Multi-interface heterojunction high-entropy sulfide, preparation method and application thereof, and chloramphenicol concentration detection method

Abstract

The invention provides a multi-interface heterojunction high-entropy sulfide, a preparation method and application thereof, and a chloramphenicol concentration detection method. According to the preparation method disclosed by the invention, (CdZnCoNiMn) Sx and PtRuFeCoMn high-entropy alloy nano-enzymes are prepared by adopting a solvothermal method and an oil bath method; wherein the (CdZnCoNiMn) Sx has optical activity, and the high-entropy alloy nano enzyme shows excellent peroxidase activity. Due to the fact that multiple different metal elements in the (CdZnCoNiMn) Sx are embedded into crystal lattices, the crystal lattice distortion effect is caused, and the photoelectric activity of the material is remarkably improved; and further, signal amplification is successfully realized through biological catalytic precipitation catalyzed by the high-entropy alloy nano-enzyme. The system shows ultrahigh sensitivity and linear response characteristics in chloramphenicol concentration detection, chloramphenicol can be sensitively detected within the range of 0.001-100 ng mL <-1 >, and the detection limit is as low as 0.7454 pg mL <-1 >.

Description

Technical Field

[0001] The present invention relates to the technical field of chloramphenicol concentration detection, and in particular to a multi-interface heterojunction high-entropy sulfide, a preparation method and application thereof, and a chloramphenicol concentration detection method. Background Art

[0002] Chloramphenicol is a broad-spectrum antibiotic. It has the activity of inhibiting a variety of Gram-positive and Gram-negative bacteria, and is also effective against certain anaerobic bacteria, Rickettsia, and Mycoplasma. In clinical applications, chloramphenicol is widely used to treat a variety of infectious diseases. However, its use is accompanied by certain toxic and side effects, including gastrointestinal discomfort, allergic reactions, nervous system toxicity, etc. The most serious one may cause aplastic anemia.

[0003] At present, the technologies for detecting chloramphenicol mainly include high performance liquid chromatography (GC-MS), enzyme-linked immunosorbent assay (ELISA), electrochemical sensors and surface enhanced Raman spectroscopy (SERS). Although these methods have excellent performance in detection accuracy, they are insufficient in operation convenience, cost control and detection speed, which limits their application in rapid detection. In contrast, the photoelectrochemical (PEC) detection technology based on high entropy sulfide has gradually become a powerful tool for rapid detection of chloramphenicol due to its simplicity, rapidity and high cost-effectiveness. However, the photocurrent intensity and stability of high entropy sulfide with high photoelectric activity in multi-interface heterojunction often limit the widespread application of this technology, which is a technical bottleneck that needs to be overcome urgently.

[0004] In recent years, transition metal sulfides with high entropy effect have become ideal candidates for constructing photoelectrochemical (PEC) sensors due to their unique multi-element synergistic effect, high thermodynamic stability and wide light absorption range. High entropy materials show remarkable electronic structure regulation ability and abundant active sites by introducing five or more metal elements, giving them excellent electrochemical performance and photoelectric conversion efficiency. In particular, high entropy transition metal sulfides have attracted extensive attention due to their relatively simple synthesis process, high thermodynamic stability and high specific surface area. By utilizing the diversified element design of high entropy materials, researchers have successfully developed sulfide materials with excellent photoelectric conversion performance. Such materials can not only enhance the charge separation efficiency, but also reduce the loss of active sites, thereby improving the detection sensitivity and stability. Among them, transition metal sulfides with high entropy effect have been successfully developed. However, the photocurrent intensity and stability of the material still need to be improved. Therefore, it is necessary to explore new improvement methods to improve the stability of photoactive materials, thereby improving their sensitivity and accuracy in chloramphenicol detection. Summary of the invention

[0005] The purpose of the present invention is to solve the problems of low photocurrent intensity and poor stability of photoactive materials in the background technology, and to propose a multi-interface heterojunction high-entropy sulfide with high photoelectric activity, a preparation method and application thereof, and a chloramphenicol concentration detection method.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing a multi-interface heterojunction high entropy sulfide, comprising the following steps:

[0008] Dissolving Cd salt, Zn salt, Co salt, Ni salt, Mn salt and thioacetamide in ethylene glycol to obtain a mixed solution;

[0009] The mixed solution is subjected to a solvothermal reaction, and the reaction product is collected, washed, and dried to obtain a multi-interface heterojunction high entropy sulfide.

[0010] Preferably, the Cd salt includes at least one of Cd(CH3COO)2, CdCl2, and Cd(NO3)2;

[0011] And / or, the Zn salt includes at least one of Zn(CH3COO)2, Zn(NO3)2, ZnSO4, and ZnCl2;

[0012] And / or, the Co salt includes at least one of Co(CH3COO)2, Co(NO3)2, CoSO4, and CoCl2;

[0013] And / or, the Ni salt includes at least one of Ni(CH3COO)2, Ni(NO3)2, NiSO4, and NiCl2;

[0014] And / or, the Mn salt includes at least one of Mn(CH3COO)2, Mn(NO3)2, MnSO4, and MnCl2;

[0015] And / or, the molar ratio of the Cd salt, Zn salt, Co salt, Ni salt, Mn salt and thioacetamide is (1-1.2):(1-1.2):(1-1.2):(1-1.2):(1-1.2):(1-1.2);

[0016] The molar volume ratio of the Co salt to the ethylene glycol is (1×10 -3 ~3×10 -3 )mol:(30~40)mL;

[0017] And / or, the solvent thermal reaction temperature is 160-170° C. and the time is 10-15 hours.

[0018] In a second aspect, the present invention also provides a multi-interface heterojunction high entropy sulfide, which is prepared by the preparation method.

[0019] In a third aspect, the present invention also provides a method for preparing a high entropy alloy nanozyme, comprising the following steps:

[0020] Pt salt, Ru salt, Fe salt, Co salt, Mn salt and ascorbic acid are dissolved in a mixture of oleylamine and octadecene, reacted, filtered, washed and dried to obtain a high entropy alloy nanozyme.

[0021] Preferably, the Pt salt is Pt(acac)2;

[0022] And / or, the Ru salt is Ru(acac)3;

[0023] And / or, the Fe salt is Fe(acac)3;

[0024] And / or, the Co salt is Co(acac)2;

[0025] And / or, the Mn salt is Mn(acac)2;

[0026] And / or, the mass ratio of the Pt salt, Ru salt, Fe salt, Co salt, Mn salt and ascorbic acid is (10-11):(5-6):(5-6):(5-6):(5-6):(5-6):(53-54);

[0027] The mass volume ratio of the Pt salt, oleylamine and octadecene is (10-11) mg: (2-3) mL: (2-3) mL;

[0028] And / or, Pt salt, Ru salt, Fe salt, Co salt, Mn salt and ascorbic acid are dissolved in a mixture of oleylamine and octadecene, and in the steps of reaction, filtering and washing, the reaction temperature is 180-200°C, the reaction time is 5-10 hours, and washing is performed using a mixed solution of ethanol and cyclohexane, and the volume ratio of ethanol to cyclohexane is (1-3):(1-3).

[0029] In a fourth aspect, the present invention also provides an application of the multi-interface heterojunction high entropy sulfide in detecting the concentration of chloramphenicol.

[0030] In a fifth aspect, the present invention also provides an application of a high entropy alloy nanozyme prepared by the preparation method in a catalytic reaction for a photoelectrochemical sensor.

[0031] In a sixth aspect, the present invention further provides a biosensor, wherein the method for constructing the biosensor comprises the following steps:

[0032] S1, coating the multi-interface heterojunction high entropy sulfide on the surface of the electrode, and drying to form a modified electrode;

[0033] S2, dropping polymethacrylate sodium salt solution on the surface of the modified electrode, incubating, and washing the electrode with PBS buffer;

[0034] S3, drop the EDC-NHS mixed solution onto the electrode surface in S2, incubate, and wash the electrode with PBS buffer;

[0035] S4, drop the cDNA solution onto the electrode surface in S3, incubate, and wash the electrode with PBS buffer;

[0036] S5, drop the BSA solution onto the electrode surface in S4, incubate, and wash the electrode with PBS buffer;

[0037] S6, adding the aptamer Apt solution dropwise to the electrode surface in S5, incubating, and washing the electrode with PBS buffer;

[0038] S7, dropping the PtRuFeCoMn-SA solution onto the electrode surface in S6, incubating, and washing the electrode with PBS buffer to obtain a biosensor;

[0039] Wherein, the gene sequence of the cDNA is 5'-NH2-TTTTTCTACCACCGACTC-3';

[0040] The gene sequence of the aptamer Apt is 5'-Bio-ACTTCAGTGAGTTGTCCCACGGTCGGCGAGTCGGTGGTAG-3';

[0041] The preparation method of the PtRuFeCoMn-SA solution is:

[0042] Adding the high entropy alloy nanozyme prepared by the preparation method into water to obtain a high entropy alloy nanozyme solution;

[0043] The streptavidin solution was mixed with the high entropy alloy nanozyme solution, the supernatant was removed by centrifugation, and the precipitate was collected; PBS buffer was added to the precipitate to obtain a PtRuFeCoMn-SA solution.

[0044] Preferably, the method for constructing the biosensor comprises the following steps:

[0045] S1. Add the multi-interface heterojunction high entropy sulfide into water to obtain 1-3 mg mL -1 A multi-interface heterojunction high entropy sulfide solution is coated on the electrode surface and dried to form a modified electrode;

[0046] S2, dripping polymethacrylate sodium salt solution onto the surface of the modified electrode, incubating at 36-37°C for 0.5-1h, and washing the electrode with PBS buffer; the mass concentration of the polymethacrylate sodium salt solution is 0.05-0.1%;

[0047] S3, dripping the EDC-NHS mixed solution onto the electrode surface in S2, incubating at 36-37°C for 1-2 hours, and washing the electrode with PBS buffer; the preparation method of the EDC-NHS mixed solution is as follows: adding EDC and NHS into PBS buffer to obtain an EDC-NHS mixed solution, wherein the concentration of EDC is 10-12 mM, and the concentration of NHS is 2-4 mM;

[0048] S4, dropping the cDNA solution onto the electrode surface in S3, incubating at 4-5°C for 12-15h, and washing the electrode with PBS buffer; the preparation method of the cDNA solution is: adding cDNA to PBS buffer to obtain a cDNA solution, wherein the concentration of the cDNA solution is 1.0-1.2 μM;

[0049] S5, dropping the BSA solution onto the electrode surface in S4, incubating at 36-37°C for 1-2 hours, and washing the electrode with PBS buffer; the preparation method of the BSA solution is: adding BSA to PBS buffer to obtain a BSA solution; the mass concentration of the BSA solution is 1.0-2.0%;

[0050] S6, adding the aptamer Apt solution dropwise to the electrode surface in S5, incubating at 36-37° C. for 1-2 hours, and washing the electrode with PBS buffer; the preparation method of the aptamer Apt solution is: adding the aptamer Apt to the PBS buffer to obtain the aptamer Apt solution, wherein the concentration of the aptamer Apt solution is 1.0-1.2 μM;

[0051] S7, adding the PtRuFeCoMn-SA solution to the electrode surface in S6, incubating at 36-37°C for 1-2h, and washing the electrode with PBS buffer to obtain a biosensor;

[0052] The concentration of the streptavidin solution is 1-2 mg / mL, and the concentration of the high entropy alloy nanozyme solution is 1-2 mg / mL;

[0053] The streptavidin solution is mixed with the high entropy alloy nanozyme solution, the supernatant is removed by centrifugation, and the precipitate is collected; in the step of adding PBS buffer to the precipitate, the volume ratio of the streptavidin solution, the high entropy alloy nanozyme solution, and the PBS buffer solution is (200-220):(400-420):(600-620);

[0054] The pH of the PBS buffer is 7.4-7.5.

[0055] In a seventh aspect, the present invention also provides a method for detecting chloramphenicol concentration, comprising the following steps:

[0056] Providing the biosensor;

[0057] Add chloramphenicol test solution to the electrode surface of the biosensor, incubate at 36-37°C for 1-2 hours, and wash the electrode with PBS buffer;

[0058] DAB solution containing H2O2 was added dropwise to the electrode surface, incubated at 36-37°C for 10-30 min, and the electrode was washed with PBS buffer;

[0059] The electrode was placed in a PBS buffer containing ascorbic acid, and the photoresponse current signal of the electrode was detected;

[0060] Detecting chloramphenicol concentration based on the light-responsive current signal;

[0061] The concentration of the DAB solution is 1.0-1.2 mM, and the concentration of H2O2 contained in the DAB solution is 0.2-0.3 mM;

[0062] The concentration of ascorbic acid in the PBS buffer containing ascorbic acid is 0.1 to 0.2 M.

[0063] The multi-interface heterojunction high entropy sulfide and its preparation method and application, and chloramphenicol concentration detection method of the present invention have the following beneficial effects compared with the prior art:

[0064] The present invention adopts solvent thermal method and oil bath method to prepare (CdZnCoNiMn)S x and PtRuFeCoMn high entropy alloy nanozymes. Among them, (CdZnCoNiMn)S x The (CdZnCoNiMn)S nanozyme has excellent peroxidase activity. Based on this, the present invention constructs a highly selective photoelectrochemical (PEC) aptamer biosensor specifically for the analysis and detection of chloramphenicol concentration. x The embedding of various metal elements into the lattice leads to a lattice distortion effect, which significantly improves the photoelectric activity of the material. The signal amplification is further achieved by biocatalytic precipitation catalyzed by PtRuFeCoMn high entropy alloy nanozymes. The system exhibits ultrahigh sensitivity and linear response characteristics in the detection of chloramphenicol concentration, and has excellent stability and reliability. Through photoelectrochemical (PEC) detection, this method can detect chloramphenicol concentrations in the range of 0.001 to 100 ng mL -1 Sensitive detection of chloramphenicol in the range of 2.5-6.5 μg / mL -1At the same time, the sensor showed good selectivity for chloramphenicol. Further applied in the fields of food safety and environmental testing, this method obtained satisfactory results, demonstrating its broad potential in practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art descriptions. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0066] Figure 1 The present invention is based on (CdZnCoNiMn)S x Schematic diagram of the constructed photoelectrochemical (PEC) biosensor for the detection of chloramphenicol;

[0067] Figure 2 (CdZnCoNiMn)S prepared in step S4 of Example 1 x Performance and structural characterization of multi-interface heterojunctions;

[0068] Figure 3 (CdZnCoNiMn)S prepared in step S4 of Example 1 x XRD patterns of multi-interface heterojunctions;

[0069] Figure 4 (CdZnCoNiMn)S prepared in step S4 of Example 1 x XPS spectra of multi-interface heterojunctions;

[0070] Figure 5 Structural characterization of the PtRuFeCoMn high entropy alloy nanozyme prepared in step S4 of Example 2;

[0071] Figure 6 The catalytic performance of the PtRuFeCoMn high entropy alloy nanozyme prepared in step S4 of Example 2;

[0072] Figure 7 Based on the (CdZnCoNiMn)S prepared in step S4 of Example 1 x Schematic diagram of the feasibility of the constructed photoelectrochemical (PEC) sensor;

[0073] Figure 8 According to the method in Example 4, the chloramphenicol concentration detection condition optimization diagram is optimized;

[0074] Fig. 9 Schematic diagram of photoelectrochemical (PEC) sensing analysis of chloramphenicol;

[0075] Fig.10 This is a comparison chart of the selectivity for detecting chloramphenicol. DETAILED DESCRIPTION

[0076] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0077] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0078] It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of the embodiments. In addition, in the description of the present application, the term "including" means "including but not limited to". Various embodiments of the present invention may be in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single values ​​within the range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the numbered ranges, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated in this article, it is meant to include any quoted numbers (fractions or integers) within the indicated range.

[0079] The present invention provides a method for preparing a multi-interface heterojunction high entropy sulfide, comprising the following steps:

[0080] S1, dissolving Cd salt, Zn salt, Co salt, Ni salt, Mn salt and thioacetamide in ethylene glycol to obtain a mixed solution;

[0081] S2. subjecting the mixed solution to a solvothermal reaction, collecting the reaction products, washing them, and drying them to obtain a multi-interface heterojunction high entropy sulfide.

[0082] In some embodiments, the Cd salt includes at least one of Cd(CH3COO)2, CdCl2, and Cd(NO3)2.

[0083] In some embodiments, the Zn salt includes at least one of Zn(CH3COO)2, Zn(NO3)2, ZnSO4, and ZnCl2.

[0084] In some embodiments, the Co salt includes at least one of Co(CH3COO)2, Co(NO3)2, CoSO4, and CoCl2.

[0085] In some embodiments, the Ni salt includes at least one of Ni(CH3COO)2, Ni(NO3)2, NiSO4, and NiCl2.

[0086] In some embodiments, the Mn salt includes at least one of Mn(CH3COO)2, Mn(NO3)2, MnSO4, and MnCl2.

[0087] In some embodiments, the molar ratio of Cd salt, Zn salt, Co salt, Ni salt, Mn salt and thioacetamide is (1-1.2):(1-1.2):(1-1.2):(1-1.2):(1-1.2):(1-1.2); preferably, the molar ratio is 1:1:1:1:1:1.

[0088] In some embodiments, the molar volume ratio of the Co salt to the ethylene glycol is (1×10 -3 ~3×10 -3 )mol:(30~40)mL; Preferably, the molar volume ratio of Co salt and ethylene glycol is 1×10 -3 mol:35mL.

[0089] In some embodiments, the solvothermal reaction temperature is 160-170° C. and the reaction time is 10-15 h.

[0090] The present invention uses a solvent thermal method to prepare (CdZnCoNiMn)S x Multi-interface heterojunction, specifically, Cd salt, Zn salt, Co salt, Ni salt, Mn salt and thioacetamide are dissolved in ethylene glycol to obtain a mixed solution, the mixed solution is subjected to a solvothermal reaction at 160°C for 12 hours in a 50 mL Teflon-lined stainless steel autoclave, cooled to room temperature, filtered to obtain a product, washed with deionized water and ethanol, and dried at 60°C to obtain a multi-interface heterojunction high-entropy sulfide with high photoelectric activity.

[0091] Based on the same inventive concept, the present invention also provides a multi-interface heterojunction high-entropy sulfide, which is prepared by the above-mentioned preparation method.

[0092] Based on the same inventive concept, the present invention also provides a method for preparing a high entropy alloy nanozyme, comprising the following steps:

[0093] Pt salt, Ru salt, Fe salt, Co salt, Mn salt and ascorbic acid are dissolved in a mixture of oleylamine and octadecene, reacted, filtered, washed and dried to obtain a high entropy alloy nanozyme.

[0094] In some embodiments, the Pt salt is Pt(acac)2 (platinum diacetylacetonate).

[0095] In some embodiments, the Ru salt is Ru(acac)3 (ruthenium(III) acetylacetonate).

[0096] In some embodiments, the Fe salt is Fe(acac)3 (iron triacetylacetonate).

[0097] In some embodiments, the Co salt is Co(acac)2 (cobalt(II) acetylacetonate).

[0098] In some embodiments, the Mn salt is Mn(acac)2 (manganese(II) acetylacetonate).

[0099] In some embodiments, the mass ratio of Pt salt, Ru salt, Fe salt, Co salt, Mn salt and ascorbic acid is (10-11):(5-6):(5-6):(5-6):(5-6):(5-6):(53-54); preferably, the mass ratio is 10:5:5:5:5:53.6.

[0100] In some embodiments, the mass volume ratio of Pt salt, oleylamine and octadecene is (10-11) mg:(2-3) mL:(2-3) mL; preferably, the mass volume ratio is 10 mg:2.5 mL:2.5 mL.

[0101] In some embodiments, Pt salt, Ru salt, Fe salt, Co salt, Mn salt and ascorbic acid are dissolved in a mixture of oleylamine and octadecene. In the steps of reaction, filtering and washing, the reaction temperature is 180-200°C and the reaction time is 5-10 hours. A mixed solution of ethanol and cyclohexane is used for washing, and the volume ratio of ethanol to cyclohexane is (1-3):(1-3). Preferably, the volume ratio of ethanol to cyclohexane is 1:1.

[0102] In some embodiments, Pt salt, Ru salt, Fe salt, Co salt, Mn salt and ascorbic acid are dissolved in a mixture of oleylamine and octadecene, reacted in an oil bath in a 30 mL sample bottle for 5 hours, cooled to room temperature, filtered to obtain a precipitate, and the reaction product is washed with a mixture of ethanol and cyclohexane in a volume ratio of 1:1, and dried to obtain a high entropy alloy nanozyme.

[0103] Based on the same inventive concept, the present invention also provides an application of the above-mentioned multi-interface heterojunction high-entropy sulfide in detecting the concentration of chloramphenicol.

[0104] Based on the same inventive concept, the present invention also provides an application of a high entropy alloy nanozyme prepared by the above-mentioned preparation method in a catalytic reaction for a photoelectrochemical sensor.

[0105] Based on the same inventive concept, the present invention also provides a biosensor, and the method for constructing the biosensor comprises the following steps:

[0106] S1, coating the multi-interface heterojunction high entropy sulfide on the electrode surface, and drying to form a modified electrode;

[0107] S2, dropping a polymethacrylate sodium salt solution onto the surface of the modified electrode, incubating, and washing the electrode with a PBS buffer; functionalizing the electrode with a polymethacrylate sodium (PAA) solution;

[0108] S3, dripping the EDC-NHS mixed solution onto the electrode surface in S2, incubating, and washing the electrode with PBS buffer; using the EDC-NHS mixed solution to remove the carboxyl groups on the electrode surface;

[0109] S4, dropping the cDNA solution onto the electrode surface in S3, incubating, and washing the electrode with PBS buffer; fixing the cDNA molecules on the electrode surface;

[0110] S5, dropping BSA (bovine serum albumin (BSA)) solution on the electrode surface in S4, incubating, and washing the electrode with PBS buffer; using BSA solution to block nonspecific binding;

[0111] S6, adding the aptamer Apt solution to the electrode surface in S5, incubating, and washing the electrode with PBS buffer; hybridizing the aptamer Apt connected to the nanozyme with the cDNA to modify the electrode surface

[0112] S7. Add the PtRuFeCoMn-SA solution dropwise onto the electrode surface in S6, incubate, and wash the electrode with PBS buffer to obtain a biosensor.

[0113] In some embodiments, the gene sequence of the cDNA is 5'-NH2-TTTTTCTACCACCGACTC-3', as shown in SEQ ID NO:1.

[0114] In some embodiments, the gene sequence of the aptamer Apt is 5′-Bio-ACTTCAGTGAGTTGTCCCACGGTCGGCGAGTCGGTGGTAG-3′, as shown in SEQ ID NO:2.

[0115] In some embodiments, the preparation method of the PtRuFeCoMn-SA solution is:

[0116] Adding the high entropy alloy nanozyme prepared by the above preparation method into water to obtain a high entropy alloy nanozyme solution;

[0117] The streptavidin solution was mixed with the high entropy alloy nanozyme solution, the supernatant was removed by centrifugation, and the precipitate was collected; PBS buffer was added to the precipitate to obtain a PtRuFeCoMn-SA solution.

[0118] In some embodiments, the method for constructing a biosensor comprises the following steps:

[0119] S1. Add the multi-interface heterojunction high entropy sulfide into water to obtain 1-3 mg mL -1 A multi-interface heterojunction high entropy sulfide solution is coated on the electrode surface and dried to form a modified electrode;

[0120] S2, dripping polymethacrylate sodium salt solution onto the surface of the modified electrode, incubating at 36-37°C for 0.5-1h, and washing the electrode with PBS buffer; the mass concentration of polymethacrylate sodium salt solution is 0.05-0.1%;

[0121] S3, dripping the EDC-NHS mixed solution onto the electrode surface in S2, incubating at 36-37°C for 1-2 hours, and washing the electrode with PBS buffer; the preparation method of the EDC-NHS mixed solution is as follows: adding EDC and NHS into PBS buffer to obtain an EDC-NHS mixed solution, wherein the concentration of EDC is 10-12 mM, and the concentration of NHS is 2-4 mM;

[0122] S4, dropping the cDNA solution onto the electrode surface in S3, incubating at 4-5°C for 12-15h, and washing the electrode with PBS buffer; the preparation method of the cDNA solution is: adding cDNA to PBS buffer to obtain a cDNA solution, the concentration of the cDNA solution is 1.0-1.2 μM;

[0123] S5, dropping the BSA solution onto the electrode surface in S4, incubating at 36-37°C for 1-2 hours, and washing the electrode with PBS buffer; the preparation method of the BSA solution is: adding BSA to PBS buffer to obtain a BSA solution; the mass concentration of the BSA solution is 1.0-2.0%;

[0124] S6, adding the aptamer Apt solution dropwise to the electrode surface in S5, incubating at 36-37° C. for 1-2 hours, and washing the electrode with PBS buffer; the preparation method of the aptamer Apt solution is as follows: adding the aptamer Apt to the PBS buffer to obtain the aptamer Apt solution, wherein the concentration of the aptamer Apt solution is 1.0-1.2 μM;

[0125] S7, adding the PtRuFeCoMn-SA solution to the electrode surface in S6, incubating at 36-37°C for 1-2h, and washing the electrode with PBS buffer to obtain a biosensor;

[0126] The concentration of the streptavidin solution is 1-2 mg / mL, and the concentration of the high entropy alloy nanozyme solution is 1-2 mg / mL;

[0127] The streptavidin solution is mixed with the high entropy alloy nanozyme solution, the supernatant is removed by centrifugation, and the precipitate is collected; in the step of adding PBS buffer to the precipitate, the volume ratio of the streptavidin solution, the high entropy alloy nanozyme solution, and the PBS buffer solution is (200-220):(400-420):(600-620);

[0128] The pH of PBS buffer is 7.4-7.5.

[0129] In some embodiments, the electrode is a FTO electrode.

[0130] In the above embodiment, EDC is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, NHS is N-hydroxysuccinimide, and the streptavidin solution is prepared by adding streptavidin to PBS buffer to obtain a streptavidin solution.

[0131] In some embodiments, the polymethacrylate sodium salt solution is also called sodium polymethacrylate, sodium polyacrylate, and polyacrylic acid sodium salt.

[0132] The biosensor constructed by the present invention can be used to detect the concentration of chloramphenicol in the range of 0.001 to 100 ng mL -1 Sensitive detection of chloramphenicol in the range of 2.5-6.5 μg / mL -1 Meanwhile, the sensor exhibited good selectivity for chloramphenicol.

[0133] Based on the same inventive concept, the present invention also provides a method for detecting chloramphenicol concentration, comprising the following steps:

[0134] Providing the above-mentioned biosensor;

[0135] Add chloramphenicol (CAP) test solution to the electrode surface of the biosensor, incubate at 36-37°C for 1-2 hours, and wash the electrode with PBS buffer;

[0136] DAB solution containing H2O2 was added dropwise to the electrode surface, incubated at 36-37°C for 10-30 min, and the electrode was washed with PBS buffer;

[0137] The electrode was placed in a PBS buffer containing ascorbic acid, and the photoresponse current signal of the electrode was detected;

[0138] Detecting chloramphenicol concentration based on the light-responsive current signal;

[0139] The concentration of the DAB solution is 1.0-1.2 mM, and the concentration of H2O2 contained in the DAB solution is 0.2-0.3 mM; DAB is 3,3'-diaminobenzidine; the preparation method of the DAB solution is: adding DAB to water to obtain the DAB solution;

[0140] The concentration of ascorbic acid in the PBS buffer containing ascorbic acid is 0.1 to 0.2 M.

[0141] In some embodiments, the electrode is placed in a PBS buffer containing ascorbic acid, and the light response of the detection electrode is induced to generate a current signal at 0V.

[0142] Specifically, in the above embodiment, the electrode is placed in a PBS buffer solution containing ascorbic acid to form a three-electrode system, the PBS buffer solution containing ascorbic acid is used as the electrolyte, the platinum electrode is used as the counter electrode, the silver-silver chloride electrode is used as the reference electrode, the above-mentioned modified electrode is used as the working electrode, and an electrochemical workstation is used. The xenon lamp is irradiated for 10 seconds, turned off for 10 seconds, and a cycle of 20 seconds is performed for 400 seconds to detect the light response current signal of the electrode.

[0143] The principle of the chloramphenicol concentration detection method of the present invention is as follows: Figure 1 As shown, the sensor uses (CdZnCoNiMn)S xAs a multi-interface heterojunction high entropy sulfide with high photoelectric activity, it effectively amplifies the initial photoelectrochemical (PEC) signal. The cDNA with amino group modified at the 5' end and the chloramphenicol aptamer Apt modified with biotin at the 5' end hybridize on the electrode surface to form a double-stranded structure. Subsequently, the electrode is incubated with the PtRuFeCoMn high entropy alloy nanozyme labeled with streptavidin, and streptavidin specifically binds to the biotin on the aptamer, so that the PtRuFeCoMn high entropy alloy nanozyme is connected to the electrode surface to form a complete detection probe. In the detection solution, when hydrogen peroxide (H2O2) is present, the PtRuFeCoMn high entropy alloy nanozyme catalyzes the oxidation of DAB (3,3'-diaminobenzidine) through its peroxidase-like activity to generate a non-conductive precipitate, resulting in a significant decrease in the photocurrent. However, when chloramphenicol is present in the solution, the chloramphenicol molecule specifically binds to the aptamer, causing the aptamer to fall off the electrode surface, and then the PtRuFeCoMn high entropy alloy nanozyme is removed. This process prevents the oxidation reaction of DAB, reduces the generation of precipitate, and leads to the enhancement of photocurrent. By monitoring the change of photocurrent, the present invention realizes the high sensitivity and high selectivity detection of chloramphenicol in solution. When the concentration of chloramphenicol increases, the photocurrent gradually increases, thereby achieving the purpose of specific and quantitative detection of chloramphenicol.

[0144] The present invention provides a multi-interface heterojunction high entropy sulfide with high photoelectric activity, a preparation method and application thereof, and a chloramphenicol concentration detection method. Specifically, the present invention adopts a solvothermal method and an oil bath method to prepare a multi-interface heterojunction high entropy sulfide (CdZnCoNiMn)S x and high entropy alloy nanozymes PtRuFeCoMn. Among them, (CdZnCoNiMn)S x The (CdZnCoNiMn)S nanozyme has excellent peroxidase activity. Based on this, the present invention constructs a highly selective photoelectrochemical (PEC) aptamer sensor specifically for the analysis and detection of chloramphenicol concentration. x In the multi-interface heterojunction, a variety of different metal elements are embedded in the lattice, resulting in a lattice distortion effect, which significantly improves the photoelectric activity of the material; further, the signal amplification is successfully achieved through the biocatalytic precipitation catalyzed by the PtRuFeCoMn high entropy alloy nanozyme. The system shows ultra-high sensitivity and linear response characteristics in the detection of chloramphenicol concentration, with excellent stability and reliability. Through photoelectrochemical (PEC) detection, this method can detect chloramphenicol concentrations in the range of 0.001 to 100 ng mL -1 Sensitive detection of chloramphenicol in the range of 2.5-6.5 μg / mL -1At the same time, the sensor showed good selectivity for chloramphenicol. Further applied in the fields of food safety and environmental testing, this method obtained satisfactory results, demonstrating its broad potential in practical applications.

[0145] The following further describes the multi-interface heterojunction high entropy sulfide of the present application and its preparation method and application, and the chloramphenicol concentration detection method with specific examples. This section further illustrates the content of the present invention in conjunction with specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0146] In the following examples, the polymethacrylate sodium salt solution was prepared by adding water to a commercially available polymethacrylate sodium salt solution (purchased from Sigma-Aldrich, product number 434507) to prepare a polymethacrylate sodium salt solution with a mass concentration of 0.05 wt %.

[0147] Example 1

[0148] The present invention provides a method for preparing a multi-interface heterojunction high entropy sulfide, comprising the following steps:

[0149] S1. Cd salt (Cd(CH3COO)2·2H2O), Zn salt (Zn(CH3COO)2·2H2O), Co salt (Co(CH3COO)2·2H2O), Ni salt (Ni(CH3COO)2·2H2O), Mn salt (Mn(CH3COO)2·2H2O) and thioacetamide were dissolved in ethylene glycol at a molar ratio of 1:1:1:1:1:1, and ultrasonicated for 30 min to obtain a mixed solution; wherein the molar volume ratio of Cd(CH3COO)2·2H2O to ethylene glycol was 1×10 -3 mol:35mL;

[0150] S2, transfer the mixed solution in S1 to a 50 mL Teflon-lined stainless steel autoclave and perform a solvothermal reaction at 160 °C for 12 hours;

[0151] S3. After the reaction is completed, the reactor is cooled to room temperature, the reaction product is centrifuged at 6000 rpm for 15 minutes, the supernatant is removed, and the precipitate is collected;

[0152] S4. The collected precipitate was washed three times with deionized water and ethanol respectively to remove unreacted precursors and by-products; the washed product was vacuum dried at 60°C to finally obtain a light green multi-interface heterojunction high entropy sulfide (denoted as (CdZnCoNiMn)S x ).

[0153] Example 2

[0154] The present invention provides a method for preparing a high entropy alloy nanozyme, comprising the following steps:

[0155] S1. Dissolve Pt salt (Pt(acac)2), Ru salt (Ru(acac)3), Fe salt (Fe(acac)3), Co salt (Co(acac)2), Mn salt (Mn(acac)2) and ascorbic acid (AA) in oleylamine (OA) and octadecene (ODE) in a mass ratio of 10:5:5:5:5:53.6, and ultrasonicate for 1 h to obtain a uniform solution; wherein the mass volume ratio of Pt(acac)2 to oleylamine and octadecene is 10 mg:2.5 mL:2.5 mL;

[0156] S2, placing the homogeneous solution in an oil bath and reacting at 180°C for 5 hours;

[0157] S3. After the reaction is completed, the product is washed with ethanol and cyclohexane solution in a volume ratio of 1:1 until the solution is colorless;

[0158] S4. The washed product is vacuum dried at 60°C to finally obtain PtRuFeCoMn high entropy alloy nanozyme (denoted as PtRuFeCoMn).

[0159] 400 μL of the PtRuFeCoMn high entropy alloy nanozyme prepared in Example 2 (concentration 1 mg / mL, solvent is water) was mixed with 200 μL of streptavidin (concentration 1 mg / mL, solvent is PBS), gently shaken overnight at 4°C, centrifuged and the supernatant was removed, 600 μL of PBS was added to dissolve, and stored in a 4°C refrigerator for use.

[0160] Example 3

[0161] The present invention provides a method for preparing a biosensor, comprising the following steps:

[0162] S1. Ultrasonic clean the FTO electrode (effective area 5×5 mm) and dry it; add 20 μL (CdZnCoNiMn)S x The solution was dripped onto the surface of the FTO electrode and dried naturally to form (CdZnCoNiMn)S x / FTO modified electrode; (CdZnCoNiMn)S x The solution preparation method is to prepare the (CdZnCoNiMn)S prepared in Example 1. x Add to water to obtain 2.5 mg mL -1 (CdZnCoNiMn)S x Solution;

[0163] S2, add 20 μL of polymethacrylate sodium salt solution (PAA, 0.05 wt%, solvent is water) to the electrode surface in S1, and incubate at 37°C for 30 minutes, and wash the electrode with PBS buffer (pH=7.4);

[0164] S3, on the surface of the electrode in S2, add 20 μL of EDC-NHS mixed solution (10 mM: 2.0 mM, the solvent is PBS buffer solution with a pH of 7.4), and incubate at 37°C for 1 hour to activate the carboxyl group, and use PBS buffer (pH = 7.4) to clean the electrode; the preparation method of the EDC-NHS mixed solution is: add EDC and NHS into PBS buffer to obtain an EDC-NHS mixed solution, wherein the concentration of EDC is 10 mM and the concentration of NHS is 2 mM;

[0165] S4. On the electrode surface in S3, 20 μL of cDNA solution (1.0 μM, the solvent is PBS buffer with a pH of 7.4, and the cDNA sequence number is 5'-NH2-TTTTTCTACCACCGACTC-3') was added dropwise, and incubated at 4°C for 12 h. The electrode was washed with PBS buffer (pH = 7.4); the preparation method of the cDNA solution was as follows: cDNA was added to PBS buffer (pH = 7.4) to obtain a cDNA solution, and the concentration of the cDNA solution was 1.0 μM;

[0166] S5. On the electrode surface in S4, 20 μL of BSA solution (mass concentration 1.0 wt%) was added dropwise to the electrode surface, and incubated at room temperature (37° C.) for 1 hour to block nonspecific binding sites, and the electrode was cleaned with PBS buffer (pH=7.4); the BSA solution was prepared by adding BSA to PBS buffer (pH=7.4) to obtain a BSA solution with a mass concentration of 1%;

[0167] S6. On the electrode surface in S5, 20 μL of aptamer Apt solution (1.0 μM, the solvent is PBS buffer solution with a pH of 7.4, and the sequence number of aptamer Apt is 5'-Bio-ACTTCAGTGAGTTGTCCCACGGTCGGCGAGTCGGTGGTAG-3') was added dropwise, and incubated at 37°C for 1 hour to allow it to fully hybridize with the fixed cDNA molecules, and the electrode was washed with PBS buffer (pH=7.4); the preparation method of the aptamer Apt solution is as follows: the aptamer Apt is added to PBS buffer (pH=7.4) to obtain the aptamer Apt solution, and the concentration of the aptamer Apt solution is 1.0 μM;

[0168] S7, dropping 20 μL of PtRuFeCoMn-SA solution on the electrode surface in S6, incubating at 37°C for 1-2 h, and washing the electrode with PBS buffer to obtain a biosensor;

[0169] The configuration method of PtRuFeCoMn-SA solution is:

[0170] 200 μL of streptavidin solution (SA, 1 mg / mL) and 400 μL of PtRuFeCoMn solution (1 mg / mL, solvent: water) were shaken overnight at 4°C, the supernatant was removed by centrifugation, and the precipitate was collected; 600 μL of PBS buffer (pH=7.4) was added to the precipitate, and the solution was evenly dissolved by shaking to obtain a PtRuFeCoMn-SA solution;

[0171] The streptavidin solution was prepared by adding streptavidin to PBS buffer (pH=7.4) to obtain a streptavidin solution with a concentration of 1 mg / mL;

[0172] The preparation method of the PtRuFeCoMn solution is as follows: the PtRuFeCoMn prepared in Example 2 is added into water to obtain a PtRuFeCoMn solution with a concentration of 1 mg / mL.

[0173] Example 4

[0174] This embodiment provides a method for detecting chloramphenicol concentration, comprising the following steps:

[0175] S1. constructing a biosensor according to the method in Example 3;

[0176] S2. Add 20 μL of different concentrations (0.001-100 ng mL -1 ) of chloramphenicol (CAP) test solution, incubate at 37°C for 1 h, and wash the electrode with PBS buffer (pH = 7.4);

[0177] S3, add 20 μL of 1.0 mM DAB solution containing 0.2 mM H2O2 (DAB solution is prepared by adding DAB to water to obtain a DAB solution with a concentration of 1.0 mM) to the electrode surface, incubate at 37°C for 25 min, and wash the electrode with PBS buffer (pH = 7.4);

[0178] S4, placing the electrode in S3 (as the working electrode) in PBS buffer (pH = 7.4) containing 0.1 M ascorbic acid (AA, as a sacrificial electron donor), using a platinum electrode as a counter electrode and a silver-silver chloride electrode as a reference electrode, using an electrochemical workstation, irradiating with a xenon lamp for 10 s, turning off for 10 s, and 20 s in a cycle, performing photoelectrochemical detection at 0 V voltage for 400 s, and detecting the light response current signal of the working electrode;

[0179] S5, detecting the concentration of chloramphenicol according to the light response current signal;

[0180] Performance Characterization

[0181] Figure 2 The multi-interface heterojunction high entropy sulfide (CdZnCoNiMn)S prepared in Example 1 x performance and structural characterization;

[0182] Figure 2 A in it is: (CdZnCoNiMn)S x Photocurrent measured for the multi-interface heterojunction in PBS buffer (pH=7.4) containing 0.1 M ascorbic acid.

[0183] Figure 2 Where B is (CdZnCoNiMn)S prepared in step S4 of Example 1 x SEM image of multi-interface heterojunction.

[0184] Figure 2 C is (CdZnCoNiMn)S prepared in step S4 of Example 1 x TEM image of multi-interface heterojunction.

[0185] Figure 2 Where D is (CdZnCoNiMn)S prepared in step S4 of Example 1 x STEM image of a multi-interface heterojunction.

[0186] Figure 2 Where E is (CdZnCoNiMn)S prepared in step S4 of Example 1 x HAADF-STEM-EDS elemental mapping of multi-interface heterojunction.

[0187] from Figure 2 As can be seen in A, in PBS buffer containing 0.1 M ascorbic acid at pH = 7.4, (CdZnCoNiMn)S xThe photocurrent response of the multi-interface heterojunction under 20 switching cycles of a xenon lamp; the photoelectric performance of the material remains stable under long-term optical switching operation without obvious attenuation, and has good cycle stability and photoelectric response characteristics. x Can be used to detect chloramphenicol concentration.

[0188] from Figure 2 The transmission electron microscope images show that the present invention successfully prepared (CdZnCoNiMn)S with various lattice arrangements. x Multi-interface heterojunction.

[0189] Figure 3 (CdZnCoNiMn)S prepared in step S4 of Example 1 x XRD patterns of multi-interface heterojunctions.

[0190] from Figure 3 It can be seen that the diffraction peak is similar to (Cd 0.8 Co 0.2 )S standard card, but due to the doping of various metal elements, its lattice has undergone certain adjustments and distortions, confirming the multi-component characteristics of the material, which confirms that (CdZnCoNiMn)S x Successful synthesis of multi-interface heterojunctions.

[0191] Figure 4 is (CdZnCoNiMn)S prepared in step S4 in Example 1 x XPS spectra of multi-interface heterojunctions, where A is the XPS spectrum of Cd 3d, B is the XPS spectrum of Zn 2p, C is the XPS spectrum of Co 2p; D is the XPS spectrum of Ni 2p; E is the XPS spectrum of Mn 2p; and F is the XPS spectrum of S2p.

[0192] XPS analysis revealed the chemical state of each element in the material and its binding energy, and further confirmed its surface chemical state.

[0193] Figure 5 This is the structural characterization of the PtRuFeCoMn high entropy alloy nanozyme prepared in step S4 in Example 2.

[0194] Specifically, Figure 5A is the TEM image of the PtRuFeCoMn high entropy alloy nanozyme prepared in step S4 in Example 2, B is the TEM image of the PtRuFeCoMn high entropy alloy nanozyme prepared in step S4 in Example 2, C is the STEM image of the PtRuFeCoMn high entropy alloy nanozyme prepared in step S4 in Example 2, D is the STEM image of the PtRuFeCoMn high entropy alloy nanozyme prepared in step S4 in Example 2, and E is the HAADF-STEM-EDS element mapping image of the PtRuFeCoMn high entropy alloy nanozyme prepared in step S4 in Example 2.

[0195] from Figure 5 It can be seen that the present invention successfully prepared PtRuFeCoMn high entropy alloy nanozyme.

[0196] Figure 6 This is the UV spectrum measured when the PtRuFeCoMn high entropy alloy nanozyme prepared in step S4 in Example 2 reacts with TMB+H2O2 in an acetic acid-sodium acetate buffer solution at pH = 4.0.

[0197] Specifically, Figure 6 In a, PtRuFeCoMn high entropy alloy nanozyme solution (concentration 0.5 mg / mL, the solvent used is water), H2O2 solution (concentration 50 mM, the solvent used is water), TMB solution (concentration 10 mM, the solvent used is water, TMB is 3,3',5,5'-tetramethylbenzidine), and acetic acid-sodium acetate buffer solution with a pH of 4.0 were mixed at room temperature in a volume ratio of 1:1:1:17, incubated for 5 minutes, and the absorbance at 652 nm was tested; Figure 6 b: H2O2 solution (concentration 50 mM, the solvent used is water), TMB solution (concentration 10 mM, the solvent used is water), and acetic acid-sodium acetate buffer solution of pH = 4.0 were mixed at room temperature in a volume ratio of 1:1:18, incubated for 5 minutes, and the absorbance at 652 nm was tested; Figure 6 In c, TMB solution (concentration 10 mM, solvent used is water) and acetic acid-sodium acetate buffer solution of pH=4.0 were mixed at room temperature in a volume ratio of 1:19, incubated for 5 minutes, and the absorbance at 652 nm was tested.

[0198] from Figure 6 It can be seen that after mixing the PtRuFeCoMn high entropy alloy nanozyme, acetic acid-sodium acetate buffer solution with pH = 4.0, H2O2, and TMB, it has an extremely high absorbance at 652nm. Therefore, the PtRuFeCoMn high entropy alloy nanozyme of the present invention can be used for photoelectrochemical (PEC) sensor biocatalytic precipitation.

[0199] Figure 7is (CdZnCoNiMn)S prepared in step S4 in Example 1 x Schematic diagram of feasibility analysis of detection of multi-interface heterojunctions obtained according to the method in Example 3;

[0200] Figure 7 A in it is: (CdZnCoNiMn)S x The photoresponse current measured by the multi-interface heterojunction in PBS buffer (pH=7.4) containing 0.1M ascorbic acid (AA). Specifically, Figure 7 a in A is 20 μL (CdZnCoNiMn)S x The solution (concentration 2.5 mg / mL, the solvent used was water) was naturally dried on the surface of a clean FTO electrode (effective area 5×5 mm) to form (CdZnCoNiMn)S x / FTO modified electrode, and its photoresponse current at 0V voltage was tested; b was adding 20μL polymethacrylate sodium salt solution (PAA, concentration 0.05wt%) to the above electrode surface, incubating at 37℃ for 30 minutes, and then dropping 20μL EDC-NHS mixed solution (EDC concentration 10mM, NHS concentration 2.0mM, solvent is PBS buffer solution with pH 7.4), and incubating at 4℃ for 1 hour to form (CdZnCoNiMn)S x / PAA / FTO modified electrode, and its photoresponse current at 0V voltage was tested; c is 20μL cDNA solution (concentration 1μM) was added to the surface of the above electrode and incubated at 4℃ for 12h to form (CdZnCoNiMn)S x / PAA / cDNA / FTO modified electrode, and its photoresponse current at 0V voltage was tested; d is to drop 20μL BSA solution (concentration 1.0wt%) onto the surface of the above electrode and incubate at 4℃ for 1 hour to form (CdZnCoNiMn)S x / PAA / cDNA / BSA / FTO modified electrode, and its photoresponse current at 0V voltage was tested; e is 20μL of aptamer Apt solution (concentration 1μM) was dropped onto the surface of the above electrode, incubated at 37℃ for 1 hour, and then 20μL of PtRuFeCoMn-SA solution was added, and incubated at 37℃ for 1 hour to form (CdZnCoNiMn)S x / PAA / cDNA / BSA / PtRuFeCoMn-SA / FTO modified electrode, testing its photoresponse current at 0V voltage; f is placing the above electrode in a 1.0mM DAB (3,3'-diaminobenzidine) solution containing 0.2mM H2O2, incubating it at 37°C for 25 minutes to test its photoresponse current at 0V voltage.

[0201] Figure 7 Where B is: (CdZnCoNiMn)S x Electrochemical impedance of multi-interface heterojunction measured in 10mM K4[Fe(CN)6] solution. Specifically, a in A is the electrochemical impedance of 20μL (CdZnCoNiMn)S x The solution (concentration 2.5 mg / mL, the solvent used was water) was dripped onto the surface of a clean FTO electrode (effective area 5×5 mm) and dried naturally to form (CdZnCoNiMn)S x / FTO modified electrode, and its electrochemical impedance was tested; b was adding 20 μL polymethacrylate sodium salt solution (PAA, concentration 0.05 wt%) to the above electrode surface, incubating at 37 ° C for 30 minutes, and then dropping 20 μL EDC-NHS mixed solution (EDC concentration 10 mM, NHS concentration 2.0 mM, solvent is PBS buffer solution with a pH of 7.4), and incubating at 4 ° C for 1 hour to form (CdZnCoNiMn)S x / PAA / FTO modified electrode, and its electrochemical impedance was tested; c is 20 μL cDNA solution (concentration 1 μM) was added to the surface of the above electrode and incubated at 4°C for 12 h to form (CdZnCoNiMn)S x / PAA / cDNA / FTO modified electrode, and its electrochemical impedance was tested; d is to drop 20 μL BSA solution (concentration 1.0 wt%) onto the surface of the above electrode and incubate at 4 °C for 1 hour to form (CdZnCoNiMn)S x / PAA / cDNA / BSA / FTO modified electrode, and its electrochemical impedance was tested; e is to drop 20 μL of aptamer Apt solution (concentration 1 μM) onto the surface of the above electrode, incubate at 37°C for 1 hour, and then drop 20 μL of PtRuFeCoMn-SA solution, incubate at 37°C for 1 hour to form (CdZnCoNiMn)S x / PAA / cDNA / BSA / PtRuFeCoMn-SA / FTO modified electrode, and its electrochemical impedance was tested; f is placing the above electrode in a 1.0 mM DAB (3,3'-diaminobenzidine) solution containing 0.2 mM H2O2, incubating at 37°C for 25 min, and testing its electrochemical impedance.

[0202] from Figure 7 It can be seen from Figure A that during the gradual assembly process of the biosensor, the light response current signal decreases. The PEC results show that the construction of the PEC sensor is successful. Therefore, the (CdZnCoNiMn)S x Multi-interface heterojunctions can be used to detect chloramphenicol concentration.

[0203] from Figure 7It can be seen from Figure B that the electrochemical impedance increases during the gradual assembly of the biosensor. The results show that the construction of the PEC sensor is successful. Therefore, the (CdZnCoNiMn)S x Multi-interface heterojunctions can be used to detect chloramphenicol concentration.

[0204] Further, according to the method in Example 4, the chloramphenicol concentration detection conditions were optimized, and the results were as follows: Figure 8 shown. Figure 8 A in it is: different (CdZnCoNiMn)S x The photoresponse currents of the solution concentrations (concentrations were 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, and 3.0 mg / mL) in PBS solution containing 0.1 M ascorbic acid (AA); B is the ... x The photoresponse current of the modified electrode measured in PBS solution containing 0.1 M ascorbic acid (AA) at a multi-interface heterojunction concentration of 2.5 mg / mL.

[0205] from Figure 8 It can be seen that by changing (CdZnCoNiMn)S x The photoresponse current of the multi-interface heterojunction measured in a PBS solution containing 0.1 M ascorbic acid (AA) reached the highest value when the concentration was 2.5 mg / mL. x The optimal concentration of the multi-interface heterojunction is 2.5 mg / mL; the incubation time of the biocatalytic precipitation was changed. When the incubation time was 25 min, the photoresponse current measured by the modified electrode in a PBS solution containing 0.1 M ascorbic acid (AA) reached the lowest, and there was no obvious change in the photoresponse current as the incubation time continued to increase. Therefore, the optimal incubation time for the biocatalytic precipitation was 25 min.

[0206] Fig. 9 The photoelectrochemical (PEC) sensing analysis of chloramphenicol was carried out according to the method in Example 4 under the optimal conditions: (CdZnCoNiMn)S x The concentration of the multi-interface heterojunction was 2.5 mg / mL, and the incubation time for biocatalytic precipitation was 25 min; Fig. 9 As shown in the figure, with the increase of chloramphenicol concentration, the photocurrent of the system in PBS solution containing 0.1M ascorbic acid (AA) increased significantly. When the chloramphenicol concentration ranged from 0.001ng / mL to 100ng / mL, specifically, Fig. 9a~f in A represent the photocurrent values ​​tested when the concentration of chloramphenicol is 1pg / mL, 10pg / mL, 100pg / mL, 1ng / mL, 10ng / mL, and 100ng / mL respectively; Fig. 9 B is the relationship between the change in photocurrent (ΔI) and the logarithm of chloramphenicol concentration. Specifically, Fig. 9 ΔI in B is the photocurrent value minus the photocurrent value when the concentration of chloramphenicol is 0.001 ng / mL to 100 ng / mL Figure 7 The photocurrent value corresponding to f in LogC is the corresponding chloramphenicol concentration; a good linear relationship is obtained between the change in photocurrent (ΔI) and the logarithm of the chloramphenicol concentration. These results show that the (CdZnCoNiMn)S x Multi-interface heterojunction photoelectrochemical (PEC) sensors are feasible for the determination of chloramphenicol.

[0207] Fig.10 To verify the selectivity of (CdZnCoNiMn)S x Specificity of photoelectrochemical (PEC) biosensors built with multi-interface heterostructures.

[0208] like Fig.10 As shown, from left to right are tetracycline, amoxicillin, streptomycin, oxytetracycline, kanamycin, chloramphenicol and mixture. At the same concentration, the photocurrent difference of chloramphenicol was significantly higher than that of these control groups. In the mixed group, the photocurrent difference ΔI was not significantly different from that of the chloramphenicol group. This shows that the constructed photoelectrochemical (PEC) detection system has good selectivity for chloramphenicol. Among them, the calculation method of the photocurrent difference ΔI is the same as above, which is to test the photocurrent value corresponding to different antibiotics minus Figure 7 The photocurrent value corresponding to f in .

[0209] The test methods for tetracycline, amoxicillin, streptomycin, oxytetracycline, kanamycin and chloramphenicol are as follows (chloramphenicol is used as an example for explanation, and other substances are tested with reference to chloramphenicol). Taking chloramphenicol as an example, Fig.10 The specific test method is as follows: 20μL (CdZnCoNiMn)S x The multi-interface heterojunction suspension (concentration 2.5 mg / mL, the solvent used was water) was naturally dried on the surface of a clean FTO electrode (effective area 5×5 mm) to form (CdZnCoNiMn)S x / FTO modified electrode; 20 μL of polymethacrylate sodium salt solution (PAA, concentration 0.05wt%) was added to the surface of the above electrode and incubated at 37°C for 30 minutes, followed by dropwise addition of 20 μL of EDC-NHS mixed solution (EDC concentration 10 mM, NHS concentration 2.0 mM, solvent is PBS buffer solution with a pH of 7.4), and incubated at 4°C for 1 hour to form (CdZnCoNiMn)S x / PAA / FTO modified electrode; 20 μL cDNA solution (concentration 1 μM) was added dropwise to the surface of the electrode and incubated overnight at 4°C to form (CdZnCoNiMn)S x / PAA / cDNA / FTO modified electrode; 20 μL BSA solution (concentration 1.0 wt%) was added dropwise to the surface of the electrode and incubated at 4°C for 1 hour to form (CdZnCoNiMn)S x / PAA / cDNA / BSA / FTO modified electrode; 20 μL of aptamer Apt solution (concentration 1 μM) was added to the surface of the above electrode and incubated at 37°C for 1 hour, followed by adding 20 μL of PtRuFeCoMn-SA solution and incubating at 37°C for 1 hour to form (CdZnCoNiMn)S x / PAA / cDNA / BSA / PtRuFeCoMn-SA / FTO modified electrode; 20μL chloramphenicol solution (concentration 100ng / mL) was added to the surface of the electrode and incubated at 37°C for 1 hour; finally, the electrode was placed in a 1.0mM DAB (3,3'-diaminobenzidine) solution containing 0.2mM H2O2 and incubated at 37°C for 25 minutes to test its photoresponse current in a PBS solution containing 0.1M ascorbic acid (AA). To test other antibiotics, chloramphenicol was replaced with other antibiotics at a concentration of 100 times that of chloramphenicol; when testing mixed antibiotics, chloramphenicol was replaced with a mixed antibiotic of tetracycline, amoxicillin, streptomycin, oxytetracycline, kanamycin, and chloramphenicol, where, except for chloramphenicol, the concentrations of other antibiotics were 100 times that of chloramphenicol.

[0210] Example 5

[0211] Detection of Chloramphenicol in Milk Samples

[0212] In order to verify the xThe accuracy and practicality of the photoelectrochemical (PEC) biosensor built with multi-interface heterostructure in practical applications were tested by the standard addition method on milk samples. Milk pretreatment: Add 200 μL of glacial acetic acid to 20 mL of pure milk; then heat in a water bath at 70°C for 2 hours; then centrifuge at 12000 rpm for 15 minutes; take the supernatant, filter it with a 0.22 μm ultrafiltration membrane, dilute it 10 times with PBS, and put it in a 4°C refrigerator for standby use; add different concentrations of chloramphenicol (100 pg / mL, 1 ng / mL, 10 ng / mL) to the treated milk sample to obtain a test solution containing chloramphenicol; the specific test method is tested with reference to the method in Example 4. The test results are shown in Table 1.

[0213] Table 1 - Detection results of chloramphenicol in milk samples

[0214]

[0215]

[0216] According to the data in Table 1, the developed chloramphenicol biosensor performed well in terms of recovery, ranging from 99.21% to 103.49%, while the relative standard deviation was less than 3.89%. This shows that the accuracy and precision of the sensor in chloramphenicol analysis are effectively guaranteed.

[0217] Example 6

[0218] Detection of Chloramphenicol in Yangtze River Water Samples

[0219] Yangtze River water pretreatment: The water sample was filtered with a 0.22 μm ultrafiltration membrane, 5 pg / mL chloramphenicol was added to the treated sample to obtain a test solution, and the concentration of chloramphenicol in the Yangtze River water sample to be tested without and with chloramphenicol was tested. The specific test method was tested with reference to the method in Example 4. The test results are shown in Table 2.

[0220] Table 2- Detection results of chloramphenicol in Yangtze River water samples

[0221]

[0222] In Table 2, Yangtze River water samples 1 to 2 are from the same batch of samples, and Yangtze River water samples 3 to 4 are from another batch of samples.

[0223] According to the data in Table 2, the developed chloramphenicol biosensor performed well in terms of recovery, ranging from 101.33% to 105.30%, while the relative standard deviation was less than 4.01%. This shows that the accuracy and precision of the sensor in chloramphenicol analysis are effectively guaranteed.

[0224] According to Examples 1 to 6, the (CdZnCoNiMn)S x The photoelectrochemical (PEC) biosensing analysis method based on multi-interface heterostructure is not only suitable for actual sample detection, but also has low detection limit, excellent selectivity and reliability. In addition, the method is easy to operate, saving time and cost.

[0225] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing multi-interface heterojunction high entropy sulfide, characterized in that: The following steps are involved: Dissolving Cd salt, Zn salt, Co salt, Ni salt, Mn salt and thioacetamide in ethylene glycol to obtain a mixed solution; The mixed solution is subjected to a solvothermal reaction, and the reaction product is collected, washed, and dried to obtain a multi-interface heterojunction high entropy sulfide.

2. The method for preparing a multi-interface heterojunction high entropy sulfide according to claim 1, characterized in that: The Cd salt includes at least one of Cd(CH3COO)2, CdCl2, and Cd(NO3)2; And / or, the Zn salt includes at least one of Zn(CH3COO)2, Zn(NO3)2, ZnSO4, and ZnCl2; And / or, the Co salt includes at least one of Co(CH3COO)2, Co(NO3)2, CoSO4, and CoCl2; And / or, the Ni salt includes at least one of Ni(CH3COO)2, Ni(NO3)2, NiSO4, and NiCl2; And / or, the Mn salt includes at least one of Mn(CH3COO)2, Mn(NO3)2, MnSO4, and MnCl2; And / or, the molar ratio of the Cd salt, Zn salt, Co salt, Ni salt, Mn salt and thioacetamide is (1-1.2):(1-1.2):(1-1.2):(1-1.2):(1-1.2):(1-1.2); The molar volume ratio of the Co salt to the ethylene glycol is (1×10 -3 ~3×10 -3 )mol:(30~40)mL; And / or, the solvent thermal reaction temperature is 160-170° C. and the time is 10-15 hours.

3. A multi-interface heterojunction high entropy sulfide, characterized in that: The preparation is obtained by the preparation method according to any one of claims 1 to 2.

4. A method for preparing a high entropy alloy nanozyme, characterized in that: The following steps are involved: Pt salt, Ru salt, Fe salt, Co salt, Mn salt and ascorbic acid are dissolved in a mixture of oleylamine and octadecene, reacted, filtered, washed and dried to obtain a high entropy alloy nanozyme.

5. The method for preparing a high entropy alloy nanozyme according to claim 4, characterized in that: The Pt salt is Pt(acac)2; And / or, the Ru salt is Ru(acac)3; And / or, the Fe salt is Fe(acac)3; And / or, the Co salt is Co(acac)2; And / or, the Mn salt is Mn(acac)2; And / or, the mass ratio of the Pt salt, Ru salt, Fe salt, Co salt, Mn salt and ascorbic acid is (10-11):(5-6):(5-6):(5-6):(5-6):(5-6):(53-54); The mass volume ratio of the Pt salt, oleylamine and octadecene is (10-11) mg: (2-3) mL: (2-3) mL; And / or, Pt salt, Ru salt, Fe salt, Co salt, Mn salt and ascorbic acid are dissolved in a mixture of oleylamine and octadecene, and in the steps of reaction, filtering and washing, the reaction temperature is 180-200°C, the reaction time is 5-10 hours, and washing is performed using a mixed solution of ethanol and cyclohexane, and the volume ratio of ethanol to cyclohexane is (1-3):(1-3).

6. Use of the multi-interface heterojunction high entropy sulfide as claimed in claim 3 in detecting the concentration of chloramphenicol.

7. Use of a high entropy alloy nanozyme prepared by the preparation method as described in any one of claims 4 to 5 in a catalytic reaction for a photoelectrochemical sensor.

8. A biosensor, characterized in that: The method for constructing the biosensor comprises the following steps: S1. coating the multi-interface heterojunction high entropy sulfide according to claim 3 on the surface of the electrode and drying to form a modified electrode; S2, dropping polymethacrylate sodium salt solution on the surface of the modified electrode, incubating, and washing the electrode with PBS buffer; S3, drop the EDC-NHS mixed solution onto the electrode surface in S2, incubate, and wash the electrode with PBS buffer; S4, drop the cDNA solution onto the electrode surface in S3, incubate, and wash the electrode with PBS buffer; S5, drop the BSA solution onto the electrode surface in S4, incubate, and wash the electrode with PBS buffer; S6, adding the aptamer Apt solution dropwise to the electrode surface in S5, incubating, and washing the electrode with PBS buffer; S7, dropping the PtRuFeCoMn-SA solution onto the electrode surface in S6, incubating, and washing the electrode with PBS buffer to obtain a biosensor; Wherein, the gene sequence of the cDNA is 5'-NH2-TTTTTCTACCACCGACTC-3'; The gene sequence of the aptamer Apt is 5'-Bio-ACTTCAGTGAGTTGTCCCACGGTCGGCGAGTCGGTGGTAG-3'; The preparation method of the PtRuFeCoMn-SA solution is: Adding the high entropy alloy nanozyme prepared by the preparation method according to any one of claims 4 to 5 into water to obtain a high entropy alloy nanozyme solution; The streptavidin solution was mixed with the high entropy alloy nanozyme solution, the supernatant was removed by centrifugation, and the precipitate was collected; PBS buffer was added to the precipitate to obtain a PtRuFeCoMn-SA solution.

9. The biosensor according to claim 8, wherein The method for constructing the biosensor comprises the following steps: S1. Add the multi-interface heterojunction high entropy sulfide into water to obtain 1-3 mg mL -1 A multi-interface heterojunction high entropy sulfide solution is coated on the electrode surface and dried to form a modified electrode; S2, dripping polymethacrylate sodium salt solution onto the surface of the modified electrode, incubating at 36-37° C. for 0.5-1 h, and washing the electrode with PBS buffer; the polymethacrylate sodium salt solution, the mass concentration of the polymethacrylate sodium salt solution is 0.05-0.1%; S3, dripping the EDC-NHS mixed solution onto the electrode surface in S2, incubating at 36-37°C for 1-2 hours, and washing the electrode with PBS buffer; the preparation method of the EDC-NHS mixed solution is as follows: adding EDC and NHS into PBS buffer to obtain an EDC-NHS mixed solution, wherein the concentration of EDC is 10-12 mM, and the concentration of NHS is 2-4 mM; S4, dropping the cDNA solution onto the electrode surface in S3, incubating at 4-5°C for 12-15h, and washing the electrode with PBS buffer; the preparation method of the cDNA solution is: adding cDNA to PBS buffer to obtain a cDNA solution, wherein the concentration of the cDNA solution is 1.0-1.2 μM; S5, dropping a BSA solution onto the electrode surface in S4, incubating at 36-37°C for 1-2 hours, and washing the electrode with a PBS buffer solution; the BSA solution is prepared by adding BSA to a PBS buffer solution to obtain a BSA solution; the mass concentration of the BSA solution is 1.0-2.0%; S6, adding the aptamer Apt solution dropwise to the electrode surface in S5, incubating at 36-37° C. for 1-2 hours, and washing the electrode with PBS buffer; the preparation method of the aptamer Apt solution is: adding the aptamer Apt to the PBS buffer to obtain the aptamer Apt solution, wherein the concentration of the aptamer Apt solution is 1.0-1.2 μM; S7, adding the PtRuFeCoMn-SA solution to the electrode surface in S6, incubating at 36-37°C for 1-2h, and washing the electrode with PBS buffer to obtain a biosensor; The concentration of the streptavidin solution is 1-2 mg / mL, and the concentration of the high entropy alloy nanozyme solution is 1-2 mg / mL; The streptavidin solution is mixed with the high entropy alloy nanozyme solution, the supernatant is removed by centrifugation, and the precipitate is collected; in the step of adding PBS buffer to the precipitate, the volume ratio of the streptavidin solution, the high entropy alloy nanozyme solution, and the PBS buffer solution is (200-220):(400-420):(600-620); The pH of the PBS buffer is 7.4-7.

5.

10. A method for detecting chloramphenicol concentration, characterized in that: The following steps are involved: Providing a biosensor as claimed in any one of claims 8 to 9; Add chloramphenicol test solution to the electrode surface of the biosensor, incubate at 36-37°C for 1-2 hours, and wash the electrode with PBS buffer; DAB solution containing H2O2 was added dropwise to the electrode surface, incubated at 36-37°C for 10-30 min, and the electrode was washed with PBS buffer; The electrode was placed in a PBS buffer containing ascorbic acid, and the photoresponse current signal of the electrode was detected; Detecting chloramphenicol concentration based on the light-responsive current signal; The concentration of the DAB solution is 1.0-1.2 mM, and the concentration of H2O2 contained in the DAB solution is 0.2-0.3 mM; The concentration of ascorbic acid in the PBS buffer containing ascorbic acid is 0.1 to 0.2 M.

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