Preparation method and application of a photoelectrochemical-surface enhanced Raman spectroscopy sensor

Abstract

The present invention belongs to the field of analytical chemistry and biosensor technology, and relates to a preparation method and application of a photoelectrochemical-surface enhanced Raman spectroscopy sensor. The sensor utilizes the excellent light absorption ability and Raman scattering characteristics of Zr-MOF, and can simultaneously generate obvious PEC and SERS signals under low-power 532nm Raman excitation light. And compared with porphyrin molecules, the Raman signal stability of Zr-MOF is better. Prepare AuNDs@AgNPs as a dual-functional enhanced substrate to enhance the photoelectric and Raman signals of Zr-MOF; use aptamers to connect the dual-functional nanoprobe Zr-MOF and AuNDs@AgNPs to construct a photoelectrochemical-surface enhanced Raman spectroscopy sensor, and use the dual signals to collect simultaneously on the same electrode and refer to each other, thereby achieving high sensitivity and high precision detection of penicillin, with a detection linear range of 1pg mL ‑1 ~100ng mL ‑1 .

Description

Technical Field

[0001] The present invention belongs to the field of analytical chemistry and biosensing technology, and relates to a preparation method and application of a photoelectrochemical-surface enhanced Raman spectroscopy sensor; specifically, it relates to the preparation of a novel photoelectrochemical-surface enhanced Raman spectroscopy (PEC-SERS) sensor, which uses porphyrin-based metal-organic frameworks (ZrMOFs) as a dual-function nanoprobe and can be used for the efficient and sensitive detection of patulin in agricultural products. Background Art

[0002] Food safety testing is a major global public health issue. Detection of patulin, a mycotoxin widely found in grains and other agricultural products, is crucial for preventing food safety incidents. Traditional patulin detection methods, such as high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA), while highly sensitive and accurate, are often complex, time-consuming, and costly.

[0003] With the development of nanotechnology and analytical chemistry, sensors based on nanomaterials have shown great potential in food safety testing due to their unique physicochemical properties. In particular, photoelectrochemical (PEC) and surface-enhanced Raman spectroscopy (SERS) techniques have been widely studied for food safety testing due to their high sensitivity, label-free nature, and real-time monitoring. However, these techniques typically require specific photoactive materials and complex nanostructures, which limits their practical application.

[0004] Existing PEC and SERS sensors face challenges in detecting small molecule pollutants such as patulin, primarily due to a lack of effective signal enhancement mechanisms and stability. Furthermore, existing sensors often cannot provide both photoelectric and Raman signals simultaneously, limiting detection accuracy and information content.

[0005] To address these issues, researchers have been exploring novel signal enhancement strategies and nanomaterials. Metal-organic frameworks (MOFs) are considered an ideal signal enhancement platform due to their highly tunable pore structure, large specific surface area, and easy functionalization. Summary of the Invention

[0006] The present invention provides a dual-function nanoprobe based on Zr-MOF for constructing a photoelectrochemical-surface enhanced Raman spectroscopy (PEC-SERS) sensor. The sensor utilizes the excellent light absorption ability and Raman scattering properties of Zr-MOF to simultaneously generate significant photoelectrochemical (PEC) and surface enhanced Raman (SERS) signals under low-power 532nm Raman excitation light. Compared with porphyrin molecules, the Raman signal stability of Zr-MOF is better. Silver-coated gold nanodumbbell nanostructures (AuNDs@AgNPs) are prepared as dual-function enhanced substrates to enhance the photoelectric and Raman signals of Zr-MOF. The dual-function nanoprobe Zr-MOF and AuNDs@AgNPs are connected with aptamers to construct a photoelectrochemical-surface enhanced Raman spectroscopy (PEC-SERS) sensor, which achieves highly sensitive and high-precision detection of penicillin.

[0007] In order to achieve the above technical objectives, the specific solutions of the present invention are as follows:

[0008] A photoelectrochemical-surface enhanced Raman spectroscopy sensor based on a porphyrin-based metal-organic framework dual-functional nanoprobe is used to detect patulin, comprising the following steps:

[0009] (1) Preparation of Zr-MOF dual-functional nanoprobes:

[0010] TCPP (tetrakis-(4-carboxyphenyl)porphine), ZrOCl2·8H2O, and benzoic acid were dissolved in DMF (N,N-dimethylformamide), and the resulting mixed solution was heated for a period of time under stirring conditions. After heating, the solution was centrifuged to collect the precipitate, which was washed several times with DMF and then centrifuged again to obtain the Zr-MOF dual-functional nanoprobe.

[0011] (2) Preparation of AuNRs seed solution:

[0012] S1. Mix ultrapure water, CTAB solution, and HAuCl4 solution, and add glacial NaBH4 solution under stirring to obtain a mixed solution; incubate the mixed solution to obtain an Au seed solution;

[0013] S2, CTAB solution, HAuCl4 solution, AgNO3 solution, HCl solution, AA (ascorbic acid) solution were mixed, and Au seed solution was added. After stirring, the solution changed from dark orange to colorless, and then incubated. After incubation, the precipitate was collected by centrifugation, washed with ultrapure water, and the collected precipitate was dispersed in ultrapure water again to obtain AuNRs seed solution;

[0014] (3) Preparation of AuNDs solution:

[0015] HAuCl4 solution, PEI (polyethyleneimine) solution, and HA (hydroxylamine hydrochloride) solution are sequentially added to the Au NRs seed solution prepared in step (2) to obtain a mixed solution; the mixed solution is incubated at room temperature for a period of time, and the precipitated product is collected by centrifugation after incubation, and then washed with CTAC solution. Finally, the washed precipitate is dispersed in deionized water to obtain an AuNDs solution;

[0016] (4) Preparation of AuNDs@AgNPs solution:

[0017] Dissolve AgNO3 in CTAC solution, add the AuNDs solution prepared in step (3), stir evenly, add AA solution dropwise, and continue stirring for a period of time to obtain a brown-yellow solution, which is the AuNDs@AgNPs solution;

[0018] (5) EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) were added to phosphate buffered saline (PBS) and stirred to dissolve to obtain a PBS mixture; the Zr-MOF solution and the PBS mixture were then stirred and mixed, and then an amino-modified Apt solution was added after stirring for a second stirring, and the stirred mixture was centrifuged to collect the precipitate, which was redissolved with PBS to obtain a Zr-MOF-Apt solution;

[0019] (6) firstly, pre-treating an indium tin oxide glass electrode to obtain a pre-treated indium tin oxide glass electrode; modifying the AuNDs@AgNPs solution prepared in step (4) onto the surface of the indium tin oxide glass electrode, and drying at room temperature; the product obtained after drying is marked as AuNDs@AgNPs / ITO;

[0020] (7) The Zr-MOF-Apt solution prepared in step (5) is modified onto the electrode surface of AuNDs@AgNPs / ITO in step (6) and dried at room temperature. The obtained product is labeled as Zr-MOF-Apt / AuNDs@AgNPs / ITO, which is a photoelectrochemical-surface enhanced Raman spectroscopy sensor based on a porphyrin-based metal organic framework dual-functional nanoprobe.

[0021] Preferably, the dosage of DMF, TCPP, ZrOCl2·8H2O and benzoic acid in step (1) is 100 mL: 100 mg: 300 mg: 2.8 g, wherein the concentration of TCPP is 0.13 mM, the concentration of ZrOCl2·8H2O is 0.93 mM, and the concentration of benzoic acid is 23 mM;

[0022] The heating temperature is 90-100°C, the heating time is 4-5 hours, and the stirring speed is 250-300 rpm;

[0023] The centrifugation conditions are 15000 rpm, 30 min; the washing times are 3-5 times.

[0024] Preferably, the dosage relationship of the ultrapure water, CTAB solution, HAuCl4 solution and glacial NaBH4 solution in S1 of step (2) is 4.5 mL: 4.5 mL: 500 μL: 610 μL, wherein the concentration of HAuCl4 solution is 5 mM, the concentration of glacial NaBH4 solution is 10 mM, and the concentration of CTAB solution is 0.2 M; the incubation operation is: standing at 30°C for 2 hours to complete the incubation.

[0025] Preferably, in S2 of step (2), the dosage relationship of CTAB solution, HAuCl4 solution, AgNO3 solution, HCl solution, AA (ascorbic acid) solution and Au seed solution is 24mL:5mL:50μL:32μL:2.8mL:48μL, wherein the concentration of CTAB solution is 0.2M, the concentration of HAuCl4 solution is 5mM, the concentration of AgNO3 solution is 100mM, the concentration of HCl solution is 1.2M, and the concentration of AA (ascorbic acid) solution is 10mM; the stirring time is 10-20s; the incubation operation is: standing at 30°C for 20h to complete the incubation; the centrifugation conditions are: 8000rpm, 10min; the number of washings with ultrapure water is 3 times; the concentration of AuNRs seed solution is 400pM.

[0026] Preferably, in step (3), the dosage relationship of HAuCl4 solution, PEI solution, HA solution, Au NRs seed solution and deionized water is 40 μL: 40 μL: 40 μL: 50 μL: 50 μL, wherein the concentration of HAuCl4 solution is 5 mM, the concentration of PEI solution is 0.15% (w / v), the concentration of HA solution is 50 mM, and the concentration of AuNRs seed solution is 400 pM;

[0027] The incubation period was 15 minutes; the centrifugation condition was 6000 rpm, 3 minutes; the concentration of the CTAC solution was 1 mM, and the number of washes was 2-3 times.

[0028] Preferably, the dosage of AgNO3, CTAC solution, AuNDs solution and AA solution in step (4) is 1 mg: 5 mL: 610 μL: 1 mL, wherein the concentration of CTAC solution is 0.1 mM and the concentration of AA solution is 2 g mL -1 ;

[0029] The AA solution was added at a rate of 1 mL / min and stirred for 1 h.

[0030] Preferably, the dosage of the Zr-MOF solution, PBS mixture, amino-modified Apt solution and reconstituted PBS in step (5) is 800 μL: 400 μL: 800 μL: 2000 μL, wherein the concentration of the Zr-MOF solution is 2 mg mL -1 , the concentration of PBS buffer was 10 mM; the concentration of amino-modified Apt solution was 2.5 μM;

[0031] The concentrations of EDC and NHS in the PBS mixture were 10 mM and 5 mM, respectively; the stirring time was 30 min, and the second stirring time was 2 h.

[0032] Preferably, the pretreatment step in step (6) is: boiling an indium tin oxide glass electrode with a diameter of 6 mm in a 1M NaOH solution for 20-30 minutes, then taking out the indium tin oxide glass electrode and ultrasonically treating it in anhydrous ethanol and ultrapure water in sequence, and finally drying it in air to obtain a pretreated indium tin oxide glass electrode;

[0033] The modification amount of the AuNDs@AgNPs solution was 20 μL.

[0034] Preferably, the modification amount of the Zr-MOF-Apt solution in step (7) is 20 μL.

[0035] The use of the photoelectrochemical-surface enhanced Raman spectroscopy sensor to detect patulin comprises the following steps:

[0036] (1) The surface of the Zr-MOF-Apt / AuNDs@AgNPs / ITO sensor was modified with a standard solution of patulin of different concentrations in volume V1 (one concentration of solution was used to modify one sensor, and the solution concentration and the sensor were in a one-to-one correspondence), and then incubated. After incubation, the sensor was cleaned with ultrapure water to obtain a cleaned sensor; the cleaned sensor was used as the working electrode, the saturated Ag / AgCl electrode was used as the reference electrode, and the platinum wire was used as the counter electrode to detect the photoelectric signal and the Raman signal, and the photoelectric signal and the Raman signal were recorded; then, the photoelectric signal and the Raman signal were used as the vertical coordinates, respectively, and the lg value of the patulin standard solution concentration was used as the horizontal coordinate to construct a standard curve based on the photoelectric signal and the Raman signal;

[0037] (2) Unknown sample detection:

[0038] A volume of V2 of the sample solution to be tested is modified on the surface of the Zr-MOF-Apt / AuNDs@AgNPs / ITO sensor. After incubation, the sensor is cleaned with ultrapure water to obtain a cleaned sensor. The cleaned sensor is then used as a working electrode, an Ag / AgCl electrode is used as a reference electrode, and a platinum wire is used as a counter electrode to detect photoelectric signals and Raman signals, and the photoelectric signals and Raman signals are recorded. Finally, the obtained photoelectric signals and Raman signals are correspondingly substituted into the standard curve constructed in step (1) to detect the sample patulin.

[0039] Preferably, the concentration of the patulin standard solution in step (1) is 1 pg mL -1 ~100ng mL -1 .

[0040] Preferably, the volumes of V1 and V2 in steps (1)-(2) are both 20 μL; the incubation conditions are: time 10-50 min, temperature 37°C; the specific method for detecting the photoelectric signal and Raman signal is as follows: a 532 nm laser in the Optiancheng ATP5020 system is used as an excitation light source to simultaneously excite the photoelectric signal and the Raman signal, and collect the Raman signal; then a Princeton VersaSTAT 3F electrochemical workstation is used to detect and record the photoelectrochemical signal; a 0.1 mol -1 AA 0.01 M PBS (pH = 7.4) buffer was used as the electrolyte. Raman signal and photoelectrochemical tests were performed in the electrolyte with an external bias voltage of 0V.

[0041] Beneficial effects of the present invention:

[0042] (1) The Zr-MOF dual-function nanoprobe can simultaneously generate relatively obvious photocurrent signals and surface-enhanced Raman signals under the excitation of a low-power 532nm Raman laser.

[0043] (2) The present invention selects AuNDs@AgNPs as an effective dual-functional enhanced substrate. Due to its plasma coupling effect, it can simultaneously cause changes in photoelectrochemical and Raman signals, realizing the photoelectrochemical-surface enhanced Raman synergistic effect.

[0044] (3) The present invention introduces patulin aptamer as a specific recognition element, which can improve the selectivity of patulin sensor, reduce the interference of other fungal toxins, and realize the specific analysis of patulin in moldy food.

[0045] (4) The present invention simultaneously acquires Raman and photoelectric signals from the same electrode under a single excitation light source, with the two serving as a reference for each other, effectively reducing interference from the solution matrix and environmental factors on the electrode and improving sensor accuracy. No separate light source is required, and the simultaneous acquisition of two signals improves detection efficiency, eliminating the need for multiple signal preparation and acquisition.

[0046] (5) The photoelectrochemical-surface enhanced Raman spectroscopy sensor based on the porphyrin-based metal organic framework dual-functional nanoprobe constructed in the present invention is used to detect penicillin, with good accuracy and sensitivity and a wide linear range of 1 pg mL -1 ~100ng mL -1 . BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 (A) is the TEM image of the Zr-MOF dual-functional nanoprobe, (B) is the stability of the Raman signal of the Zr-MOF dual-functional nanoprobe, (C) is the TEM image of AuNDs, (D) is the TEM image of AuNDs@AgNPs, (E) is the SERS signal of Zr-MOF and Zr-MOF / AuNDs@AgNPs, and (F) is the photocurrent of Zr-MOF and Zr-MOF / AuNDs@AgNPs.

[0048] Figure 2 (A) is a schematic diagram of the detection of patulin based on a three-electrode system; (B) is a schematic diagram of the sensor detecting 1 ng mL -1 and 10 ng mL -1 Feasibility of photoelectric signal response of patulin, (C) sensor detects 1ng mL -1 and 10 ng mL -1 Feasibility of SERS signal response of patulin.

[0049] Figure 3 (A) Response curves of the photoelectric mode (right) and surface-enhanced Raman mode (left) sensors to different concentrations of patulin; (B) Linear regression curve for the detection of patulin PAT (photocurrent, Raman signal, and the logarithm of patulin PAT concentration); (C) Selectivity of the photoelectrochemical-surface-enhanced Raman spectroscopy sensor, where the upper and lower figures correspond to the surface-enhanced Raman mode and the photoelectric mode, respectively. DETAILED DESCRIPTION

[0050] The present invention will be further explained below with reference to specific embodiments and accompanying drawings.

[0051] Notes on reagents involving sequences: This invention does not involve a sequence listing, so a sequence listing is not required. All reagents used were commercially available. The amino-modified aptamer (Apt) was purchased from Sangon Biotech (Shanghai) Co., Ltd. and has the following sequence: 5′-HS-SH-GATCGTACCACACCCTTACTTTCTTGATCGGATGGACACGGT-NH2-3′.

[0052] Example 1:

[0053] (1) Preparation of Zr-MOF dual-functional nanoprobes:

[0054] TCPP (100 mg, 0.13 mM), ZrOCl2·8H2O (300 mg, 0.93 mM) and benzoic acid (2.8 g, 23 mM) were dissolved in 100 mL DMF to obtain a mixed solution, which was stirred (300 rpm) at 90 °C for 5 h. After stirring, the solution was centrifuged (15000 rpm, 30 min) to collect the precipitate, which was washed three times with DMF and then centrifuged again (15000 rpm, 30 min) to collect the precipitate, which is the Zr-MOF dual-functional nanoprobe;

[0055] (2) Preparation of AuNRs seed solution:

[0056] In a 25 mL glass bottle, 4.5 mL of ultrapure water, 5 mL of CTAB solution (0.2 M), and 500 μL of HAuCl4 solution (5 mM) were added in sequence, and 610 μL of ice NaBH4 solution (10 mM) was added under vigorous stirring to obtain a mixed solution. The mixed solution was placed in an incubator at 30 °C for 2 h to obtain an Au seed solution.

[0057] 24 mL of CTAB solution (0.2 M), 5 mL of HAuCl4 solution (5 mM), 50 μL of AgNO3 solution (100 mM), 32 μL of HCl solution (1.2 mM), and 2.8 mL of AA solution (10 mM) were mixed in a 50 mL glass bottle, and 48 μL of Au seed solution was added. After stirring for 15 s, the solution changed from dark orange to colorless. Then, the solution was placed in a 30 ° C incubator. After incubation for 20 h, the solution turned brick red and was collected by centrifugation (8000 rpm, 10 min). The precipitate was washed three times with ultrapure water, and the collected precipitate was dispersed in ultrapure water again to obtain an AuNRs seed solution with a concentration of 400 pM.

[0058] (3) Preparation of AuNDs solution:

[0059] 40 μL of HAuCl4 (5 mM), 40 μL of PEI solution (0.15%, w / v), and 40 μL of HA solution (50 mM) were sequentially added to 50 μL of Au NRs solution (400 pM) and mixed for 10 seconds to obtain a mixed solution. The resulting mixed solution was incubated at room temperature for 15 minutes. After incubation, the product was collected by centrifugation (6000 rpm, 3 minutes), washed twice with 1 mM CTAC solution, and finally dispersed in 50 μL of deionized water to obtain an Au NRs solution.

[0060] (4) Preparation of AuNDs@AgNPs solution:

[0061] 1 mg of AgNO3 was dissolved in 5 mL of CTAC solution (0.1 mM), and then 400 μL of AuNDs solution was added. The mixture was stirred evenly, and 1 mL of AA solution (2 g mL -1 ), and then stirred for 1 h to obtain a brown-yellow solution, which is the AuNDs@AgNPs solution.

[0062] (5) The Zr-MOF dual-functional nanoprobe is labeled on the aptamer and labeled as Zr-MOF-Apt. The specific operation is as follows:

[0063] EDC and NHS were added to PBS (pH 7.4, 10 mM) and stirred to dissolve to obtain a PBS mixture. The concentrations of EDC and NHS in the PBS mixture were 10 mM and 5 mM, respectively. Then 800 μL of Zr-MOF solution (2 mg mL -1 ) was stirred with 400 μL of PBS mixture for 30 min to activate the carboxyl groups of Zr-MOF, and then 800 μL of amino-modified aptamer (2.5 μM) solution was added and stirred for further 2 h. After stirring, the mixture was centrifuged to remove unreacted EDC / NHS, and the precipitate was collected and redissolved with 2000 μL of PBS (pH 7.4, 10 mM) to obtain Zr-MOF-Apt solution;

[0064] (6) boiling an indium tin oxide glass electrode with a diameter of 6 mm in a 1 M NaOH solution for 30 min, then ultrasonically treating it in anhydrous ethanol and ultrapure water in sequence, and finally drying it in air to obtain a pretreated indium tin oxide glass electrode; modifying the surface of the indium tin oxide glass electrode with 20 μL of AuNDs@AgNPs solution prepared in step (4) and drying it at room temperature, the obtained product is labeled as AuNDs@AgNPs / ITO;

[0065] (7) 20 μL of the Zr-MOF-Apt solution prepared in step (5) was modified onto the surface of the modified electrode in step (6) and dried at room temperature. The resulting product was labeled as Zr-MOF-Apt / AuNDs@AgNPs / ITO; that is, a photoelectrochemical-surface enhanced Raman spectroscopy sensor based on a porphyrin-based metal organic framework dual-functional nanoprobe.

[0066] The morphology of Zr-MOF nanoparticles was studied by transmission electron microscopy (TEM). Figure 1 (A) is the TEM image of the Zr-MOF dual-functional nanoprobe, (B) is the stability of the Raman signal of the Zr-MOF dual-functional nanoprobe, (C) is the TEM image of AuNDs, (D) is the TEM image of AuNDs@AgNPs, (E) is the SERS signal of Zr-MOF and Zr-MOF / AuNDs@AgNPs, and (F) is the photocurrent of Zr-MOF and Zr-MOF / AuNDs@AgNPs.

[0067] TEM images show that the synthesized Zr-MOF dual-functional nanoprobe is circular ( Figure 1 A). Raman intensity analysis of individual porphyrins and Zr-MOFs revealed significant differences in stability, as shown in the box plots; Zr-MOF exhibited enhanced stability, with a relative standard deviation (RSD) of 7.9%, significantly lower than the 39.2% observed for individual porphyrins ( Figure 1 B) This enhanced stability is due to the orderly arrangement of porphyrins in Zr-MOF, which improves the aggregation tendency of porphyrins. Figure 1 As shown in C, the TEM image shows that the synthesized AuNDs are dumbbell-shaped. The TEM image of a single AuNDs@AgNPs confirms that the AuNDs are completely wrapped by a uniform Ag shell ( Figure 1 D) Using Zr-MOF as nanotag, the SERS and PEC performance of AuNDs@AgNPs and their enhancement mechanism were studied. -1 Taking the peak at as an example, the SERS enhancement intensity based on AuNDs@AgNPs is 2.7 times that of Zr-MOF ( Figure 1 E). Figure 1 Figure F shows the photocurrent responses of Zr-MOF and Zr-MOF / AuNDs@AgNPs under 100 mW 532 nm laser. Obviously, the photocurrent of Zr-MOF / AuNDs@AgNPs (103 nA) is 1.8 times higher than that of Zr-MOF.

[0068] Based on the use of the sensor to detect patulin, the steps are as follows;

[0069] S1, 20 μL of different concentrations of patulin standard solution (concentration of 10 pg mL-1 , 100 pg mL -1 , 1ng mL -1 , 10 ng mL -1 , 100ng mL -1 ) were modified on the surface of the Zr-MOF-Apt / AuNDs@AgNPs / ITO sensor (one concentration of solution was used to modify one sensor, and the solution concentration and the sensor were in a one-to-one correspondence), incubated at 37°C for 40 min after modification, and then cleaned with ultrapure water to obtain a cleaned sensor; the cleaned sensor was used as the working electrode, the saturated Ag / AgCl electrode was used as the reference electrode, and the platinum wire was used as the counter electrode to detect the photoelectric signal and the Raman signal, and the photoelectric signal and the Raman signal were recorded; then, the photoelectric signal and the Raman signal were used as the vertical coordinates, respectively, and the lg value of the concentration of the penicillin standard solution was used as the horizontal coordinate to construct a standard curve based on the photoelectric signal and the Raman signal;

[0070] The photoelectric and Raman signal detections were performed by using a 532nm laser in the Optiancheng ATP5020 system as the excitation light source to simultaneously excite the photoelectric and Raman signals and collect the Raman signals. A Princeton VersaSTAT 3F electrochemical workstation was used to record and detect the photoelectrochemical signals. -1 0.01 M PBS (pH = 7.4) buffer solution of AA was used as the electrolyte. Raman signal and photoelectrochemical tests were performed in the electrolyte with an external bias voltage of 0V.

[0071] This approach led to the construction of an aptamer-based interfacial biosensing system, with the patulin aptamer acting as the recognition element and the Zr-MOF as a dual-functional nanoprobe, acting as a transduction element to convert binding events into measurable photocurrent and SERS signals. AuNDs@AgNPs served as a matrix to stabilize the immobilization of Zr-MOF-Apt via the formation of Ag-S bonds. This resulted in a photoelectrochemical-surface-enhanced Raman sensing platform capable of simultaneously providing photocurrent and SERS spectroscopy measurements.

[0072] Figure 2 Middle (A) is a schematic diagram of the detection of patulin based on a three-electrode system; Figure 2 Middle (B) is the sensor detection 1ngmL -1 and 10 ng mL -1 Feasibility of photoelectric signal response of patulin, Figure 2 Middle (C) is the sensor detection of 1ng mL -1 and 10 ng mL -1 Feasibility of SERS signal response of patulin.

[0073] Using a three-electrode system ( Figure 2 A), including a working electrode, a reference electrode and a counter electrode, which are essential components in electrochemical measurements. AuNDs@AgNPs are deposited on an indium tin oxide (ITO) electrode as a working electrode and assembled in a photoelectrochemical-surface enhanced Raman electrolysis cell. A laser probe with an operating wavelength of 532 nm is coupled to a Raman spectrometer to simultaneously excite photocurrent and SERS signals, and collect SERS signals on the electrode surface. The working electrode is connected to an electrochemical workstation to measure the photocurrent. Figure 2 As shown in (B) and (C), with the increase of PAT concentration, the photocurrent (I PEC ) gradually decreases; the surface enhanced Raman response gradually increases.

[0074] Figure 3 (A) shows the response curves of the photoelectric mode (right) and surface-enhanced Raman mode (left) sensors to different concentrations of patulin; (B) Linear regression curve for the detection of patulin PAT (photocurrent, Raman signal, and the logarithm of patulin PAT concentration); (C) Selectivity of the photoelectrochemical-surface-enhanced Raman spectroscopy sensor, the upper and lower figures correspond to the surface-enhanced Raman mode and the photoelectric mode, respectively.

[0075] The PAT concentration was quantified based on the photocurrent intensity and surface enhanced Raman intensity ( Figure 3 A). is the linear regression curve of photocurrent and SERS signal with the logarithm of PAT concentration ( Figure 3 B) The results show that the linear range of the sensor for PAT detection is 1pgmL -1 to 100ng mL -1 The linear regression equation is I = 2830.0 + 1130.2 lgC PAT (R 2 =0.996) and I = 165.2–28.81 gC PAT (R 2 =0.999), corresponding to the photoelectric mode and surface enhanced Raman spectroscopy mode respectively. Figure 3 As can be seen from Figure C, the sensor has no obvious response to other interferents, proving that the sensor has good selectivity.

[0076] S2. Sample testing: (sample selection is moldy apples)

[0077] Sample testing: Take physically damaged apples and place them at room temperature until they rot. Remove the rotten part of one rotten apple and use a juicer to squeeze the fruit near the rotten part into a puree. Then take 1 mL of puree and add it to a centrifuge tube. Then add 4 mL of acetonitrile to mix and obtain a mixed solution. After shaking the mixed solution for 30 minutes, filter it using a 0.22 μm aqueous filter membrane and use the filtered solution as the puree sample extract. Add 0, 25, and 50 ng mL of acetonitrile to the sample extract, respectively. -1 Patulin was tested with a test solution.

[0078] 20 μL of detection solution was modified on the Zr-MOF-Apt / AuNDs@AgNPs / ITO electrode. After incubation at 37°C for 40 min, the product was washed with ultrapure water to obtain a cleaned sensor. According to the detection method in step S1, in a three-electrode system, the cleaned sensor was used as the working electrode, the Ag / AgCl electrode was used as the reference electrode, and the platinum wire was used as the counter electrode to perform photoelectric and Raman signal detection, and record the photoelectric and Raman signals. Finally, the obtained photoelectric and Raman signals were correspondingly substituted into the constructed standard curve to detect the test sample patulin.

[0079] Table 1: Detection levels of spiked moldy apple samples and verification of the national standard method (HPLC-MS / MS).

[0080]

[0081] “ND”: Not Detected

[0082] As can be seen from Table 1, the method provided by the present invention is basically consistent with the national standard method (HPLC-MS / MS), indicating that the method of the present invention is reliable and accurate, can sensitively and quantitatively detect patulin in the test sample, does not require professional training, and is easy to operate.

[0083] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although this specification has described the present invention in detail with reference to the above embodiments, it should be understood by those skilled in the art that the present invention may still be modified or replaced by equivalents. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A method for preparing a photoelectrochemical-surface enhanced Raman spectroscopy sensor, characterized in that: Here are the steps: (1) Preparation of Zr-MOF dual-functional nanoprobes: TCPP, ZrOCl2·8H2O, and benzoic acid are dissolved in DMF, and the obtained mixed solution is heated under stirring for a period of time. After heating, the mixed solution is centrifuged to collect the precipitate, which is washed several times with DMF. After washing, the precipitate is collected by centrifugation again to obtain the Zr-MOF dual-functional nanoprobe; the amount of DMF, TCPP, ZrOCl2·8H2O, and benzoic acid is 100 mL: 100 mg: 300 mg: 2.8 g, wherein the concentration of TCPP is 0.13 mM, the concentration of ZrOCl2·8H2O is 0.93 mM, and the concentration of benzoic acid is 23 mM; the heating temperature is 90-100°C, the heating period is 4-5 hours, and the stirring rate is 250-300 rpm; (2) Preparation of AuNRs seed solution: S1. Mix ultrapure water, CTAB solution, and HAuCl4 solution, and add glacial NaBH4 solution under stirring to obtain a mixed solution; incubate the mixed solution to obtain an Au seed solution; the dosage of the ultrapure water, CTAB solution, HAuCl4 solution, and glacial NaBH4 solution is 4.5 mL: 4.5 mL: 500 μL: 610 μL, wherein the concentration of the HAuCl4 solution is 5 mM, the concentration of the glacial NaBH4 solution is 10 mM, and the concentration of the CTAB solution is 0.2 M; the incubation operation is: standing at 30°C for 2 hours to complete the incubation; S2, CTAB solution, HAuCl4 solution, AgNO3 solution, HCl solution, and AA solution were mixed, and Au seed solution was added. After stirring, the solution changed from dark orange to colorless, and then incubated. After incubation, the precipitate was collected by centrifugation, washed with ultrapure water, and the collected precipitate was dispersed in ultrapure water again to obtain AuNRs seed solution; The dosage of the CTAB solution, HAuCl4 solution, AgNO3 solution, HCl solution, AA solution, and Au seed solution is in the following order: 24 mL: 5 mL: 50 μL: 32 μL: 2.8 mL: 48 μL, wherein the concentration of the CTAB solution is 0.2 M, the concentration of the HAuCl4 solution is 5 mM, the concentration of the AgNO3 solution is 100 mM, the concentration of the HCl solution is 1.2 M, and the concentration of the AA solution is 10 mM. The incubation step is as follows: standing at 30°C for 20 h to complete the incubation. The concentration of the AuNRs seed solution is 400 pM. (3) Preparation of AuNDs solution: HAuCl4 solution, PEI solution, and HA solution are sequentially added to the Au NRs seed solution prepared in step (2) to obtain a mixed solution; The mixed solution was incubated at room temperature, and after incubation, the precipitate was collected by centrifugation and washed with a CTAC solution. Finally, the washed precipitate was dispersed in deionized water to obtain an AuNDs solution. The dosage of the HAuCl4 solution, PEI solution, HA solution, AuNRs seed solution, and deionized water used for dispersion was 40 μL: 40 μL: 40 μL: 50 μL: 50 μL, wherein the concentration of the HAuCl4 solution was 5 mM, the concentration of the PEI solution was 0.15% (w / v), the concentration of the HA solution was 50 mM, and the concentration of the Au NRs seed solution was 400 pM. (4) Preparation of AuNDs@AgNPs solution: Dissolve AgNO3 in CTAC solution, add the AuNDs solution prepared in step (3), stir evenly, add AA solution dropwise, and continue stirring for a period of time to obtain a brown-yellow solution, which is the AuNDs@AgNPs solution; the dosage relationship of AgNO3, CTAC solution, AuNDs solution and AA solution is 1 mg: 5 mL: 610 μL: 1 mL, where the concentration of CTAC solution is 0.1 mM and the concentration of AA solution is 2 g mL -1 ; (5) EDC and NHS were added to PBS and stirred to dissolve to obtain a PBS mixture; the Zr-MOF solution and the PBS mixture were then stirred and mixed, and then an amino-modified Apt solution was added and stirred for a second time. The stirred mixture was centrifuged, the precipitate was collected, and redissolved with PBS to obtain a Zr-MOF-Apt solution; the dosage of the Zr-MOF solution, PBS mixture, amino-modified Apt solution, and redissolved PBS was 800 μL: 400 μL: 800 μL: 2000 μL, where the concentration of the Zr-MOF solution was 2 mg mL -1 , the concentration of PBS buffer was 10 mM; the concentration of amino-modified Apt solution was 2.5 μM; the concentrations of EDC and NHS in the PBS mixture were 10 mM and 5 mM, respectively; (6) First, an indium tin oxide glass electrode is pretreated to obtain a pretreated indium tin oxide glass electrode; the AuNDs@AgNPs solution prepared in step (4) is modified onto the surface of the indium tin oxide glass electrode, and dried at room temperature. The product obtained after drying is marked as AuNDs@AgNPs / ITO; (7) The Zr-MOF-Apt solution prepared in step (5) was modified onto the electrode surface of AuNDs@AgNPs / ITO in step (6) and dried at room temperature. The obtained product was labeled as Zr-MOF-Apt / AuNDs@AgNPs / ITO, which is a photoelectrochemical-surface enhanced Raman spectroscopy sensor based on a porphyrin-based metal organic framework dual-functional nanoprobe.

2. The preparation method according to claim 1, characterized in that The centrifugation conditions in step (1) are 15000 rpm, 30 min; the washing times are 3-5 times.

3. The preparation method according to claim 1, characterized in that In step (2) S2, the stirring time is 10-20 seconds; the centrifugal conditions are: 8000 rpm, 10 minutes; and the number of washing times with ultrapure water is 3 times.

4. The preparation method according to claim 1, characterized in that The incubation period in step (3) is 15 minutes; the centrifugation condition is 6000 rpm, 3 minutes; the concentration of the CTAC solution is 1 mM, and the number of washing times is 2-3 times.

5. The preparation method according to claim 1, characterized in that The AA solution in step (4) was added at a rate of 1 mL / min and stirred for 1 h.

6. The preparation method according to claim 1, characterized in that The stirring time in step (5) is 30 min, and the second stirring time is 2 h.

7. The preparation method according to claim 1, characterized in that The pretreatment steps in step (6) are as follows: boiling an indium tin oxide glass electrode with a diameter of 6 mm in a 1 M NaOH solution for 20-30 minutes, then taking out the indium tin oxide glass electrode and ultrasonically treating it in anhydrous ethanol and ultrapure water in sequence, and finally drying it in air to obtain a pretreated indium tin oxide glass electrode; the modification amount of the AuNDs@AgNPs solution is 20 μL.

8. The preparation method according to claim 1, characterized in that The modification amount of the Zr-MOF-Apt solution in step (7) is 20 μL.

9. Use of the sensor prepared by the method according to any one of claims 1 to 8 for detecting patulin, characterized in that: The steps include: (1) The surface of the Zr-MOF-Apt / AuNDs@AgNPs / ITO sensor was modified with a standard solution of patulin of different concentrations in volume V1. One concentration of solution was used to modify one sensor, and the solution concentration and the sensor were in a one-to-one correspondence. After modification, the sensor was incubated and cleaned with ultrapure water to obtain a cleaned sensor. The cleaned sensor was used as the working electrode, the saturated Ag / AgCl electrode was used as the reference electrode, and the platinum wire was used as the counter electrode to detect the photoelectric signal and Raman signal, and the photoelectric signal and Raman signal were recorded. Then, the photoelectric signal and Raman signal were used as the vertical coordinates, respectively, and the lg value of the patulin standard solution concentration was used as the horizontal coordinate to construct a standard curve based on the photoelectric signal and the Raman signal. (2) Unknown sample detection: A volume of V2 of the sample solution to be tested is modified on the surface of the Zr-MOF-Apt / AuNDs@AgNPs / ITO sensor. After incubation, the sensor is cleaned with ultrapure water to obtain a cleaned sensor. The cleaned sensor is then used as a working electrode, an Ag / AgCl electrode as a reference electrode, and a platinum wire as a counter electrode to detect photoelectric signals and Raman signals, and the photoelectric signals and Raman signals are recorded. Finally, the obtained photoelectric signals and Raman signals are correspondingly substituted into the standard curve constructed in step (1) to detect the sample patulin.

10. The use according to claim 9, characterized in that The concentration of the patulin standard solution in step (1) is 1 pg mL -1 ~100 ng mL -1 ; The volumes of V1 and V2 in steps (1)-(2) are both 20 μL, and the incubation conditions are: time is 10-50 min, temperature is 37°C; the specific method of detecting the photoelectric signal and Raman signal is as follows: the 532 nm laser in the Optiancheng ATP5020 system is used as the excitation light source to simultaneously excite the photoelectric signal and Raman signal, and collect the Raman signal; then the electrochemical workstation model Princeton VersaSTAT 3F is used to detect and record the photoelectrochemical signal; a 0.1 mol -1 PBS buffer with 0.01 M AA and pH = 7.4 was used as the electrolyte. Raman signals and photoelectrochemical tests were carried out in the electrolyte with an external bias voltage of 0 V.

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