Photoelectrochemical sensor based on monatomic material and enzyme-driven cascade hairpin amplification and application of photoelectrochemical sensor in detection of tetracycline

Abstract

The invention discloses a photoelectrochemical sensor based on a monatomic material and enzyme-driven cascade hairpin amplification and application of the photoelectrochemical sensor in detection of tetracycline, and belongs to the field of application of photoelectric materials. The monatomic-based photoactive material ZnIn2S4 / Co / NH2-MIL-125 (Ti) prepared by the invention is used as a photosensitive material, visible light absorption is enhanced by introducing a proper amount of Co monatomic atoms, and the separation and transfer capabilities of current carriers are improved. An enzyme-driven cascade hairpin amplification method is provided for triggering an HCR reaction, and a hemin / G-quadruplex with peroxidase characteristics is formed on the surface of an electrode to realize in-situ oxidation of 4-CN to form a precipitate, so that the photoelectrochemical sensor with high sensitivity and good stability is constructed, and efficient detection of tetracycline can be realized.

Description

Technical Field

[0001] The present invention relates to the application field of photoelectric materials, and in particular to a photoelectric chemical sensor based on single-atom materials and enzyme-driven cascade hairpin amplification, and its application in detecting tetracycline. Background Art

[0002] Single-atom semiconductor materials, due to their unique electronic states and coordination environments, exhibit unprecedented catalytic activity and excellent atom utilization. In recent years, research on single-atom materials in fields such as photoelectrocatalysis has made significant progress. With a deeper understanding of their catalytic mechanisms and properties, researchers have begun applying single-atom materials to photoelectrochemical sensing. Their highly active sites and enhanced light absorption make single-atom materials show great potential in photoelectrochemical sensing.

[0003] Tetracycline is a broad-spectrum antibiotic widely used in healthcare, livestock farming, agricultural production, and other industries due to its inhibitory and bactericidal abilities. However, excessive use of tetracycline can lead to high levels of toxic residues, and excessive intake by humans from the environment can cause serious side effects (such as liver and gastrointestinal damage, anaphylactic shock, and even death). Therefore, there is an urgent need to explore new, efficient, and sensitive strategies for tetracycline detection.

[0004] Currently, the main analytical methods for tetracycline residues in food are instrumental analysis, such as capillary electrophoresis, liquid chromatography-mass spectrometry, and high-performance liquid chromatography. Although these methods have high sensitivity, they often require a long time, complex sample pretreatment, and expensive laboratory equipment, making them inadequate for real-time analysis of large-scale samples. Photoelectrochemical biosensors have the advantages of low background signal, high sensitivity, and fast detection speed, and have been widely used in the field of food safety. Due to the relatively low concentrations of antibiotics in food and the environment, their detection is usually carried out by trace analysis. Therefore, it is crucial to establish a rapid, simple, low-cost, and highly specific method for the detection of antibiotics. Summary of the Invention

[0005] The purpose of the present invention is to provide a photoelectrochemical sensor based on single-atom materials and enzyme-driven cascade hairpin amplification and its application in detecting tetracycline, so as to solve the problems existing in the above-mentioned prior art. The prepared single-atom-based photoactive material ZnIn2S4 / Co / NH2-MIL-125 (Ti) is used as a photosensitive material, and the visible light absorption is enhanced by introducing an appropriate amount of Co single atoms, thereby improving the separation and transfer ability of carriers; and based on ZnIn2S4 / Co / NH2-MIL-125 (Ti) combined with enzyme-driven cascade hairpin amplification technology, by triggering the HCR reaction, a hemin / G-quadruplex with peroxidase characteristics is formed on the electrode surface, and the in situ oxidation of 4-CN is realized to form a precipitate, thereby forming a photoelectrochemical sensor with high sensitivity and good stability, which can realize efficient detection of tetracycline.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a photoelectrochemical sensor based on single-atom materials and enzyme-driven cascade hairpin amplification. The photoelectrochemical sensor comprises a ZnIn2S4 / Co / NH2-MIL-125(Ti) photoelectrode prepared with a ZnIn2S4 / Co / NH2-MIL-125(Ti) single-atom-based material as a substrate material, and a capture antibody and a protein for blocking nonspecific active sites that are dropwise added to the surface of the ZnIn2S4 / Co / NH2-MIL-125(Ti) photoelectrode.

[0008] The ZnIn2S4 / Co / NH2-MIL-125(Ti) single-atom-based material is prepared by introducing Co single atoms into ZnIn2S4 to prepare Co / ZnIn2S4, and then introducing NH2-MIL-125(Ti) into the Co / ZnIn2S4, so that NH2-MIL-125(Ti) is attached to the surface of the Co / ZnIn2S4 nanoflower ball in the form of nanosheets.

[0009] Preferably, the nucleotide sequence of the capture antibody is as shown in SEQ ID NO. 4, and the protein for blocking non-specific active sites includes bovine serum albumin.

[0010] The present invention also provides application of the photoelectrochemical sensor in detecting tetracycline.

[0011] The present invention also provides a method for detecting tetracycline using the photoelectrochemical sensor, which is characterized by comprising the following steps:

[0012] (1) After the tetracycline aptamer and primer P1 are incubated to form Apt / P1, the target analyte tetracycline to be detected is added for incubation. After the incubation is completed, the supernatant is collected, and primer P2 and circular template DNA are added for heating reaction and annealing, and then T4 ligase is added for incubation to form the RCA reaction precursor;

[0013] (2) adding phi 29 DNA polymerase and Nb.BbvCI endonuclease to the RCA reaction precursor solution, performing amplification and enzyme cleavage reactions simultaneously, and terminating the reaction to form an RCA reaction solution containing a large amount of output hairpin DNA H1 and H2;

[0014] (3) dropping the RCA reaction solution onto the photoelectrochemical sensor for incubation, and then adding the hemin solution to react, thereby forming a hemin / G-quadruplex structure with peroxidase-like activity on the electrode surface of the obtained photoelectrochemical sensor;

[0015] (4) placing the photoelectrochemical sensor obtained by the reaction in step (3) in a 4-chloro-1-naphthol solution containing H2O2 to perform a catalytic oxidation reaction to form a precipitate;

[0016] (5) Under the irradiation of an excitation light source, the photoelectrochemical sensor that has undergone the reaction in step (4) is used as a working electrode, a three-electrode system is used to record the photocurrent signal, and a linear relationship curve between the photocurrent signal and the tetracycline concentration is prepared, thereby realizing the detection of tetracycline.

[0017] Preferably, in step (1), the nucleotide sequence of the tetracycline aptamer is shown as SEQ ID NO.5; and / or the nucleotide sequence of the primer P1 is shown as SEQ ID NO.2; and / or the nucleotide sequence of the primer P2 is shown as SEQ ID NO.3; and / or the nucleotide sequence of the circular template DNA is shown as SEQ ID NO.1;

[0018] and / or the volume ratio of the tetracycline adaptor to the primer P1 is 1:1;

[0019] and / or the volume ratio of the supernatant, primer P2 and circular template DNA is 1:1:1;

[0020] and / or adding primer P2 and circular template DNA to the supernatant, heating at 90-95° C. for 5 min and then annealing;

[0021] And / or after adding T4 ligase to the annealed solution, incubate at 20-25° C. for 1 hour.

[0022] Preferably, in step (2), the amplification reaction solution comprises 4.5-5.5 μL dNTP, 0.5-1.5 μL 10×Phi29 DNA polymerase and 1.5-2.5 μL Nb.BbvCI;

[0023] And / or the conditions of the amplification and enzyme digestion reaction are: incubation at 45° C. for 1 hour and incubation at 65° C. for 10 minutes to inactivate the enzyme.

[0024] Preferably, in step (3), the volume ratio of the RCA reaction solution to the hemin solution is 1:4; the reaction solution is dropped onto the surface of the photoelectrochemical sensor and incubated at 35-40°C for 2 hours, and the hemin is added and the reaction is continued at 35-40°C for 50 minutes.

[0025] And / or in step (4), the 4-chloro-1-naphthol solution containing H2O2 is a 4-chloro-1-naphthol solution containing 1 mM H2O2, and the reaction time of the catalytic oxidation reaction is 20 min.

[0026] Preferably, the preparation method of the photoelectrochemical sensor comprises the following steps:

[0027] (a) Indium chloride tetrahydrate, zinc chloride and thioacetamide are added to a glycerol aqueous solution, heated and stirred for reaction, washed and dried to obtain ZnIn2S4;

[0028] (b) ZnIn2S4 is mixed with methanol and aqueous solution, and then cobalt (II) nitrate hexahydrate is added and stirred for reaction, followed by low-temperature photodeposition, washing, and drying to obtain Co / ZnIn2S4;

[0029] (c) 2-aminoterephthalic acid and hexadecyltrimethylammonium bromide were ultrasonically dissolved in a mixture of N,N-dimethylformamide and methanol, and tetrabutyl titanate was added. After ultrasonic dispersion, Co / ZnIn2S4 was added to the reaction solution and reacted at high temperature to obtain ZnIn2S4 / Co / NH2-MIL-125(Ti);

[0030] (d) ZnIn2S4 / Co / NH2-MIL-125(Ti) was uniformly dispersed in an ethanol aqueous solution, mixed with a Nafion solution, and dropped onto the conductive surface of the electrode, dried, and rinsed; chitosan solution was then added dropwise, dried, and rinsed; glutaraldehyde solution was then added dropwise, allowed to stand at room temperature, and rinsed to obtain a photoelectrode;

[0031] (e) dropping a capture antibody modified with an amino group onto the surface of a photoelectrode for incubation, and after the incubation is complete, dropping bovine serum albumin onto the surface of the electrode, and continuing the incubation to obtain the photoelectrochemical sensor.

[0032] Preferably, in step (a), the ratio of indium chloride tetrahydrate, zinc chloride, thioacetamide and glycerol is 2 mmol:1 mmol:8 mmol:80 mL; the heating and stirring reaction is: stirring in an oil bath at 75° C. for 1 h;

[0033] And / or in step (b), the ratio of ZnIn2S4, methanol aqueous solution and cobalt (II) nitrate hexahydrate is 50 mg:30 mL:3 mL; the volume ratio of methanol to water in the methanol aqueous solution is 1:1; the stirring reaction is: stirring at 10°C for 30 minutes with a 300W xenon lamp;

[0034] And / or in step (c), the ratio of 2-aminoterephthalic acid, hexadecyltrimethylammonium bromide, N,N-dimethylformamide and methanol is 0.2-0.4g:1-2g:5-8mL:2-5mL; the ratio of 2-aminoterephthalic acid and tetrabutyl titanate is 0.2-0.4g:0.1-0.3mL; the ratio of 2-aminoterephthalic acid and Co / ZnIn2S4 is 0.2-0.4g:60-90mg;

[0035] and / or in step (d), the concentration of the ZnIn2S4 / Co / NH2-MIL-125(Ti) solution is 2.0 mg mL -1 The volume ratio of the ZnIn2S4 / Co / NH2-MIL-125(Ti) solution to the Nafion solution is 50:(1-3); the addition amount of the chitosan solution and the glutaraldehyde solution is 15-25 μL;

[0036] And / or in step (e), the amount of the amino-modified capture antibody added is 15-25 μL, and the incubation conditions are 37° C. for 1-2 hours; the amount of the bovine serum albumin added is 5-15 μL, and the incubation conditions are room temperature for 30-40 minutes.

[0037] Preferably, the tetracycline is derived from food.

[0038] The present invention discloses the following technical effects:

[0039] The ZnIn2S4 / Co / NH2-MIL-125(Ti) single-atom-based material prepared by the present invention is prepared by introducing an appropriate amount of Co single atoms to form an O-Co-N coordination structure, providing a new charge transfer channel for the Z-type heterostructure, thereby enhancing visible light absorption, improving the separation and transfer ability of carriers, having a higher photocurrent response, and further expanding the application range of single-atom-based materials.

[0040] An enzyme-driven cascade hairpin amplification method is proposed to trigger the HCR reaction. By forming a peroxidase-like hemin / G-quadruplex on the electrode surface, 4-CN is oxidized in situ to form a precipitate, enabling the construction of a photoelectrochemical sensor for ultrasensitive detection of tetracycline (TET). This photoelectrochemical sensor exhibits a wide linear range and high selectivity for tetracycline detection, providing a new strategy for food safety testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] 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. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0042] Figure 1 This is a scanning electron microscope image of the ZnIn2S4 / Co / NH2-MIL-125(Ti) sample prepared in Example 1;

[0043] Figure 2 This is the standard curve of the PEC sensing platform for different concentrations of tetracycline;

[0044] Figure 3 The selective analysis results of the biosensor;

[0045] Figure 4 Comparison results of the photocurrent responses of ZnIn2S4 / Co / NH2-MIL-125(Ti) and ZnIn2S4 / NH2-MIL-125(Ti) samples. DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0048] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0049] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0050] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0051] The names of some compounds involved in the present invention, and their corresponding chemical formulas or abbreviations are:

[0052] Indium chloride tetrahydrate: InCl3·4H2O;

[0053] Zinc chloride: ZnCl2;

[0054] Thioacetamide: C2H5NS;

[0055] Glycerol: C3H8O3;

[0056] Methanol: MeOH;

[0057] Ethanol: EtOH;

[0058] Cobalt(II) nitrate hexahydrate: Co(NO3)2·6H2O;

[0059] N,N-dimethylformamide:DMF;

[0060] Cetyltrimethylammonium bromide: CTAB;

[0061] 2-aminoterephthalic acid: NH2-BDC;

[0062] Tetrabutyl titanate: TBOT.

[0063] Example 1 Preparation method of ZnIn2S4 / Co / NH2-MIL-125(Ti) single atom-based material

[0064] (1) InCl3·4H2O (2 mmol), ZnCl2 (1 mmol), and C2H5NS (8 mmol) were added to a glycerol aqueous solution (80 mL, 20 vol%, pH = 2.5) and stirred for 30 min. The resulting solution was placed in a 75°C oil bath and stirred for 1 h. After cooling to room temperature, the yellow precipitate was collected by centrifugation, washed several times with deionized water and ethanol, and dried in a vacuum at 60°C overnight to obtain indium zinc sulfide (ZnIn2S4) microspheres.

[0065] (2) 50 mg of ZnIn2S4 microspheres were uniformly dispersed in 30 mL of methanol / water (V:V = 1:1) solution to obtain a ZnIn2S4 suspension. 22 μL of Co(NO3)2·6H2O solution (10 mM) was then diluted to 3 mL and added to the ZnIn2S4 suspension. The mixture was stirred at 10°C for 30 min using a 300 W xenon lamp. The Co / ZnIn2S4 sample was obtained by centrifugation, washed several times with deionized water and ethanol, and dried in a vacuum oven at 60°C overnight.

[0066] (3) NH2-BDC (0.34 g) and CTAB (1.12 g) were dissolved in a mixed solution of DMF (7.0 mL) and MeOH (3 mL) using ultrasound. 0.25 mL of TBOT was added to the above solution and ultrasound was continued. Then, 60-90 mg of synthesized Co / ZnIn2S4 (70 mg was selected in this example) was added to the solution. The suspension was transferred to a 50 mL polytetrafluoroethylene autoclave (PTFE) and stored at 150°C for 8 h. After cooling to room temperature, it was washed several times with DMF and MeOH and vacuum dried at 60°C overnight to obtain ZnIn2S4 / Co / NH2-MIL-125(Ti) single atom-based material.

[0067] The SEM images of the prepared samples are as follows Figure 1 As shown in the figure, it can be seen that NH2-MIL-125(Ti) is attached to the surface of Co / ZnIn2S4 nanoflower ball in the form of nanosheets.

[0068] Example 2

[0069] Using the ZnIn2S4 / Co / NH2-MIL-125(Ti) single-atom-based material prepared in Example 1 as the substrate material, an ultrasensitive photoelectrochemical sensor based on single-atom material and enzyme-driven cascade hairpin amplification was prepared, specifically comprising the following steps:

[0070] (1) Ultrasonic cleaning of FTO conductive glass was performed with acetone, ethanol and ultrapure water in sequence, and then dried in an oven.

[0071] (2) 50 μL ZnIn2S4 / Co / NH2-MIL-125(Ti) suspension (2.0 mg mL-1 ) was mixed with Nafion solution (2 μL) and dropped onto the conductive surface of the FTO electrode (fixed area 1.0 cm 2 ) and dried at 45°C.

[0072] (3) 15-25 μL of chitosan solution (0.1 wt%, 20 μL selected in this embodiment) was dripped onto the electrode surface, and the electrode was dried in a 60°C oven. After rinsing with PBS, 15-25 μL of glutaraldehyde solution (2.5 v / v%, 20 μL selected in this embodiment) was dripped onto the electrode surface, and the electrode was allowed to stand at room temperature for 1 h. The electrode was rinsed with PBS to obtain a photoelectrode.

[0073] (4) 15 to 25 μL of amino-modified capture probe Cp (1 mM, 20 μL selected in this example) was dropped onto the electrode surface and incubated at 37°C for 1 h. After the incubation, 5 to 15 μL of bovine serum albumin (1 wt%, 10 μL selected in this example) was added to the electrode surface and incubated at room temperature for 40 min to block nonspecific binding sites and obtain a photoelectrochemical sensor.

[0074] Example 3

[0075] In this example, the photoelectrochemical sensor prepared in Example 2 was used for the high-sensitivity determination of tetracycline (TET), and the specific steps were as follows:

[0076] (1) After the tetracycline aptamer (aptamer, 2 μM, 10-20 μL, 10 μL was selected in this example) was incubated with its recognition chain P1 (2 μM, 10-20 μL, 10 μLL was selected in this example) to form Apt / P1, different concentrations of tetracycline (2 μL) were added to the solution and incubated at 37°C for 1 h. The aptamer recognized TET and released P1, which served as a primer for the rolling circle amplification reaction.

[0077] (2) Take 2-3 μL of the supernatant (P1, 2.5 μL selected in this example) from the reaction solution in step (1), add 2-3 μL of Circle DNA (10 μM, 2.5 μL selected in this example) and 2-3 μL of P2 (10 μM, 2.5 μL selected in this example). Heat the mixed solution at 95°C for 5 minutes and then gradually cool to room temperature. After annealing, add 10×T4 DNA ligase buffer and 1.5-2.5 μL of T4 ligase (2 μL selected in this example) to the reaction system and incubate at 22°C for 1 hour to form the RCA reaction precursor.

[0078] (3) Then, add 4.5-5.5 μL of 25 mM dNTP, 0.5-1.5 μL of Hi 29 DNA polymerase (10 U / μL), and 1.5-2.5 μL of 10 U / μL Nb.BbvCI (nicking endonuclease), and add 2 μL of the corresponding nuclease buffer to each enzyme.

[0079] Amplification was initiated by incubation at 45°C for 1 hour and terminated by incubation at 65°C for 10 minutes, yielding output hairpin DNAs H1 and H2 (partial base pairing between H2 and H1). Finally, the product was stored at 4°C for subsequent analysis.

[0080] (4) H1 hybridizes with the capture DNA (Cp) modified on the electrode surface and exposes its fulcrum, further triggering the subsequent hybridization chain reaction to generate double-stranded DNA nanowires. The sensing electrode was placed in 5μL RCA reaction solution (containing 50mM KCl) and incubated at 37°C for 2h. Then, 20μL hemin (0.2mM hemin) was added and reacted for 50min. A hemin / G-quadruplex structure with peroxidase-like activity was formed on the electrode surface.

[0081] (5) Finally, the electrode was incubated in a 10 mM 4-chloro-1-naphthol (4-CN) solution containing 1 mM H2O2 for 20 min to perform a catalytic oxidation reaction, in situ catalytic oxidation of 4-CN to form 4-CD precipitate. After each incubation, the electrode was rinsed with PBS buffer.

[0082] (6) Under the irradiation of an excitation light source, the obtained sensor electrode is used as the working electrode, and a three-electrode system is used to record the photocurrent signal. A linear relationship curve between the photocurrent signal and the tetracycline concentration is prepared to achieve high-sensitivity detection of tetracycline.

[0083] The above-mentioned related DNA base sequence designs are shown in Table 1. The detection photocurrent results are shown in Table 1. Figure 2 As shown, the photocurrent change value is plotted against the tetracycline concentration. The sensor shows a good linear relationship in the concentration range of 0.1fM-10nM, indicating that the sensor can achieve quantitative detection of tetracycline within a certain range.

[0084] Table 1 DNA sequence design

[0085]

[0086] Example 4

[0087] In order to verify the accuracy and practicality of the ultrasensitive photoelectrochemical sensor based on single-atom materials and enzyme-driven cascade hairpin amplification in practical applications, chloramphenicol (CHL), kanamycin (KAN), carbamazepine (CBZ), and amoxicillin (AMX) were used as interfering substances to evaluate its anti-interference ability. Figure 3 As shown in the figure, compared with the blank sample, the change in the photocurrent intensity of non-target analytes was negligible, while the detection signal changed significantly regardless of whether the target analyte TET was present alone or with these interferents, demonstrating that the prepared PEC biosensor has strong TET specificity.

[0088] Comparative Example 1

[0089] (1) ZnIn2S4 / NH2-MIL-125(Ti) was prepared and compared with ZnIn2S4 / Co / NH2-MIL-125(Ti) to demonstrate that the present invention has superior photoelectric performance. The specific steps are as follows: InCl3·4H2O (2mmol), ZnCl2 (1mmol), and C2H5NS (8mmol) were added to a glycerol aqueous solution (80mL, 20vol%, pH=2.5) and stirred for 30min. The resulting solution was placed in a 75℃ oil bath and stirred for 1h. After cooling to room temperature, the yellow precipitate was collected by centrifugation, washed several times with deionized water and ethanol, and dried in a vacuum at 60℃ overnight.

[0090] (2) NH2-BDC (0.34 g) and CTAB (1.12 g) were dissolved in a mixed solution of DMF (7.0 mL) and MeOH (3 mL) using ultrasound. 0.25 mL of TBOT was added to the above solution and ultrasound was continued. Then, a certain amount of synthesized ZnIn2S4 was added to the solution. The suspension was transferred to a 50 mL polytetrafluoroethylene autoclave (PTFE) and stored at 150°C for 8 h. After cooling to room temperature, it was washed several times with DMF and MeOH and dried in vacuum at 60°C overnight to obtain ZnIn2S4 / NH2-MIL-125(Ti).

[0091] Under the irradiation of the excitation light source, ZnIn2S4 / NH2-MIL-125(Ti) and ZnIn2S4 / Co / NH2-MIL-125(Ti) were used as photosensitive materials to prepare working electrodes, and a three-electrode system was used to record the photocurrent signal. The time and photocurrent density were plotted. Figure 4 It can be seen that the present invention has a more excellent photoelectric response ability by introducing Co single atoms.

[0092] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A photoelectrochemical sensor based on single-atom materials and enzyme-driven cascade hairpin amplification, characterized in that: The photoelectrochemical sensor includes a ZnIn2S4 / Co / NH2-MIL-125(Ti) photoelectrode made of a ZnIn2S4 / Co / NH2-MIL-125(Ti) single-atom-based material as a substrate, and a capture antibody and a protein for blocking nonspecific active sites that are dropwise added to the surface of the ZnIn2S4 / Co / NH2-MIL-125(Ti) photoelectrode; The ZnIn2S4 / Co / NH2-MIL-125(Ti) single-atom-based material is prepared by introducing Co single atoms into ZnIn2S4 to prepare Co / ZnIn2S4, and then introducing NH2-MIL-125(Ti) into the Co / ZnIn2S4, so that NH2-MIL-125(Ti) is attached to the surface of the Co / ZnIn2S4 nanoflower ball in the form of nanosheets.

2. The photoelectrochemical sensor according to claim 1, wherein The nucleotide sequence of the capture antibody is shown in SEQ ID NO. 4, and the protein used to block non-specific active sites includes bovine serum albumin.

3. Use of the photoelectrochemical sensor according to claim 1 or 2 in detecting tetracycline.

4. A method for detecting tetracycline using the photoelectrochemical sensor according to claim 1 or 2, characterized in that: The following steps are involved: (1) After the tetracycline aptamer and primer P1 are incubated to form Apt / P1, the target analyte tetracycline to be detected is added for incubation. After the incubation is completed, the supernatant is collected, and primer P2 and circular template DNA are added for heating reaction and annealing, and then T4 ligase is added for incubation to form the RCA reaction precursor; (2) adding a reaction enzyme to the RCA reaction precursor, performing amplification and enzyme cleavage reactions simultaneously, and terminating the reaction to form an RCA reaction solution containing output hairpin DNA H1 and H2; (3) dropping the RCA reaction solution onto the surface of the photoelectrochemical sensor for incubation, and then adding the hemin solution to continue the incubation reaction, thereby forming a hemin / G-quadruplex structure with peroxidase-like activity on the electrode surface of the obtained photoelectrochemical sensor; (4) placing the photoelectrochemical sensor obtained by the reaction in step (3) in a 4-chloro-1-naphthol solution containing H2O2 to perform a catalytic oxidation reaction to form a precipitate; (5) Under the irradiation of an excitation light source, the photoelectrochemical sensor that has undergone the reaction in step (4) is used as a working electrode, a three-electrode system is used to record the photocurrent signal, and a linear relationship curve between the photocurrent signal and the tetracycline concentration is prepared, thereby realizing the detection of tetracycline.

5. The method according to claim 4, wherein In step (1), the nucleotide sequence of the tetracycline aptamer is shown as SEQ ID NO.5; and / or the nucleotide sequence of the primer P1 is shown as SEQ ID NO.2; and / or the nucleotide sequence of the primer P2 is shown as SEQ ID NO.3; and / or the nucleotide sequence of the circular template DNA is shown as SEQ ID NO.1; and / or the volume ratio of the tetracycline adaptor to the primer P1 is 1:1; and / or the volume ratio of the supernatant, primer P2 and circular template DNA is 1:1:1; and / or adding primer P2 and circular template DNA to the supernatant, heating at 90-95° C. for 5 min and then annealing; And / or T4 ligase is added to the reaction system after annealing, and incubated at 20-25° C. for 1 hour.

6. The method according to claim 4, wherein In step (2), the amplification reaction solution includes 4.5-5.5 μL dNTP, 0.5-1.5 μL 10×Phi 29 DNA polymerase and 1.5-2.5 μL Nb.BbvCI; And / or the conditions of the amplification and enzyme digestion reaction are: incubation at 45° C. for 1 hour and incubation at 65° C. for 10 minutes to inactivate the enzyme.

7. The method according to claim 4, wherein In step (3), the volume ratio of the RCA reaction solution to the hemin solution is 1:4; the reaction solution is dropped onto the surface of the photoelectrochemical sensor and incubated at 35-40°C for 2 hours, and the hemin is added and the reaction is continued at 35-40°C for 50 minutes; And / or in step (4), the 4-chloro-1-naphthol solution containing H2O2 is a 4-chloro-1-naphthol solution containing 1 mM H2O2, and the reaction time of the catalytic oxidation reaction is 20 min.

8. The method according to claim 4, wherein The preparation method of the photoelectrochemical sensor comprises the following steps: (a) Indium chloride tetrahydrate, zinc chloride and thioacetamide are added to a glycerol aqueous solution, heated and stirred for reaction, washed and dried to obtain ZnIn2S4; (b) ZnIn2S4 is mixed with methanol and aqueous solution, and then cobalt (II) nitrate hexahydrate is added and stirred for reaction, followed by low-temperature photodeposition, washing, and drying to obtain Co / ZnIn2S4; (c) 2-aminoterephthalic acid and hexadecyltrimethylammonium bromide were ultrasonically dissolved in a mixture of N,N-dimethylformamide and methanol, and tetrabutyl titanate was added. After ultrasonic dispersion, Co / ZnIn2S4 was added to the reaction solution and reacted at high temperature to obtain ZnIn2S4 / Co / NH2-MIL-125(Ti); (d) ZnIn2S4 / Co / NH2-MIL-125(Ti) was uniformly dispersed in an ethanol aqueous solution, mixed with a Nafion solution, and dropped onto the conductive surface of the electrode, dried, and rinsed; chitosan solution was then added dropwise, dried, and rinsed; glutaraldehyde solution was then added dropwise, allowed to stand at room temperature, and rinsed to obtain a photoelectrode; (e) dropping a capture antibody modified with an amino group onto the surface of a photoelectrode for incubation, and after the incubation is complete, dropping bovine serum albumin onto the surface of the electrode, and continuing the incubation to obtain the photoelectrochemical sensor.

9. The method according to claim 8, wherein In step (a), the ratio of indium chloride tetrahydrate, zinc chloride, thioacetamide and glycerol is 2 mmol:1 mmol:8 mmol:80 mL; the heating and stirring reaction is: stirring in an oil bath at 75° C. for 1 hour; And / or in step (b), the ratio of ZnIn2S4, methanol aqueous solution and cobalt (II) nitrate hexahydrate is 50 mg:30 mL:3 mL; the volume ratio of methanol to water in the methanol aqueous solution is 1:1; the stirring reaction is: stirring at 10°C for 30 minutes with a 300W xenon lamp; And / or in step (c), the ratio of 2-aminoterephthalic acid, hexadecyltrimethylammonium bromide, N,N-dimethylformamide and methanol is 0.2-0.4g:1-2g:5-8mL:2-5mL; the ratio of 2-aminoterephthalic acid and tetrabutyl titanate is 0.2-0.4g:0.1-0.3mL; the ratio of 2-aminoterephthalic acid and Co / ZnIn2S4 is 0.2-0.4g:60-90mg; and / or in step (d), the concentration of the ZnIn2S4 / Co / NH2-MIL-125(Ti) solution is 2.0 mg mL -1 The volume ratio of the ZnIn2S4 / Co / NH2-MIL-125(Ti) solution to the Nafion solution is 50:(1-3); the addition amount of the chitosan solution and the glutaraldehyde solution is 15-25 μL; And / or in step (e), the amount of the amino-modified capture antibody added is 15-25 μL, and the incubation conditions are 37° C. for 1-2 hours; the amount of the bovine serum albumin added is 5-15 μL, and the incubation conditions are room temperature for 30-40 minutes.

10. The method according to claim 4, wherein The tetracyclines are derived from food.

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