mixed solution, calcining the aged product under an inert atmosphere, and soaking the calcined product in a H2SO4 solution to obtain a FeCo@NC material;

[0009] S2, the FeCo@NC material obtained in step S1 was dispersed in MES buffer solution, and a mixed solution of EDC and NHS was added for activation. After activation, a PBS solution containing anti-MCLR antibody was added for coupling, and then coated on the electrode surface. After blocking treatment, a FeCo-Anti-MCLR modified electrode was obtained;

[0010] S3. The FeCo-Anti-MCLR modified electrode obtained in step S2 is used as a working electrode, and is constructed with a reference electrode and a counter electrode to obtain an electrochemical sensor based on FeCo-Anti-MCLR.

[0011] In some specific embodiments, in step S1, the mass ratio of the iron salt, cobalt salt, polyvinyl pyrrolidone and water is 1:(0.2-0.3):(22-23):(744-745), and the volume ratio of the potassium cobalt cyanide solution to the mixed solution is 1:1.

[0012] In some specific embodiments, in step S1, the iron salt is ferrous chloride tetrahydrate, the cobalt salt is cobalt chloride, and the initial concentration of the potassium cobalt cyanide solution is 5 mM.

[0013] In some specific embodiments, in step S1, the aging time is 20 hours;

[0014] The calcination temperature is 650°C, the inert atmosphere during calcination is N2, and the calcination time is 1 hour;

[0015] The calcined product was immersed in the H2SO4 solution for 1 h at a temperature of 80°C, and the concentration of the H2SO4 solution was 1M.

[0016] In some specific embodiments, in step S2, the FeCo@NC material is uniformly dispersed in a MES buffer solution at a mass concentration of 1 g / L, wherein the concentration of the MES buffer solution is 50 mmol / L and the pH is 6.0.

[0017] In some specific embodiments, in step S2, the activation reagents used during activation include EDC solution and NHS solution, wherein the mass concentration of the EDC solution is 5 g / L, and the mass concentration of the NHS solution is 25 g / L.

[0018] In some embodiments, in step S2, the mass concentration of the Anti-MCLR antibody in the PBS solution containing the Anti-MCLR antibody is 5 mg / L, the coupling temperature is 4° C., and the coupling time is 10 h.

[0019] In some embodiments, in step S2, the blocking solution used is bovine serum albumin with a concentration of 1 g / L.

[0020] In some specific embodiments, in step S3, the reference electrode is a silver chloride electrode, and the counter electrode is a platinum wire electrode.

[0021] The second technical solution of the present invention is to provide an electrochemical sensor based on FeCo-Anti-MCLR obtained by the preparation method described in one of the above technical solutions.

[0022] The third technical solution of the present invention is to provide an application of the electrochemical sensor based on FeCo-Anti-MCLR as described in the second technical solution above, wherein the electrochemical sensor based on FeCo-Anti-MCLR is used to detect microcystin-LR.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The electrochemical sensor for detecting microcystin-LR provided by the present invention has a simple preparation method. By combining FeCo nanoparticles with microcystin antibodies (Anti-MCLR), the antibodies provide highly specific recognition functions, effectively identifying target toxin molecules, thereby significantly reducing interference caused by nonspecific adsorption and improving the selectivity and accuracy of the detection system. The sensor is used to construct a working electrode with electrochemical immunoassay capabilities, namely an FeCo-Anti-MCLR modified electrode, which can achieve rapid on-site detection of MC-LR with high sensitivity, a detection range of 0.02-0.09 nmol / L, and a minimum detection limit of 0.02 nmol / L, meeting the World Health Organization (WHO) interim guidance limit of 1 μg / L (1.01 nmol / L) for MC-LR in drinking water. This technology provides a powerful tool for sensitive and rapid monitoring of MC-LR in the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a performance comparison chart of different modified electrodes in Example 1.

[0026] Figure 2 This is a graph showing the detection performance of the electrode in comparative example 1 that has not been blocked with BSA for different concentrations of MC-LR.

[0027] Figure 3This is a graph showing the detection performance of the electrode treated with BSA in Example 1 for different concentrations of MC-LR.

[0028] Figure 4 This is a graph showing the detection performance of the antibody electrode treated with BSA for different concentrations of MC-LR in comparative example 4. DETAILED DESCRIPTION

[0029] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0030] In the following examples and comparative examples, unless otherwise specified, raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0031] Example 1

[0032] This embodiment provides a method for preparing an electrochemical sensor based on FeCo-Anti-MCLR, comprising the following steps:

[0033] (1) 53.7 mg of ferrous chloride tetrahydrate (FeCl2·4H2O, 191.81 g / mol), 11.69 mg of cobalt chloride (CoCl2, 129.84 g / mol), 1.2 g of polyvinylpyrrolidone (PVP) and 40 mL of deionized water were stirred, and an equal volume of 5 mM potassium cobalt cyanide solution was slowly added and stirred for further 30 min. The mixture was aged for 20 h, and the precipitate obtained after aging was centrifuged and washed three times with ethanol and deionized water. The product was dried at 60 °C. Then, it was calcined in a N2 atmosphere at a temperature of 650 °C for 1 h. The product was soaked in a 1M H2SO4 solution at 80 °C for 4 h. The final product was collected and washed with a large amount of deionized water and dried to obtain the product FeCo@NC.

[0034] (2) FeCo@NC was uniformly dispersed in MES buffer solution at a mass concentration of 1 g / L, shaken and incubated at a speed of 200 r / min at room temperature for 2 h, and then washed with phosphate buffered saline (PBS) to remove residual reagents. The concentration of MES buffer solution was 50 mmol / L and the pH was 6.0. Then, activation reagents were added for activation. The activation reagents included EDC and NHS, wherein the mass concentration of EDC was 5 g / L and the mass concentration of NHS was 25 g / L. The activated FeCo particles were dispersed in PBS solution with a mass concentration of 5 mg / L of Anti-MCLR (purchased from Shenzhen Kejie Industrial Development Co., Ltd., product batch: 20240824) and incubated at 4°C for 10 h to obtain FeCo-Anti-MCLR complex.

[0035] (3) After the obtained FeCo-Anti-MCLR complex was evenly mixed, 5 μL was applied to the surface of the glassy carbon electrode and dried at room temperature. It was then blocked with 1 g / L bovine serum albumin (BSA) to obtain the FeCo-Anti-MCLR modified electrode after drying.

[0036] Comparative Example 1

[0037] Compared with Example 1, most of the steps are the same, with the only difference being that the sealing treatment in step (3) is omitted, thereby obtaining an unsealed FeCo-Anti-MCLR modified electrode.

[0038] Comparative Example 2

[0039] Compared with Example 1, most of the steps are the same, except that step (2) is omitted, that is, the FeCo@NC prepared in step (1) is coated on the surface of the glassy carbon electrode and dried at room temperature to obtain a FeCo modified electrode.

[0040] Comparative Example 3

[0041] Compared with Example 1, most of the steps are the same, except that step (1) is omitted, that is, the Anti-MCLR of step (2) is directly coated on the surface of the glassy carbon electrode to obtain the Anti-MCLR modified electrode.

[0042] The performance test is as follows:

[0043] The electrodes prepared in the above examples and comparative examples 1 and 2 were tested under the following conditions:

[0044] The analysis was performed using an electrochemical workstation three-electrode system, with the working electrode being the electrode prepared in the above embodiments and comparative examples, the reference electrode being a silver chloride electrode, and the counter electrode being a platinum wire electrode.

[0045] (1) Cyclic voltammetry (CV) test

[0046] The detection system comprises a mixed solution of 0.1 mol / L KCl and 5 mmol / L K3[Fe(CN)6], preferably in a volume of 10 mL. The voltage scanning range is preferably -0.2-0.6 V.

[0047] like Figure 1 As shown in the figure, the performance comparison of different modified electrodes is shown. Figure 1It can be seen that the electrode modified with the FeCo-Anti-MCLR catalyst exhibits oxidation current peaks and reduction current peaks near 0.2V. These current peaks are all greater than those of the electrode before modification, indicating that the FeCo-Anti-MCLR catalyst can effectively increase the electron transfer rate of the electrode and improve the electrochemical performance of the electrode. Comparing the CV graphs of the electrodes modified with FeCo-Anti-MCLR and FeCo catalysts, the peak value of the electrode modified with the FeCo-Anti-MCLR catalyst decreases slightly. This is attributed to the fact that antibodies are proteins with large molecular weight, which causes the conductivity of the electrode with the added antibody to deteriorate slightly. However, modifying the electrode with MC-LR antibodies can provide specific recognition function and improve selectivity. The performance of the three different modified electrodes was investigated. The conductivity of FeCo-Anti-MCLR / GCE was significantly reduced after encapsulation. The purpose of the encapsulation here is to reduce nonspecific adsorption and improve the selectivity of the sensor.

[0048] (2) Differential Pulse Voltammetry (DPV) test

[0049] The electrolyte solution is 0.1 mol / L PBS (pH 6.0) containing 0.1 mol / L KCl and 5 mmol / L K₃[Fe(CN)₆]. The system volume is preferably 10 mL. The scanning voltage range is preferably -0.2-0.6 V. The reaction time in MC-LR solutions of varying concentrations is preferably 5 min.

[0050] like Figure 2 、 Figure 3 、 Figure 4 The following are the detection performance diagrams of the FeCo-Anti-MCLR modified electrode before and after BSA encapsulation and the Anti-MCLR antibody modified electrode after BSA encapsulation for different concentrations of MC-LR. The current-concentration linearity diagram of the electrochemical detection of microcystin-LR by encapsulated FeCo-Anti-MCLR / GCE shows that the concentration and the response current value show a linear decreasing relationship within the detection range of 0.02 to 0.09 nM, and the linear regression coefficient R 2The α-value of the FeCo-Anti-MCLR / GCE was 0.988, indicating that it has certain potential for quantitative detection and can be considered to have preliminary accuracy and applicability. The electrochemical response signal sensitivity reached 824.7 μA / nM, which is significantly superior to that of unencapsulated FeCo-Anti-MCLR / GCE (324 μA / nM) and Anti-MCLR / GCE (333.7 μA / nM). This indicates that the encapsulated FeCo-Anti-MCLR / GCE can be used for highly sensitive and rapid detection of MC-LR. Combined with the above analysis and discussion, it is clear that the electrochemical detection of MC-LR using FeCo-Anti-MCLR / GCE has significant research value and application prospects. This technology is of great significance in the fields of water quality monitoring, pollution early warning, and ecological protection, providing a powerful tool for the precise monitoring of algal toxin contamination.

[0051] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A method for preparing an electrochemical sensor based on FeCo-Anti-MCLR, characterized in that: The steps include: S1. Mixing an iron salt, a cobalt salt, polyvinyl pyrrolidone, and water to obtain a mixed solution, adding a potassium cobalt cyanide solution, and aging the mixed solution. The aged product is calcined under an inert atmosphere, and the calcined product is immersed in a H2SO4 solution to obtain a FeCo@NC material. S2, the FeCo@NC material obtained in step S1 was dispersed in MES buffer solution, and a mixed solution of EDC and NHS was added for activation. After activation, a PBS solution containing anti-MCLR antibody was added for coupling, and then coated on the electrode surface. After blocking treatment, a FeCo-Anti-MCLR modified electrode was obtained; S3. The FeCo-Anti-MCLR modified electrode obtained in step S2 is used as a working electrode, and is constructed with a reference electrode and a counter electrode to obtain an electrochemical sensor based on FeCo-Anti-MCLR.

2. The method for preparing an electrochemical sensor based on FeCo-Anti-MCLR according to claim 1, characterized in that: In step S1, the mass ratio of the iron salt, cobalt salt, polyvinyl pyrrolidone and water is 1:(0.2-0.3):(22-23):(744-745), and the volume ratio of the potassium cobalt cyanide solution to the mixed solution is 1:

1.

3. The method for preparing an electrochemical sensor based on FeCo-Anti-MCLR according to claim 2, characterized in that: In step S1, the iron salt is ferrous chloride tetrahydrate, the cobalt salt is cobalt chloride, and the initial concentration of the potassium cobalt cyanide solution is 5 mM.

4. The method for preparing an electrochemical sensor based on FeCo-Anti-MCLR according to claim 1, wherein: In step S1, the aging time is 20 to 22 hours; The calcination temperature is 650°C, the inert atmosphere during calcination is N2, and the calcination time is 1 hour; The calcined product is immersed in the H2SO4 solution for 1 hour, the temperature is 70-80°C, and the concentration of the H2SO4 solution is 1M.

5. The method for preparing an electrochemical sensor based on FeCo-Anti-MCLR according to claim 1, characterized in that: In step S2, the FeCo@NC material is uniformly dispersed in a MES buffer solution at a mass concentration of 1 g / L. The concentration of the MES buffer solution is 50 mmol / L and the pH is 6.

0.

6. The method for preparing an electrochemical sensor based on FeCo-Anti-MCLR according to claim 1, characterized in that: In step S2, the activation reagents used during activation include EDC solution and NHS solution, wherein the mass concentration of the EDC solution is 5 g / L, and the mass concentration of the NHS solution is 25 g / L.

7. The method for preparing an electrochemical sensor based on FeCo-Anti-MCLR according to claim 1, characterized in that: In step S2, the mass concentration of the Anti-MCLR antibody in the PBS solution containing the Anti-MCLR antibody is 5 mg / L, the coupling temperature is 4° C., and the coupling time is 10 h.

8. The method for preparing an electrochemical sensor based on FeCo-Anti-MCLR according to claim 1, characterized in that: In step S2, the blocking solution used is bovine serum albumin with a concentration of 1 g / L.

9. An electrochemical sensor based on FeCo-Anti-MCLR, characterized in that: Obtained according to the preparation method according to any one of claims 1 to 8.

10. An application of the electrochemical sensor based on FeCo-Anti-MCLR according to claim 9, characterized in that: The FeCo-Anti-MCLR-based electrochemical sensor is used to detect microcystin-LR.

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