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

Abstract

The application discloses a photoelectrochemical sensor based on single-atom material and enzyme-driven cascade hairpin amplification and application in detection of tetracycline, and belongs to the field of photoelectric material application. The single-atom-based photoactive material ZnIn2S4 / Co / NH2-MIL-125(Ti) prepared by the application is used as a photosensitive material, the visible light absorption is enhanced by introducing appropriate Co single atoms, and the separation and transfer capacity of carriers is improved. An enzyme-driven cascade hairpin amplification method is proposed to trigger the HCR reaction, a hemin / G-quadruplex with peroxidase characteristics is formed on the electrode surface, in-situ oxidation of 4-CN is realized to form a precipitate, and therefore, a photoelectrochemical sensor with high sensitivity and good stability is constructed, and efficient detection of tetracycline can be realized.

Description

Technical Field

[0001] This invention relates to the field of optoelectronic materials applications, and in particular to an optochemical sensor based on single-atom materials and enzyme-driven cascade hairpin amplification and its application in the detection of tetracycline. Background Technology

[0002] Single-atom semiconductor materials exhibit unprecedented catalytic activity and excellent atomic utilization due to their unique electronic states and coordination environments. In recent years, significant progress has been made in the research of single-atom materials in fields such as photoelectrocatalysis. With a deeper understanding of their catalytic mechanisms and performance, researchers have begun to apply single-atom materials to the field of photoelectrochemical sensing. Their high active sites and light-absorbing properties demonstrate the enormous potential of single-atom materials in photoelectrochemical sensing.

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

[0004] Currently, the main analytical methods for tetracycline residues in food are instrumental analyses, such as capillary electrophoresis, liquid chromatography-mass spectrometry, and high-performance liquid chromatography. While these methods offer high sensitivity, they often require lengthy processing times, complex sample pretreatment, and expensive laboratory equipment, making them unsuitable for real-time analysis of large-scale samples. Photoelectrochemical biosensors, with their advantages of low background signal, high sensitivity, and fast detection speed, have been widely used in the field of food safety. Because antibiotic concentrations in food and the environment are relatively low, trace analysis is typically employed for antibiotic detection. Therefore, establishing a rapid, simple, low-cost, and highly specific method for antibiotic detection is crucial. Summary of the Invention

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

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a photoelectrochemical sensor based on single-atom materials and enzyme-driven cascaded hairpin amplification. The photoelectrochemical sensor includes a ZnIn2S4 / Co / NH2-MIL-125(Ti) photoelectrode made with ZnIn2S4 / Co / NH2-MIL-125(Ti) single-atom-based material as the substrate, and a capture antibody and a protein for blocking non-specific active sites dropped onto 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 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 used to block nonspecific active sites includes bovine serum albumin.

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

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

[0012] (1) After incubating the tetracycline aptamer with primer P1 to form Apt / P1, add the target tetracycline to be detected and incubate. After incubation, take the supernatant, add primer P2 and circular template DNA, heat and react, then anneal. Add T4 ligase and incubate to form the RCA reaction precursor.

[0013] (2) Add phi 29 DNA polymerase and Nb.BbvCI endonuclease to the RCA reaction precursor solution, and carry out amplification and enzyme digestion reactions simultaneously. After terminating the reaction, an RCA reaction solution containing a large amount of hairpin DNA H1 and H2 is formed.

[0014] (3) After the RCA reaction solution is dropped onto the photoelectrochemical sensor for incubation, hemin chloride solution is added to react and a hemin / G-quadruplex structure with peroxidase-like activity is formed on the electrode surface of the photoelectrochemical sensor.

[0015] (4) The photoelectrochemical sensor reacted in step (3) is placed in a 4-chloro-1-naphthol solution containing H2O2 for catalytic oxidation to form a precipitate;

[0016] (5) Under the irradiation of the excitation light source, the photoelectrochemical sensor that has undergone the reaction in step (4) is used as the working electrode. The photocurrent signal is recorded using a three-electrode system, and a linear relationship curve between the photocurrent signal and the tetracycline concentration is prepared, so that the detection of tetracycline can be realized.

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

[0018] And / or the volume ratio of the tetracycline aptamer 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] After adding primer P2 and circular template DNA to the supernatant, heat at 90-95℃ for 5 min and then anneal;

[0021] 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 for the amplification and enzyme digestion reactions are: incubation at 45°C for 1 hour, followed by 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 heme chloride 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; after adding the heme chloride, 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 method for preparing the photoelectrochemical sensor includes the following steps:

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

[0028] (b) ZnIn2S4 was mixed with an aqueous methanol solution, and then cobalt(II) nitrate hexahydrate was added and stirred to react. The mixture was then subjected to low-temperature photodeposition, washed and dried 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 then 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 Nafion solution, dropped onto the conductive surface of the electrode, dried and rinsed; chitosan solution was then added, dried and rinsed; glutaraldehyde solution was then added, allowed to stand at room temperature and rinsed to obtain the photoelectrode.

[0031] (e) The amino-modified capture antibody is dropped onto the surface of the photoelectrode and incubated. After incubation, bovine serum albumin is dropped onto the electrode surface and incubation is continued 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 carried out by stirring in an oil bath at 75°C for 1 hour.

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

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

[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 dropwise addition amounts of the chitosan solution and the glutaraldehyde solution are both 15-25 μL.

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

[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 this invention introduces an appropriate amount of Co single atoms to form an O-Co-N coordination structure, which provides a new charge transfer channel for the Z-type heterostructure, thereby enhancing visible light absorption, improving the separation and transfer of charge carriers, and exhibiting a higher photocurrent response, further expanding the application range of single-atom-based materials.

[0040] An enzyme-driven cascaded hairpin amplification method was proposed to trigger the hemin / G-quadruplex reaction (HCR). This method achieves in-situ oxidation of 4-CN to form a precipitate by forming a hemin / G-quadruplex with peroxidase properties on the electrode surface, thus constructing a photoelectrochemical sensor for ultrasensitive detection of tetracycline (TET). This photoelectrochemical sensor exhibits a broad linear range and high selectivity for tetracycline detection, providing a novel strategy for food safety testing. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

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

[0043] Figure 2 Standard curves for different concentrations of tetracycline on the PEC sensing platform;

[0044] Figure 3 The results are from the selective analysis of the biosensor.

[0045] Figure 4 Comparison of photocurrent response results for ZnIn2S4 / Co / NH2-MIL-125(Ti) and ZnIn2S4 / NH2-MIL-125(Ti) samples. Detailed Implementation

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of 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 terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0048] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0049] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0051] The present invention relates to some of the compound names, and their corresponding chemical formulas or abbreviations:

[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] Hexadecyltrimethylammonium bromide: CTAB;

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

[0062] Tetrabutyl titanate (TBOT).

[0063] Example 1: A method for preparing a 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 an aqueous glycerol solution (80 mL, 20 vol%, pH = 2.5) and stirred for 30 min. The resulting solution was then placed in an oil bath at 75 °C 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 under 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. Then, 22 μL of Co(NO3)2·6H2O solution (10 mM) was 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 under vacuum 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 solution, and the mixture was continuously sonicated. Then, 60-90 mg of the synthesized Co / ZnIn2S4 (70 mg was used in this example) was added to the solution. The suspension was transferred to a 50 mL polytetrafluoroethylene (PTFE) autoclave and stored at 150 °C for 8 h. After cooling to room temperature, the mixture was washed several times with DMF and MeOH and then vacuum dried overnight at 60 °C to obtain the ZnIn2S4 / Co / NH2-MIL-125(Ti) single-atom-based material.

[0067] SEM images of the prepared samples are shown below. Figure 1 As shown in the figure, NH2-MIL-125(Ti) is attached to the surface of Co / ZnIn2S4 nanoflower in the form of nanosheets.

[0068] Example 2

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

[0070] (1) The FTO conductive glass was ultrasonically cleaned with acetone, ethanol and ultrapure water in sequence, and then dried in an oven.

[0071] (2) Add 50 μL of ZnIn2S4 / Co / NH2-MIL-125(Ti) suspension (2.0 mg / mL)-1 Mix with Nafion solution (2 μL) and drop onto the conductive surface of the FTO electrode (fixed area 1.0 cm²). 2 Dry at 45℃.

[0072] (3) Add 15-25 μL of chitosan solution (0.1 wt%, 20 μL in this example) to the electrode surface, dry it in an oven at 60°C, rinse with PBS, then add 15-25 μL of glutaraldehyde solution (2.5 v / v%, 20 μL in this example) to the electrode surface, let it stand at room temperature for 1 h, rinse with PBS, and obtain the photoelectrode.

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

[0074] Example 3

[0075] In this embodiment, the photoelectrochemical sensor prepared in Example 2 is used for the highly sensitive determination of tetracycline (TET). The specific steps are as follows:

[0076] (1) After incubating the tetracycline aptamer (2 μM, 10-20 μL, 10 μL in this example) with its recognition strand P1 (2 μM, 10-20 μL, 10 μL 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 was used as a primer for rolling circle amplification reaction.

[0077] (2) Take 2–3 μL of supernatant (P1, 2.5 μL in this example) from the reaction solution of step (1), add 2–3 μL of Circle DNA (10 μM, 2.5 μL in this example) and 2–3 μL of P2 (10 μM, 2.5 μL in this example). Heat the mixture at 95 °C for 5 min, 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 in this example) to the reaction system, and incubate at 22 °C for 1 h 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 Lhi 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 for each enzyme.

[0079] Amplification began with incubation at 45°C for 1 hour and ended with incubation at 65°C for 10 minutes, yielding hairpin DNAs H1 and H2 (H2 partially complements H1). Finally, the products were stored at 4°C for subsequent analysis.

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

[0081] (5) Finally, the electrode was incubated for 20 min in a 10 mM solution of 4-chloro-1-naphthol (4-CN) containing 1 mM H2O2 to carry out the catalytic oxidation reaction, and the 4-CN was catalytically oxidized in situ to form 4-CD precipitate. The electrode was washed with PBS buffer after each incubation.

[0082] (6) Under the irradiation of the 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 relevant DNA base sequence design mentioned above is shown in Table 1. The photocurrent detection results are as follows: Figure 2 As shown in the figure, when the photocurrent change value is plotted against the tetracycline concentration, the sensor exhibits 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] To verify the accuracy and practicality of the ultrasensitive photoelectrochemical sensor based on single-atom materials and enzyme-driven cascaded hairpin amplification in real-world applications, chloramphenicol (CHL), kanamycin (KAN), carbamazepine (CBZ), and amoxicillin (AMX) were used as interfering agents to evaluate its anti-interference capability. Figure 3 As shown, compared with the blank sample, the change in photocurrent intensity of non-target analytes is negligible, while the detection signal changes significantly regardless of whether the target analyte TET is present alone or together with these interfering substances. This demonstrates that the prepared PEC biosensor has strong TET specificity.

[0088] Comparative Example 1

[0089] (1) The ZnIn2S4 / NH2-MIL-125(Ti) material was prepared and compared with ZnIn2S4 / Co / NH2-MIL-125(Ti) to demonstrate that the present invention has superior photoelectric properties. The specific steps are as follows: 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 an oil bath at 75 °C 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 under vacuum at 60 °C 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 solution, and the mixture was continuously sonicated. Then, a certain amount of synthesized ZnIn2S4 was added to the solution. The suspension was transferred to a 50 mL polytetrafluoroethylene (PTFE) autoclave and stored at 150 °C for 8 h. After cooling to room temperature, the solution was washed several times with DMF and MeOH and then vacuum dried overnight at 60 °C to obtain ZnIn2S4 / NH2-MIL-125(Ti).

[0091] Under excitation light source irradiation, working electrodes were prepared using ZnIn2S4 / NH2-MIL-125(Ti) and ZnIn2S4 / Co / NH2-MIL-125(Ti) as photosensitive materials, respectively. A three-electrode system was used to record the photocurrent signal, and the results were plotted using time and photocurrent density. Figure 4 It is evident that the present invention, by introducing Co single atoms, possesses superior photoelectric response capabilities.

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

Claims

1. A method for detecting tetracycline using a photoelectrochemical sensor, characterized by, The photoelectrochemical sensor includes a ZnIn2S4 / Co / NH2-MIL-125(Ti) photoelectrode made with ZnIn2S4 / Co / NH2-MIL-125(Ti) single-atom-based material as the substrate, and capture DNA and proteins for blocking non-specific active sites dropped onto 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 Co / ZnIn2S4 nanoflower balls in the form of nanosheets; The nucleotide sequence of the captured DNA is shown in SEQ ID NO.4, and the protein used to block nonspecific active sites includes bovine serum albumin. The method for detecting tetracycline includes the following steps: (1) After incubating the tetracycline aptamer with primer P1 to form Apt / P1, add the target tetracycline to be detected and incubate. After incubation, take the supernatant, add primer P2 and circular template DNA, heat and react, then anneal. Add T4 ligase and incubate to form the RCA reaction precursor. (2) Add the reaction enzyme to the RCA reaction precursor and carry out amplification and enzymatic digestion reactions simultaneously. After terminating the reaction, an RCA reaction solution containing the hairpin DNA H1 and H2 is formed. (3) The RCA reaction solution is dropped onto the surface of the photoelectrochemical sensor and incubated. Then, heme chloride solution is added to continue the incubation reaction, and a hemin / G-quadruplex structure with peroxidase-like activity is formed on the electrode surface of the photoelectrochemical sensor. (4) The photoelectrochemical sensor reacted in step (3) is placed in a 4-chloro-1-naphthol solution containing H2O2 for catalytic oxidation to form a precipitate; (5) Under the irradiation of the excitation light source, the photoelectrochemical sensor that has undergone the reaction in step (4) is used as the working electrode. The photocurrent signal is recorded using a three-electrode system, and a linear relationship curve between the photocurrent signal and the tetracycline concentration is prepared, so that the detection of tetracycline can be realized. In step (1), the nucleotide sequence of the tetracycline aptamer is shown in SEQ ID NO.5; the nucleotide sequence of primer P1 is shown in SEQ ID NO.2; the nucleotide sequence of primer P2 is shown in SEQ ID NO.3; and the nucleotide sequence of the circular template DNA is shown in SEQ ID NO.

1.

2. The method of claim 1, wherein, In step (1), the volume ratio of the tetracycline aptamer to the primer P1 is 1:1; The volume ratio of the supernatant, primer P2, and circular template DNA is 1:1:1; After adding primer P2 and circular template DNA to the supernatant, the mixture was heated at 90-95℃ for 5 minutes and then annealed. Add T4 ligase to the annealed reaction system and incubate at 20-25℃ for 1 hour.

3. The method of claim 1, wherein, In step (2), the amplification reaction solution comprises 4.5-5.5 μL dNTP, 0.5-1.5 μL 10× Phi 29 DNA polymerase and 1.5-2.5 μL Nb.BbvCI; The conditions of the amplification and enzyme digestion reaction are as follows: incubation at 45°C for 1 h and incubation at 65°C for 10 min to deactivate the enzyme.

4. The method of claim 1, wherein, In step (3), the volume ratio of the RCA reaction solution to the hematin chloride solution is 1:4; the RCA reaction solution is dropped onto the surface of the photoelectrochemical sensor and incubated at 35-40°C for 2 h, and after the hematin chloride is added, the reaction is further incubated at 35-40°C for 50 min; 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.

5. The method of claim 1, wherein, The preparation method of the photoelectrochemical sensor comprises the following steps: (a) adding indium chloride tetrahydrate, zinc chloride and thioacetamide into a glycerol aqueous solution, heating and stirring to react, washing and drying to obtain ZnIn2S4; (b) mixing ZnIn2S4 with a methanol aqueous solution, adding cobalt(II) nitrate hexahydrate to stir to react, performing low-temperature photodeposition, washing and drying to obtain Co / ZnIn2S4; (c) ultrasonically dissolving 2-amino terephthalic acid and cetyltrimethylammonium bromide in a mixture of N,N-dimethylformamide and methanol, adding tetrabutyl titanate, uniformly ultrasonically dispersing, adding Co / ZnIn2S4 into the reaction solution, and performing high-temperature reaction to obtain ZnIn2S4 / Co / NH2-MIL-125(Ti); (d) uniformly dispersing ZnIn2S4 / Co / NH2-MIL-125(Ti) in an ethanol aqueous solution, mixing with a Nafion solution, dropping on the conductive surface of an electrode, drying and washing; further dropping a chitosan solution, drying and washing; further dropping a glutaraldehyde solution, standing at room temperature, and washing to obtain a photoelectrode; (e) dropping the capture DNA modified with an amino group on the surface of the photoelectrode to incubate, adding bovine serum albumin on the surface of the electrode after the incubation is completed, and continuing to incubate to obtain the photoelectrochemical sensor.

6. The method of claim 5, wherein, In step (a), the ratio of indium chloride tetrahydrate, zinc chloride, thioacetamide and glycerol is 2 mmol:1 mmol:8 mmol:80 mL; and the heating and stirring reaction is performed at 75°C in an oil bath for 1 h; In step (b), the ratio of ZnIn2S4, the 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; and the stirring reaction is performed at 10°C under the irradiation of a 300 W xenon lamp for 30 min; In step (c), the ratio of 2-amino terephthalic acid, cetyltrimethylammonium bromide, N,N-dimethylformamide and methanol is 0.2-0.4 g: 1-2 g: 5-8 mL: 2-5 mL; the ratio of 2-amino terephthalic acid and tetrabutyl titanate is 0.2-0.4 g: 0.1-0.3 mL; the ratio of 2-amino terephthalic acid and Co / ZnIn2S4 is 0.2-0.4 g: 60-90 mg; In step (d), after the ZnIn2S4 / Co / NH2-MIL-125(Ti) is uniformly dispersed in the ethanol aqueous solution, 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); and the dropwise addition amount of the chitosan solution and the glutaraldehyde solution is 15-25 μL. In step (e), the dropwise addition amount of the modified amino group capture DNA is 15-25 μL, and the incubation condition is 37°C, 1-2 h; the dropwise addition amount of the bovine serum albumin is 5-15 μL, and the incubation condition is room temperature incubation for 30-40 min.

7. The method of claim 1, wherein, The tetracycline is derived from food.

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