A photoelectrochemical sensing method for detecting oxytetracycline using a differential signal strategy

Abstract

This invention discloses a photoelectrochemical sensing method for detecting oxytetracycline using a differential signal strategy. The method includes the preparation of FeCu-BMO and Fe2O3@CuO nanospheres, the preparation of silver chloride cubes, and the construction of an oxytetracycline detection method. This invention prepares a bimetallic organic framework FeCu-MOF, using its oxide as a photoelectroactive material. It utilizes the specific recognition of oxytetracycline by an aptamer and the binding of the auxiliary chain on the silver chloride nanocube to the aptamer. Due to the steric hindrance effect of oxytetracycline and the quenching effect of silver chloride, a differential signal is calculated through two consecutive and correlated events, constructing a novel photoelectrochemical sensor for detecting oxytetracycline. This improves the specific binding of oxytetracycline to the sensor, achieving high-sensitivity detection of oxytetracycline. The detection limit of the differential signal strategy is 5 nmol / L, which is lower than the 22 nmol / L detection limit of the single-event photoelectrochemical sensor. Furthermore, the sensor exhibits excellent selectivity, stability, and reproducibility, making it suitable for oxytetracycline residue detection needs in various scenarios.

Description

Technical field:

[0001] The present invention relates to a method for detecting oxytetracycline, and in particular to a photoelectrochemical sensing method for detecting oxytetracycline using a differential signal strategy, and belongs to the technical field of photoelectrochemical sensing. Background technology:

[0002] Oxytetracycline (OTC) is an antibiotic widely used in agriculture and food industry, and is widely used to prevent and treat infectious diseases in poultry, livestock and other animals. Although oxytetracycline plays an important role in agricultural production, its residues may pose a potential threat to human health, so the rapid and accurate detection of oxytetracycline residues has become an urgent need.

[0003] Nowadays, there are many technical means to detect oxytetracycline, such as high performance liquid chromatography tandem mass spectrometry and enzyme-linked immunosorbent assay. High performance liquid chromatography tandem mass spectrometry has the advantages of high accuracy and high sensitivity, but its widespread use is limited by factors such as expensive equipment, long time consumption and the need for professional operation; enzyme-linked immunosorbent assay is simple and fast to operate, but it is prone to false positives. Therefore, it is necessary to develop a fast, sensitive and highly selective photoelectrochemical sensing detection method.

[0004] Photoelectrochemical (PEC) sensors, as a new detection technology, have the advantages of rapidity, high sensitivity, and low cost, and show great potential in the field of antibiotic residue detection. By combining the photophysical properties and chemical reaction mechanism of oxytetracycline with photoelectrochemical sensors, rapid and accurate detection of oxytetracycline can be achieved. The surface modification or functionalization of photoelectrochemical sensors can improve the specific binding of oxytetracycline to the sensor, thereby achieving high-sensitivity detection of oxytetracycline. In addition, photoelectrochemical sensors also have the characteristics of real-time monitoring, no need for expensive equipment, and easy to carry, which are suitable for the detection needs of oxytetracycline residues in different scenarios. Summary of the invention:

[0005] The technical problem to be solved by the present invention is: to overcome the shortcomings of the prior art, use a bimetallic-metal organic framework material derivative as a base material, silver chloride as a signal probe, design a differential signal strategy, and construct a photoelectrochemical sensing method for detecting oxytetracycline using a differential signal strategy.

[0006] The technical solution adopted by the present invention to solve the technical problem is as follows:

[0007] A photoelectrochemical sensing method for detecting oxytetracycline using a differential signal strategy comprises the following processing steps:

[0008] A. Preparation of FeCu-BMOF and Fe2O3@CuO nanospheres:

[0009] Preparation of bimetallic metal organic framework material FeCu-BMOF: take ferric chloride hexahydrate, copper nitrate trihydrate and 4-pyrazole carboxylic acid, dissolve them in dimethylformamide by ultrasonication, heat the mixture at 100°C for 12h, centrifuge and wash after cooling to room temperature, and dry at 60°C to obtain green powder FeCu-BMOF; take FeCu-BMOF powder and calcine it at 550°C for 90-100min to obtain brown-red Fe2O3@CuO nanospheres, cool naturally and store in the dark;

[0010] B. Preparation of silver chloride cubes:

[0011] Preparation of silver chloride cubes: dissolving polyvinyl pyrrolidone in the first portion of ethylene glycol, dissolving silver nitrate in the second portion of ethylene glycol, adding the dissolved polyvinyl pyrrolidone and silver nitrate into a 100 mL round-bottom bottle, and mixing at 1500 rpm; adding concentrated hydrochloric acid to the solution, the concentration of the concentrated hydrochloric acid being 37%, and then heating the mixture at 150° C. for 20 min, and then cooling the mixture to room temperature for purification, washing once with acetone and twice with ultrapure water to obtain silver chloride cubes; using tris (2-chloroethyl) phosphate solution to cultivate thiol-modified auxiliary chains to remove disulfide bonds; and then shaking it together with the silver chloride cubes at a constant temperature for 12 h, obtaining a silver chloride cube-auxiliary chain signal probe by combining silver with thiol, and storing it at 4° C. in the dark for later use;

[0012] C. Construction of oxytetracycline detection method:

[0013] (1) Fe2O3@CuO nanospheres were added dropwise onto the surface of the indium tin oxide electrode and dried at room temperature for 8 h in a dark environment to obtain a Fe2O3@CuO / indium tin oxide electrode;

[0014] (2) chitosan was added dropwise and reacted at room temperature for 12 h, then modified with glutaraldehyde and reacted for 30 min. After washing, chitosan / Fe2O3@CuO / indium tin oxide electrode was obtained;

[0015] (3) adding the aptamer dropwise, reacting for 2 h, and washing to obtain the aptamer / chitosan / Fe2O3@CuO / indium tin oxide electrode;

[0016] (4) bovine serum albumin was added dropwise and reacted for 1 h. After washing, a bovine serum albumin / aptamer / chitosan / Fe2O3@CuO / indium tin oxide electrode was obtained and the photoelectric signal A was measured;

[0017] (5) adding a certain concentration of oxytetracycline, reacting for a period of time, washing, obtaining an oxytetracycline / bovine serum albumin / aptamer / chitosan / Fe2O3@CuO / indium tin oxide electrode, and measuring a photoelectric signal B;

[0018] (6) adding silver chloride cube-auxiliary chain signal probe, reacting for a period of time, washing to obtain a silver chloride cube-auxiliary chain / oxytetracycline / bovine serum albumin / aptamer / chitosan / Fe2O3@CuO / indium tin oxide electrode, and measuring the photoelectric signal C;

[0019] (7) Calculate the differential signal value: |BC|-|AB|.

[0020] In step A, the usage ratio of ferric chloride hexahydrate, copper nitrate trihydrate, 4-pyrazolecarboxylic acid and dimethylformamide is 35.5 mg:114.5 mg:54.0 mg:10 mL.

[0021] In step B, the usage ratio of polyvinyl pyrrolidone, the first portion of ethylene glycol, silver nitrate, the second portion of ethylene glycol and concentrated hydrochloric acid is: 0.4 g: 30 mL: 0.4 g: 20 mL: 1.23 mL.

[0022] In step C:

[0023] The concentration of the Fe2O3@CuO nanospheres is 1 mg / mL, and the dosage is 25 μL;

[0024] The weight percentage concentration of the chitosan is 0.025wt%, and the dosage is 20μL;

[0025] The concentration of glutaraldehyde is 2.5%, and the dosage is 20 μL;

[0026] The aptamer is a nucleic acid aptamer with a concentration of 1 μmol / L and a dosage of 20 μL;

[0027] The concentration of the bovine serum albumin is 1%, and the dosage is 20 μL;

[0028] The dosage of oxytetracycline is 20 μL;

[0029] The concentration of the silver chloride cube-auxiliary chain signal probe is 1.5 μmol / L, and the dosage is 20 μL.

[0030] The aptamer is a nucleic acid aptamer having a nucleic acid sequence shown in Table 1.

[0031] The auxiliary chain is an auxiliary chain having a nucleic acid sequence shown in Table 1, is an auxiliary chain marked on a silver chloride cube, and can be combined with a nucleic acid aptamer.

[0032] Table 1 Nucleotide sequences

[0033]

[0034] The positive beneficial effects of the present invention are as follows:

[0035] 1. The present invention combines the photophysical properties and chemical reaction mechanism of oxytetracycline with a photoelectrochemical sensor to achieve rapid and accurate detection of oxytetracycline. The surface modification or functionalization of the photoelectrochemical sensor is used to improve the specific binding of oxytetracycline to the sensor, thereby achieving high-sensitivity detection of oxytetracycline.

[0036] 2. The present invention uses a bimetallic-metal organic framework material derivative as a base material and silver chloride as a signal probe, designs a differential signal strategy, and constructs a photoelectrochemical sensing method for detecting oxytetracycline using a differential signal strategy. The method has the characteristics of real-time monitoring, no need for expensive equipment, and easy to carry, and is suitable for oxytetracycline residue detection needs in different scenarios.

[0037] 3. The present invention prepares a bimetallic organic framework FeCu-MOF, uses its oxide as a photoelectrically active material, utilizes the specific recognition of oxytetracycline by an aptamer, calculates the differential signal through two consecutive and related events, and constructs a new photoelectrochemical sensor for detecting oxytetracycline. The detection limit of the differential signal strategy is 5nmol / L, which is lower than the detection limit of 22nmol / L of a single event photoelectrochemical sensor; at the same time, the sensor has excellent selective specificity, and satisfactory stability and reproducibility. The high-performance photoelectrochemical sensor of the present invention provides new research inspiration for oxytetracycline detection. Description of the drawings:

[0038] Figure 1 The schematic diagram of the photoelectrochemical sensing method for detecting oxytetracycline based on a silver chloride signal probe and a differential signal strategy of the present invention;

[0039] Figure 2 Middle: A is the scanning electron microscope image of FeCu-BMOF, B is the scanning electron microscope image of Fe2O3@CuO, C is the scanning electron microscope image of silver chloride cubes, and D is the X-ray diffraction spectrum of FeCu-BMOF and Fe2O3@CuO nanospheres;

[0040] Figure 3 It is the X-ray photoelectron spectroscopy analysis spectrum, A is the full spectrum, B is Cu p2, C is O1s, and D is Fe 2p;

[0041] Figure 4 is the nitrogen isothermal adsorption-desorption curve of Fe2O3@CuO nanospheres;

[0042] Figure 5 Middle: A is the graph of the change of AC impedance during the assembly process of the electrochemical sensor, and B is the graph of the change of photocurrent during the assembly process of the electrochemical sensor;

[0043] Figure 6Middle: A is the optimization diagram of oxytetracycline capture time, and B is the optimization diagram of silver chloride cube and aptamer binding time;

[0044] Figure 7-1 The photocurrent response diagram of different concentrations of oxytetracycline (A), the concentration of oxytetracycline (ag): 50, 100, 200, 300, 400, 500, 600nmo l / L;

[0045] Figure 7-2 is the standard curve diagram of the differential signal (B);

[0046] Figure 7-3 (C) is the standard curve of single photocurrent signal.

[0047] Figure 8 Selectivity (A), stability (B), and reproducibility (C) of the photoelectrochemical sensor. Specific implementation method:

[0048] The present invention will be further explained and illustrated below in conjunction with specific embodiments and accompanying drawings:

[0049] 1. Example

[0050] A photoelectrochemical sensing method for detecting oxytetracycline using a differential signal strategy comprises the following processing steps:

[0051] A. Preparation of FeCu-BMOF and Fe2O3@CuO nanospheres:

[0052] Preparation of bimetallic metal organic framework material FeCu-BMOF: take ferric chloride hexahydrate (35.5 mg), copper nitrate trihydrate (114.5 mg) and 4-pyrazolecarboxylic acid (54.0 mg), ultrasonically dissolve them in 10 mL of dimethylformamide; heat the mixture at 100 ° C for 12 h, centrifuge and wash after cooling to room temperature, and dry at 60 ° C to obtain green powder FeCu-BMOF; take FeCu-BMOF powder and calcine at 550 ° C for 90 min to obtain brown-red Fe2O3@CuO nanospheres, and store them in the dark after natural cooling;

[0053] B. Preparation of silver chloride cubes:

[0054] Preparation of silver chloride cubes: 0.4 g of polyvinyl pyrrolidone was dissolved in 30 mL of ethylene glycol, 0.4 g of silver nitrate was dissolved in another 20 mL of ethylene glycol, the dissolved polyvinyl pyrrolidone and silver nitrate were added to a 100 mL round-bottom flask, and mixed at 1500 rpm; 1.23 mL of concentrated hydrochloric acid (37%) was added to the solution, and the mixture was heated at 150° C. for 20 min, and then the mixture was cooled to room temperature and purified, washed once with acetone and twice with ultrapure water to obtain silver chloride cubes; tris(2-chloroethyl) phosphate solution was used to cultivate the thiol-modified auxiliary chain to remove the disulfide bond; then it was shaken at a constant temperature for 12 h together with the silver chloride cube, and the silver chloride cube-auxiliary chain signal probe was obtained by combining silver with the thiol group, and stored at 4° C. in the dark for later use;

[0055] C. Construction of oxytetracycline detection method

[0056] (1) Add 25 μL of Fe2O3@CuO nanosphere suspension (1 mg / mL) onto the surface of the indium tin oxide electrode and dry at room temperature for 8 h in the dark to obtain a Fe2O3@CuO / indium tin oxide electrode;

[0057] (2) 20 μL chitosan (0.025 wt%) was added dropwise and reacted at room temperature for 12 h; then 20 μL glutaraldehyde (2.5%) was added and reacted for 30 min. After washing, chitosan / Fe2O3@CuO / indium tin oxide electrode was obtained;

[0058] (3) 20 μL of aptamer (1 μmol / L) was added dropwise, reacted for 2 h, and washed to obtain the aptamer / chitosan / Fe2O3@CuO / indium tin oxide electrode;

[0059] (4) 20 μL of bovine serum albumin (1%) was added dropwise and reacted for 1 h. After washing, a bovine serum albumin / aptamer / chitosan / Fe2O3@CuO / indium tin oxide electrode was obtained and the photoelectric signal A was measured;

[0060] (5) 20 μL of a certain concentration of oxytetracycline was added dropwise, reacted for a period of time, and washed to obtain an oxytetracycline / bovine serum albumin / aptamer / chitosan / Fe2O3@CuO / indium tin oxide electrode, and the photoelectric signal B was measured;

[0061] (6) adding 20 μL of silver chloride cube-auxiliary chain signal probe (1.5 μmol / L) and reacting for a period of time, washing to obtain a silver chloride cube-auxiliary chain / oxytetracycline / bovine serum albumin / aptamer / chitosan / Fe2O3@CuO / indium tin oxide electrode, and measuring the photoelectric signal C;

[0062] (7) Calculate the differential signal value: |BC|-|AB|.

[0063] 2. Results

[0064] 2.1 Material characterization

[0065] like Figure 2 As shown in (A), the scanning electron microscope image of FeCu-BMOF shows a flower ball structure, and the morphology of Fe2O3@CuO obtained after oxidation does not change significantly ( Figure 2 (B)). Figure 2 In (C), polyvinyl pyrrolidone was used as a surfactant, and silver nitrate in ethylene glycol was exchanged with hydrochloric acid to synthesize silver chloride cubes with a size of about 400nm. Figure 2 As shown in (D), the characteristic diffraction peaks of FeCu-BMOF are consistent with those reported in the literature. When FeCu-BMOF is carbonized, new diffraction peaks appear. The diffraction peaks at 24.14°, 33.15°, 35.61°, 40.85°, 49.48°, 54.09°, 57.59°, 62.45°, 63.99°, 72.26°, 72.43° and 82.94° correspond to the crystal planes of Fe2O3 at (102), (104), (110), (113), (024), (116), (018), (214), (300), (119), (220) and (0210), respectively (JCPDS, PDF#33-0664); The characteristic diffraction peaks appearing at ta are 32.49, 35.69, 38.73, 48.73, 53.45, 58.30, 61.53, 65.78, 66.24, 68.08, 72.42, 75.22, and 83.10, corresponding to the (-110), (002), (111), (-202), (020), (202), (-113), (022), (-311), (-220), (311), (-222), and (222) planes of CuO, respectively (JCPDS, PDF#45-0937), indicating the successful preparation of Fe2O3@CuO nanospheres.

[0066] The full spectrum of X-ray photoelectron spectroscopy analysis of Fe2O3@CuO nanospheres is shown in Figure 3 As shown in (A), it contains Fe, Cu and O elements. Figure 3 In (B), the peaks at 934.6 eV and 954.5 eV are attributed to Cu 2p 3 / 2 and Cu 2p 1 / 2 , the peak observed at 942.9 eV is Cu 2p 3 / 2 The satellite peak of Cu 2p was observed at 962.8 eV. 1 / 2 These results indicate that the Cu element in the material has a valence of "+2". Figure 3As can be seen in (C), the high-resolution O1s spectrum with a peak at 530.58 eV corresponds to the O 2- ions, the peaks at 532.38 eV and 533.76 eV belong to oxygen vacancies and water molecules absorbed on the polyhedral surface, respectively. Figure 3 (D) is the fitted XPS spectrum of Fe 2p. The peaks at 711.8 eV and 725.2 eV are attributed to Fe 2p3 / 2 and Fe 2p1 / 2, respectively. The peaks at binding energies of 716.5 eV and 734.2 eV correspond to Fe 2p 3 / 2 and Fe 2p 1 / 2 spin orbit, these results are consistent with Fe 3+ The binding energy values ​​are consistent.

[0067] In order to study the pore structure and specific surface area of ​​Fe2O3@CuO nanospheres, nitrogen adsorption and desorption tests were carried out. Figure 4 As shown in Figure 2, the BET specific surface area and average pore size of Fe2O3@CuO nanospheres are 4.82 m 2 / g and 14.9nm, indicating that the Fe2O3@CuO nanospheres have a porous structure and a high specific surface area.

[0068] 2.2 Feasibility Analysis

[0069] Electrochemical impedance spectroscopy is an effective method to verify the preparation process of photoelectrochemical sensors. Figure 5 (A) shows the AC impedance curves of the charge transfer resistance at the electrode interface at different assembly stages. The Fe2O3@CuO / ITO electrode showed a small electron transfer resistance (curve a). However, the introduction of the aptamer to the electrode surface via chitosan and glutaraldehyde crosslinkers and the blocking of the excess binding sites with BSA resulted in increasing impedance values ​​(curves b, c, and d). When oxytetracycline was incubated with BSA / aptamer / chitosan / Fe2O3@CuO / ITO electrode, the impedance value increased (curve e), which can be attributed to the significant steric hindrance of oxytetracycline bound to the electrode. After the introduction of silver chloride cubes into the electrode, the impedance value increased further (curve f), which can be explained by the weak charge transfer of the silver chloride semiconductor.

[0070] Record the photocurrent response values ​​at different stages of the assembly process ( Figure 5(B)). The cathode photocurrent of the initial Fe2O3@CuO / indium tin oxide electrode is large (curve a). After the aptamer is assembled using a crosslinker and blocked with bovine serum albumin, the conductivity deteriorates due to enhanced steric hindrance, and the photocurrent decreases continuously (curves b, c, and d). When oxytetracycline is captured by the aptamer on the electrode, the photoelectric response decreases significantly (curve e), which may be affected by the poor conductivity of oxytetracycline, indicating that the photoelectrochemical sensor successfully captures oxytetracycline. The electron transfer process of Fe2O3@CuO is affected by the photocurrent quenching effect of silver chloride cubes, resulting in further weakening of the photocurrent (curve f). The above results show the feasibility of photoelectrochemical sensors for oxytetracycline detection.

[0071] exist Figure 5 Different modified electrodes: (a) Fe2O3@CuO / indium tin oxide electrode; (b) chitosan / Fe2O3@CuO / indium tin oxide electrode; (c) aptamer / chitosan / Fe2O3@CuO / indium tin oxide electrode; (d) bovine serum albumin / aptamer / chitosan / Fe2O3@CuO / indium tin oxide electrode; (e) oxytetracycline / bovine serum albumin / aptamer / chitosan / Fe2O3@CuO / indium tin oxide electrode; (f) silver cube-auxiliary chain / oxytetracycline / bovine serum albumin / aptamer / chitosan / Fe2O3@CuO / indium tin oxide electrode.

[0072] 2.3 Condition Optimization

[0073] The time of aptamer capture of oxytetracycline affects the photoelectric response. Figure 6 (A) It can be seen that as the capture time increases, the photoelectric response value gradually decreases and approaches a plateau at 60 min, so 60 min is selected for this operation.

[0074] The remaining aptamers bind to the auxiliary probes, fixing the silver chloride cubes on the electrode surface and affecting the photoelectric response value. As shown in 6(B), the cathode photoelectric response value decreases with time and reaches a platform at 90 minutes, indicating that the binding of the aptamer and the auxiliary probe has reached saturation. Therefore, 90 minutes is selected as the optimal binding time.

[0075] 2.4 Detection performance analysis

[0076] Under optimized experimental conditions, the developed photoelectrochemical sensing platform was applied to the determination of oxytetracycline. Figure 7-1(A) It can be seen that the cathode photoelectric response value increases with the increase of illumination time due to the consumption of ascorbic acid. Therefore, the maximum photoelectric response value is used. When oxytetracycline is captured, the cathode photocurrent decreases with the increase of oxytetracycline concentration (50-600nmol / L); on the contrary, the introduction of silver chloride causes the cathode photocurrent to increase with the increase of oxytetracycline concentration. By differentially calculating the changes in the photoelectric signals of these two consecutive and related events, Figure 7-2 (B) shows that the differential signal value has a good linear relationship with the oxytetracycline concentration in the range of 50-600 nmol / L, and the linear regression equation is I(nA)=51.802-0.102C 土霉素 (R 2 =0.996), with a low detection limit of 5nmol / L (S / N=3). Figure 7-3 As shown in (C), when the silver chloride auxiliary chain is not introduced, the photoelectrochemical signal change generated by the event of oxytetracycline being captured by the aptamer alone is constructed, and the standard curve I (nA) = -100.242 + 0.036C 土霉素 (R 2 =0.994), and the calculated detection limit was 22 nmol / L, indicating that the differential signal strategy improved the sensitivity of the photoelectrochemical sensor in detecting oxytetracycline.

[0077] 2.5 Selectivity

[0078] In order to evaluate the selectivity of the established photoelectrochemical sensor, erythromycin (EM), ciprofloxacin hydrochloride (CIP), kanamycin (Kana), tetracycline (TC) and doxycycline (DOX) were selected for anti-interference tests. Figure 8 As shown in (A), when oxytetracycline is absent, other antibiotics do not cause changes in the photocurrent response value of the photoelectrochemical sensor, and the calculated differential signal is similar to the signal generated by the blank group. However, when oxytetracycline (500nmol / L) is present, the silver chloride nanocubes fixed on the electrode are reduced due to the capture of oxytetracycline by the aptamer, and the differential signal value is significantly reduced. These results show that the constructed photoelectrochemical sensor can effectively eliminate interfering signals and has excellent selectivity.

[0079] 2.6 Stability

[0080] The photoelectric response values ​​of the sensor stored at 4°C for 0, 7, 14, and 21 days were measured to evaluate the stability of the method. Figure 8(B) shows that after the sensor was stored at 4°C for 7 days, 14 days and 24 days, the photoelectric response values ​​to oxytetracycline (500 nmol / L) were maintained at about 99.2%, 98.4% and 99.1% of the initial value, respectively. These results indicate that the constructed photoelectrochemical sensor shows good stability.

[0081] 2.7 Reproducibility

[0082] like Figure 8 As shown in (C), for 200 nmol / L oxytetracycline, the relative standard deviation (RSD) of 5 groups of measurements from the same batch after differential calculation was 4.4%, indicating that the method has satisfactory reproducibility.

[0083] 2.8 Spike recovery test

[0084] In order to further evaluate the feasibility of the constructed photoelectrochemical sensing strategy in actual sample detection, different concentrations of oxytetracycline (100, 300 and 500 nmol / L) were added to pure water for testing. The experimental results are shown in Table 2. The RSDs of the three groups of samples were 2.7%, 4.3% and 0.9%, respectively, and the average recovery rates were 99.7-105.9%.

[0085] Table 2 Detection of oxytetracycline spiked recovery (n = 3)

[0086]

[0087] 3. Conclusion

[0088] In summary, the present invention prepares a bimetallic organic framework FeCu-MOF, uses its oxide as a photoelectrically active material, utilizes the specific recognition of oxytetracycline by an aptamer, calculates the differential signal through two consecutive and related events, and constructs a new photoelectrochemical sensor for detecting oxytetracycline. The detection limit of the differential signal strategy is 5nmol / L, which is lower than the detection limit of 22nmol / L of a single event photoelectrochemical sensor. At the same time, the sensor has excellent selective specificity, as well as satisfactory stability and reproducibility.

[0089] The high-performance photoelectrochemical sensor designed in the present invention provides new research inspiration for the detection of oxytetracycline. The surface modification or functionalization of the photoelectrochemical sensor is used to improve the specific binding of oxytetracycline to the sensor. A differential strategy is designed in combination with a silver chloride signal probe to achieve high-sensitivity detection of oxytetracycline.

Claims

1. A photoelectrochemical sensing method for detecting oxytetracycline using a differential signal strategy, characterized in that: The processing steps include: A. Preparation of FeCu-BMOF and Fe2O3@CuO nanospheres: Preparation of bimetallic metal organic framework material FeCu-BMOF: take ferric chloride hexahydrate, copper nitrate trihydrate and 4-pyrazole carboxylic acid, dissolve them in dimethylformamide by ultrasonication, heat the mixture at 100°C for 12h, centrifuge and wash after cooling to room temperature, and dry at 60°C to obtain green powder FeCu-BMOF; take FeCu-BMOF powder and calcine it at 550°C for 90-100min to obtain brown-red Fe2O3@CuO nanospheres, cool naturally and store in the dark; B. Preparation of silver chloride cubes: Preparation of silver chloride cubes: dissolving polyvinyl pyrrolidone in the first portion of ethylene glycol, dissolving silver nitrate in the second portion of ethylene glycol, adding the dissolved polyvinyl pyrrolidone and silver nitrate into a 100 mL round-bottom bottle, and mixing at 1500 rpm; adding concentrated hydrochloric acid to the solution, the concentration of the concentrated hydrochloric acid being 37%, and then heating the mixture at 150° C. for 20 min, and then cooling the mixture to room temperature for purification, washing once with acetone and twice with ultrapure water to obtain silver chloride cubes; using tris (2-chloroethyl) phosphate solution to cultivate thiol-modified auxiliary chains to remove disulfide bonds; then shaking it together with the silver chloride cubes at a constant temperature for 12 h, obtaining a silver chloride cube-auxiliary chain signal probe by combining silver with thiol, and storing it at 4° C. in the dark for later use; C. Construction of oxytetracycline detection method: (1) Fe2O3@CuO nanospheres were added dropwise onto the surface of the indium tin oxide electrode and dried at room temperature for 8 h in a dark environment to obtain a Fe2O3@CuO / indium tin oxide electrode; (2) chitosan was added dropwise and reacted at room temperature for 12 h, then modified with glutaraldehyde and reacted for 30 min. After washing, chitosan / Fe2O3@CuO / indium tin oxide electrode was obtained; (3) adding the aptamer dropwise, reacting for 2 h, and washing to obtain the aptamer / chitosan / Fe2O3@CuO / indium tin oxide electrode; (4) bovine serum albumin was added dropwise and reacted for 1 h. After washing, a bovine serum albumin / aptamer / chitosan / Fe2O3@CuO / indium tin oxide electrode was obtained and the photoelectric signal A was measured; (5) adding a certain concentration of oxytetracycline, reacting for a period of time, washing, obtaining an oxytetracycline / bovine serum albumin / aptamer / chitosan / Fe2O3@CuO / indium tin oxide electrode, and measuring a photoelectric signal B; (6) adding silver chloride cube-auxiliary chain signal probe, reacting for a period of time, washing to obtain a silver chloride cube-auxiliary chain / oxytetracycline / bovine serum albumin / aptamer / chitosan / Fe2O3@CuO / indium tin oxide electrode, and measuring the photoelectric signal C; (7) Calculate the differential signal value: |BC|-|AB|.

2. The photoelectrochemical sensing method for detecting oxytetracycline using a differential signal strategy according to claim 1, characterized in that: In step A, the usage ratio of ferric chloride hexahydrate, copper nitrate trihydrate, 4-pyrazolecarboxylic acid and dimethylformamide is 35.5 mg:114.5 mg:54.0 mg:10 mL.

3. The photoelectrochemical sensing method for detecting oxytetracycline using a differential signal strategy according to claim 1, characterized in that: In step B, the usage ratio of polyvinyl pyrrolidone, the first portion of ethylene glycol, silver nitrate, the second portion of ethylene glycol and concentrated hydrochloric acid is: 0.4 g: 30 mL: 0.4 g: 20 mL: 1.23 mL.

4. The photoelectrochemical sensing method for detecting oxytetracycline by a differential signal strategy according to claim 1, characterized in that In step C: The concentration of the Fe2O3@CuO nanospheres is 1 mg / mL, and the dosage is 25 μL; The weight percentage concentration of the chitosan is 0.025wt%, and the dosage is 20μL; The concentration of glutaraldehyde is 2.5%, and the dosage is 20 μL; The aptamer is a nucleic acid aptamer with a concentration of 1 μmol / L and a dosage of 20 μL; The concentration of the bovine serum albumin is 1%, and the dosage is 20 μL; The dosage of oxytetracycline is 20 μL; The concentration of the silver chloride cube-auxiliary chain signal probe is 1.5 μmol / L, and the dosage is 20 μL.

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