An electrochemical bionic taste sensor based on molecularly imprinted polymer-target-gustducin double recognition sandwich assembly and application thereof

Abstract

The application discloses an electrochemical biomimetic taste sensor based on molecular imprinting polymer-target-umami receptor double recognition sandwich assembly and application thereof, and belongs to the technical field of electrochemical sensing and food flavor detection. The sensor takes a silk screen printed electrode as a substrate, a molecular imprinting polymer film is constructed on the surface of the substrate through electropolymerization, and after elution of a template molecule, imprinting cavities with specific recognition capability for sodium glutamate or inosine 5'-monophosphate are formed; meanwhile, a Ni-MOF is used as a conductive framework, PVP-AuNPs are loaded, and a umami receptor T1R1-VFT is further fixed, so that an AuNPs-T1R1-VFT / Ni-MOF signal probe is prepared. The sensor has high selectivity of the molecular imprinting material, high affinity recognition capability of the umami receptor and signal enhancement effect of the conductive nanocomposite material, and can be used for rapid quantitative detection of umami substances in fish, shrimps and other aquatic products, and has the advantages of strong anti-interference capability, high sensitivity, good stability, fast detection speed and convenience for on-site analysis.

Description

Technical Field

[0001] This invention relates to the field of electrochemical biomimetic sensor technology, and more particularly to an electrochemical biomimetic taste sensor based on a molecularly imprinted polymer-target-umami receptor dual-recognition sandwich assembly and its application. Background Technology

[0002] Umami, as the fifth basic taste, plays an irreplaceable role in enhancing the flavor and quality of food. Fish, shrimp, and other aquatic products are rich in various umami substances, the most representative of which are monosodium glutamate (MSG) and inosine 5'-monophosphate (IMP). MSG imparts a rich and mellow umami flavor to food, enhancing the consumer's taste experience; inosine 5'-monophosphate not only synergistically enhances umami flavor with glutamate-like substances, increasing the overall umami intensity of food, but also reflects changes in the quality of aquatic products to some extent. Therefore, developing an accurate and efficient method for the quantitative detection of umami substances is of great significance.

[0003] To date, sensory evaluation, instrumental analysis, and electronic tongue techniques have been widely applied to the detection and evaluation of umami substances in food. Although these methods have formed relatively complete operational systems, they still face limitations such as high subjectivity, cumbersome detection procedures, high costs, and difficulty in achieving rapid on-site analysis. Electrochemical sensors, with their advantages of high sensitivity, rapid response, and ease of miniaturization, have shown great application potential in the field of umami substance detection. Researchers have utilized the Venus flytrap domain (T1R1-VFT) of umami receptors as a sensing element and combined it with various nanomaterials to construct an electrochemical biomimetic taste sensor. However, previous studies have mainly focused on amplifying the response signal, broadening the detection range, and improving sensor stability, while neglecting the selectivity limitations caused by the broad-spectrum response of natural receptors themselves. Although T1R1-VFT can recognize a variety of umami compounds, it still has limitations in achieving high selective differentiation and accurate quantification of umami molecules with different structures. Furthermore, sensing interfaces constructed using common interface modification methods are susceptible to non-specific adsorption and impaired interfacial charge transport in complex food matrices, thus reducing the accuracy and reliability of the detection results.

[0004] In recent years, molecularly imprinted polymers (MIPs) have been widely used in electrochemical sensors due to their advantages such as high recognition specificity, high stability, and low cost. By constructing imprinted cavities on the electrode surface that match the spatial structure and functional group distribution of the target molecule, the selective capture capability of the target can be significantly improved. Meanwhile, conductive metal-organic frameworks (MIPs) are also used to construct high-performance electrochemical sensing interfaces due to their high carrier density, excellent electron transport performance, and large specific surface area. In particular, the composite of gold nanoparticles with conductive Ni-MOF not only improves interfacial conductivity but also provides a stable immobilization platform for the umami receptor T1R1-VFT. However, while improving selectivity, molecularly imprinted layers often introduce large interfacial resistance, affecting sensor sensitivity. Existing dual-recognition sensing systems typically rely mainly on a preset number of signal probes or fixed interfacial response modes, with the target molecule acting primarily as a recognition object rather than directly participating in the assembly and regulation of the signal probes, resulting in limited signal amplification efficiency. Therefore, developing a dual-recognition sandwich electrochemical sensor that simultaneously possesses high selective recognition capability, excellent interfacial conductivity, and target-driven signal amplification capability is an urgent problem to be solved in this field. Summary of the Invention

[0005] To overcome the shortcomings of current umami substance detection technologies, such as insufficient specificity, high interfacial resistance, limited signal amplification efficiency, and susceptibility to non-specific interference in complex food matrices, the primary objective of this invention is to provide an electrochemical sensor based on a molecularly imprinted polymer-target-umami receptor dual-recognition sandwich assembly and its applications. This electrochemical sensor possesses advantages such as high recognition specificity, good conductivity, high sensitivity, and good stability, enabling rapid quantitative detection of monosodium glutamate (MSG) and inosine 5'-monophosphate. During detection, the target molecule is first selectively captured by the molecularly imprinted layer, and then acts as a bridging medium to promote the assembly of the signal probe on the electrode surface, forming a MIP-target-T1R1-VFT dual-recognition sandwich structure, thereby achieving target concentration-dependent electrochemical signal amplification. This sensor combines the high selectivity of molecularly imprinted materials, the high affinity recognition ability of umami receptors, and the signal enhancement effect of conductive nanocomposite materials. It can be used for rapid quantitative detection of umami substances in aquatic products such as fish and shrimp, and has advantages such as strong anti-interference ability, high sensitivity, good stability, fast detection speed, and ease of on-site analysis.

[0006] The above-mentioned objective of the present invention is achieved through the following technical methods: A dual-recognition sandwich electrochemical sensor comprising a molecularly imprinted modified working electrode and a receptor-functionalized signal probe; The molecularly imprinted modified working electrode is a molecularly imprinted polymer film (MIP) modified electrode constructed on the surface of a screen-printed electrode. The molecularly imprinted polymer film uses umami substances as template molecules to form specific recognition cavities. The receptor functionalization signal probe is an AuNPs-T1R1-VFT / Ni-MOF signal probe; During detection, the target umami molecules first specifically bind to the recognition cavity of the molecularly imprinted polymer membrane, and then act as a bridging medium to promote the assembly of the receptor-functionalized signal probe onto the surface of the working electrode, forming a MIP-target-T1R1-VFT dual recognition sandwich structure, thereby achieving electrochemical signal amplification.

[0007] Preferably, the umami substance is monosodium glutamate or inosine 5'-monophosphate or a combination thereof; The functional monomer of the molecularly imprinted polymer film is o-phenylenediamine; The working electrode is a screen-printed carbon electrode.

[0008] Preferably, the molecularly imprinted modified working electrode is prepared according to the following steps: Step A1: Dissolve the template molecule and o-phenylenediamine in PBS buffer, and electropolymerize using cyclic voltammetry with a screen-printed electrode as the substrate to form a molecularly imprinted polymer film. Step A2: The obtained electrode is then placed in a methanol / acetic acid mixed elution solution for template molecule elution to obtain a molecularly imprinted modified working electrode with specific recognition cavity.

[0009] Preferably, the PBS buffer concentration is 0.05-0.3 M, and the pH is 7.1-7.5; the volume ratio of the methanol / acetic acid mixed elution buffer is (8-10):1; More preferably, the PBS buffer concentration is 0.1 M and the pH is 7.4; the volume ratio of the methanol / acetic acid mixed elution buffer is 9:1. The electropolymerization conditions are as follows: template molecule concentration of 4 mM to 5 mM, o-phenylenediamine concentration of 5 mM, scanning potential range of -0.5 V to +0.8 V, scanning rate of 50 mV / s, and number of cycles of 14 to 16; the template molecule elution time is 30 min to 35 min.

[0010] Preferably, the AuNPs-T1R1-VFT / Ni-MOF signal probe is prepared according to the following steps: Step B1: Dissolve nickel acetate in a DMF / DMA mixed solvent, add sodium acetate solution and HATP·6HCl solution, and obtain Ni-MOF through a solvothermal reaction; Step B2: Add tetrachloroauric acid to boiling ultrapure water, then add sodium citrate solution for reduction reaction, cool and add polyvinylpyrrolidone for surface modification to obtain PVP-AuNPs; Step B3: Mix Ni-MOF with PVP-AuNPs to obtain AuNPs / Ni-MOF composite material; Step B4: Incubate the AuNPs / Ni-MOF composite material with T1R1-VFT to obtain the AuNPs-T1R1-VFT / Ni-MOF signal probe.

[0011] More preferably, in step B1, the solid-liquid ratio of nickel acetate tetrahydrate to DMF / DMA mixed solvent is 7:(10-15) mg / mL, the volume ratio of DMF to DMA is (1-2):(1-2), the solid-liquid ratio of nickel acetate tetrahydrate, sodium acetate solution, and HATP·6HCl solution is 7:(7-9):(2-5) mg / mL / mL, the molar concentration of sodium acetate solution is 2-4 M, the molar concentration of HATP·6HCl solution is 9.3-9.9 μM, and the reaction conditions are 65-70 °C for 1.5-2.5 h. Preferably, the solid-liquid ratio of nickel acetate tetrahydrate to DMF / DMA mixed solvent is 7:12 mg / mL, the volume ratio of DMF to DMA is 1:1, the solid-liquid ratio of nickel acetate tetrahydrate, sodium acetate solution, and HATP·6HCl solution is 7:8:3 mg / mL, the molar concentration of sodium acetate solution is 2 M, the molar concentration of HATP·6HCl solution is 9.3 μM, and the reaction conditions are 65 °C for 2 h. More preferably, in step B2, the volume ratio of 1 wt% tetrachloroauric acid solution to boiling ultrapure water is 1:(90-110), the volume ratio of 1 wt% tetrachloroauric acid solution to 1 wt% sodium citrate solution is 1:(2-3), the volume ratio of 1 wt% tetrachloroauric acid solution to 30 mg / mL polyvinylpyrrolidone solution is 1:(4-6), the stirring time is 1-3 min, the reaction time continues for 14-16 min, and the stirring time in the dark is 23-25 ​​h. Preferably, the volume ratio of 1 wt% tetrachloroauric acid solution to boiling ultrapure water is 1:100, the volume ratio of 1 wt% tetrachloroauric acid solution to 1 wt% sodium citrate solution is 1:2.5, the volume ratio of 1 wt% tetrachloroauric acid solution to 30 mg / mL polyvinylpyrrolidone solution is 1:5, the stirring time is 2 min, the reaction time continues for 15 min, and the stirring time in the dark is 24 h. In step B3, the volume ratio of 5 mg / mL Ni-MOF dispersion to 1 wt% PVP-AuNPs solution is 3:(0.8-1.2), the ultrasonic treatment time is 4-6 min, and the stirring time at room temperature is 10-14 h (overnight). More preferably, the volume ratio of the 5 mg / mL Ni-MOF dispersion to the 1 wt% PVP-AuNPs solution is 3:1, the ultrasonic treatment time is 5 min, and the stirring time at room temperature is overnight; Preferably, in step B4, the volume ratio of AuNPs / Ni-MOF dispersion to 0.15 mg / mL T1R1-VFT protein solution is 1:(0.25-0.35), the incubation temperature is 3-5 °C, and the incubation time is 10-14 h (overnight). Preferably, the volume ratio of the AuNPs / Ni-MOF dispersion to the 0.15 mg / mL T1R1-VFT protein solution is 1:0.3, the incubation temperature is 4 °C, and the incubation time is overnight.

[0012] An application of the aforementioned dual-recognition sandwich electrochemical sensor in the quantitative detection of umami substances; The umami substance is monosodium glutamate or inosine 5'-monophosphate or a combination thereof, and the application is the rapid quantitative detection of umami substances in fish and shrimp.

[0013] A method for quantitatively detecting umami substances using the aforementioned dual-recognition sandwich electrochemical sensor, characterized by comprising the following steps: S1: Add solutions of umami substances of different concentrations to the surface of the molecularly imprinted working electrode and incubate them to allow the umami substances to bind to the imprint cavity; S2: After rinsing, AuNPs-T1R1-VFT / Ni-MOF signal probes are dropped onto the surface of the working electrode and incubated to allow them to bind with the target molecules to form a MIP-target-T1R1-VFT dual recognition sandwich structure. S3: Place the constructed electrode in a detection solution containing electrolyte for electrochemical detection; S4: A standard curve is obtained by linear fitting with the logarithm of the umami substance concentration as the abscissa and the response current value as the ordinate. S5: Detect the response current of the sample to be tested, and calculate the concentration of umami substances in the sample to be tested in combination with the standard curve.

[0014] Preferably, the volume of the umami substance solution added is 10 μL, and the incubation time is 25 min to 30 min; the volume of the signal probe solution added is 10 μL, and the incubation time is 25 min to 30 min; the detection solution contains 0.1 M KCl and 5 mM [Fe(CN)6]. 3- / 4- The mixed solution; the electrochemical detection method is cyclic voltammetry, differential pulse voltammetry and electrochemical impedance spectroscopy.

[0015] Preferably, the test samples are derived from crucian carp and whiteleg shrimp; the test samples are pretreated as follows: weigh the fish or shrimp meat sample, add trichloroacetic acid solution to homogenize, extract by shaking at 4 °C, centrifuge, collect the supernatant and adjust the pH to neutral, filter through a microporous membrane, dilute with PBS buffer and then perform electrochemical detection.

[0016] In the fabrication of the dual-recognition sandwich electrochemical sensor provided by this invention, the molecularly imprinted polymer can form a specific recognition cavity on the electrode surface that matches the target umami molecule in terms of spatial structure and functional group distribution, thereby achieving selective capture of the target analyte. Ni-MOF has good conductivity, a large specific surface area, and an open structure, which can provide sufficient sites for loading functional nanomaterials and promote interfacial charge transport. AuNPs have good biocompatibility and are easy to modify, and can achieve efficient immobilization of the umami receptor T1R1-VFT through Au-S interaction. The umami receptor T1R1-VFT can further recognize the target umami molecule. Therefore, by combining the molecularly imprinted recognition layer with the AuNPs-T1R1-VFT / Ni-MOF signal probe and using the target molecule to mediate the formation of a sandwich structure, a dual-recognition electrochemical sensor with high selectivity, high conductivity, and high signal amplification capability can be constructed.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The electrochemical sensor constructed in this invention uses molecularly imprinted polymers as the first recognition interface, which can specifically capture MSG and IMP, effectively making up for the lack of specificity caused by the broad-spectrum response of natural umami receptors and improving detection accuracy.

[0018] (2) The AuNPs-T1R1-VFT / Ni-MOF signal probe constructed in this invention has good conductivity and high acceptor loading capacity, which can reduce the interfacial charge transfer resistance and enhance the electrochemical response, thereby improving the sensitivity of the sensor.

[0019] (3) The present invention adopts a target-driven dual recognition sandwich assembly method, so that the target molecule not only serves as the recognition object, but also participates in the assembly of the signal probe as a bridging medium; as the concentration of the target molecule increases, the load of the probe on the electrode surface increases synchronously, thereby realizing concentration-dependent electrochemical signal amplification.

[0020] (4) The electrochemical sensor constructed in this invention has a wide detection range and a low detection limit, with a detection range of 10 for MSG. -9 ~10 -2 M, detection limit is 0.33 nM; detection range for IMP is 10 -10 ~10 -3 M, detection limit is 0.03nM.

[0021] (5) The electrochemical sensor constructed in this invention has good selectivity, repeatability and stability; the relative standard deviations of the six independent sensors for MSG and IMP detection are 1.22% and 1.28%, respectively. After being stored at 4 °C for 15 days, the response signals for MSG and IMP still retain 80.3% and 82.4% of the initial values, respectively.

[0022] (6) The electrochemical sensor constructed in this invention can be used for highly sensitive quantitative detection of umami substances MSG and IMP in real food matrices such as fish and shrimp, and the detection results are consistent with the trend of high performance liquid chromatography, indicating that it has good reliability and application prospects. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the electrochemical sensor detection system based on a molecularly imprinted polymer-target-umami receptor dual-recognition sandwich assembly provided by the present invention. 1. Working electrode; 2. Reference electrode; 3. Counter electrode; 4. MIP-target-T1R1-VFT sandwich recognition structure modified on the surface of the working electrode; 5. Electrolyte containing 5 mM potassium ferrocyanide / potassium ferrocyanide and 0.1 M potassium chloride.

[0024] Figure 2 This is a bar chart comparing the surface roughness of the bare screen-printed electrode, the molecularly imprinted modified screen-printed electrode (before elution), and the molecularly imprinted modified screen-printed electrode (after elution) in the embodiments.

[0025] Figure 3 This is a scanning electron microscope image of the AuNPs / Ni-MOF composite material in the examples.

[0026] Figure 4 The Fourier transform infrared spectra of the AuNPs / Ni-MOF and AuNPs-T1R1-VFT / Ni-MOF signal probes in the embodiments are shown.

[0027] Figure 5 This is a differential pulse voltammetric curve of the sensor assembly process in the embodiment; where: a represents the bare screen-printed electrode (SPCE), b represents the molecularly imprinted modified screen-printed electrode (MIP / SPCE before elution corresponding to Example 4), c represents the molecularly imprinted modified screen-printed electrode (MIP / SPCE after elution corresponding to Example 4), d represents the MSG / molecularly imprinted modified screen-printed electrode, and e represents the AuNPs-T1R1-VFT / Ni-MOF / MSG / molecularly imprinted modified screen-printed electrode.

[0028] Figure 6DPV curves of MIP / SPCE (a) and NIP / SPCE (b) after incubation with MSG and AuNPs-T1R1-VFT / Ni-MOF, respectively: a is the recognition signal of MIP / SPCE (MSG) for monosodium glutamate, and b is the recognition signal of NIP / SPCE for monosodium glutamate.

[0029] Figure 7 The graph shows the sensor's detection performance for monosodium glutamate (MSG) in this embodiment; where: A is the differential pulse voltammetric response curve for different concentrations of MSG, and B is the standard curve of MSG concentration versus response current.

[0030] Figure 8 The graph shows the detection performance of the sensor for inosine 5'-monophosphate in the embodiment; where: A is the differential pulse voltammetric response curve of inosine 5'-monophosphate at different concentrations, and B is the standard curve of inosine 5'-monophosphate concentration versus response current.

[0031] Figure 9 The graph shows the results of selective testing of monosodium glutamate (MSG) and inosine 5'-monophosphate by the sensor in this embodiment; wherein: A is a bar chart of the response current of the MSG sensing system to the target and different interfering substances, and B is a bar chart of the response current of the inosine 5'-monophosphate sensing system to the target and different interfering substances.

[0032] Figure 10 The graph shows the imprinting factor test results of the sensor in the embodiment; where: A is a bar chart of the imprinting factor of sodium glutamate molecular imprinting sensing system for sodium glutamate and different interfering substances, and B is a bar chart of the imprinting factor of inosine 5'-monophosphate molecular imprinting sensing system for inosine 5'-monophosphate and different interfering substances.

[0033] Figure 11 The above is a bar chart showing the response current of the sensor to monosodium glutamate (MSG) and inosine 5'-monophosphate in fish and shrimp samples processed by different methods (raw, steamed, boiled, and roasted) in the embodiment. Among them, A is the response result of MSG and inosine 5'-monophosphate in crucian carp samples under different processing methods, and B is the response result of MSG and inosine 5'-monophosphate in whiteleg shrimp samples under different processing methods. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to examples and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0035] In this embodiment of the invention, the standard solutions of monosodium glutamate (MSG) and inosine 5'-monophosphate (IMP) were prepared by continuously diluting the MSG or IMP stock solution with 0.01 M PBS buffer (pH 7.4) to obtain standard solutions of umami substances at different concentrations. All electrochemical tests were performed at room temperature using a CHI760F electrochemical workstation, and the electrolyte used was a mixed solution containing 5 mM potassium ferrocyanide / potassium ferrocyanide and 0.1 M potassium chloride. The screen-printed electrode (SPCE) used was an integrated three-electrode system, including a carbon working electrode, a carbon counter electrode, and an Ag / AgCl reference electrode.

[0036] Example 1 A Ni-MOF material is prepared according to the following steps: (1) Weigh 7 mg of nickel acetate tetrahydrate and dissolve it in 12 mL of DMF / DMA mixed solvent, wherein the volume ratio of DMF to DMA is 1:1; (2) Transfer the obtained mixture to a pressure-resistant reaction tube and preheat it in an oil bath at 65 °C for 10 min; (3) Add 8 mL of 2 M sodium acetate solution and 3 mL of 9.3 μM HATP·6HCl solution to the system in sequence, and stir continuously at 65 °C for 2 h; (4) After the reaction is complete, the product is collected by centrifugation and washed twice each with 100 mL of ultrapure water and 100 mL of methanol. (5) Finally, vacuum dry at 75 °C for 3 h to obtain Ni-MOF powder.

[0037] Example 2 A PVP-modified gold nanoparticle is prepared according to the following steps: (1) Add 1 mL of 1 wt% tetrachloroauric acid solution to 100 mL of vigorously boiling ultrapure water and stir for 2 min; (2) Quickly add 2.5 mL of 1 wt% sodium citrate solution, and continue the reaction for 15 min after the solution color changes from light yellow to wine red; (3) Stop heating and cool to room temperature; (4) Add 5 mL of 30 mg / mL polyvinylpyrrolidone (PVP) solution to the above solution and stir continuously for 24 h under light-protected conditions; (5) Finally, wash three times with 100 mL of methanol to obtain the PVP-modified gold nanoparticle solution.

[0038] Example 3 An AuNPs-T1R1-VFT / Ni-MOF signal probe is prepared according to the following steps: (1) Take 3 mL of Ni-MOF dispersion with a concentration of 5 mg / mL and mix it with 1 mL of PVP-modified gold nanoparticle solution with a concentration of 1 wt%. (2) After sonicating the mixture for 5 min, stir it overnight at room temperature; (3) The obtained product was washed with 50 mL of ultrapure water and 50 mL of methanol respectively to obtain AuNPs / Ni-MOF composite material; (4) Take 1 mL of AuNPs / Ni-MOF dispersion, add 300 μL of T1R1-VFT protein solution with a concentration of 0.15 mg / mL, and incubate overnight at 4 °C with shaking to obtain AuNPs-T1R1-VFT / Ni-MOF signal probe.

[0039] Example 4 An MSG molecularly imprinted modified electrode is prepared according to the following steps: (1) Prepare a 0.1 M PBS buffer containing 5 mM MSG and 5 mM o-phenylenediamine (o-PD) at pH 7.4; (2) Immerse the screen-printed electrode in the above solution and perform electropolymerization using cyclic voltammetry. The scanning potential range is -0.5 V to +0.8 V, the scanning rate is 50 mV / s, and the cycle is 14 times. (3) After polymerization, the electrode surface is gently rinsed with ultrapure water to obtain the MIP / SPCE before elution; (4) Then place the electrode in a mixed eluent with a methanol / acetic acid volume ratio of 9:1 and shake for 30 min to remove the template molecules; (5) Finally, wash thoroughly with ultrapure water and dry at room temperature to obtain the MSG molecularly imprinted modified electrode (MIP / SPCE after elution).

[0040] Example 5 An IMP molecularly imprinted modified electrode is prepared according to the following steps: (1) Prepare a 0.1 M PBS buffer containing 4 mM IMP and 5 mM o-phenylenediamine (o-PD) at pH 7.4; (2) Immerse the screen-printed electrode in the above solution and perform electropolymerization using cyclic voltammetry. The scanning potential range is -0.5 V to +0.8 V, the scanning rate is 50 mV / s, and the cycle is 16 times. (3) After polymerization, the electrode surface is gently rinsed with ultrapure water to obtain the MIP / SPCE before elution; (4) Then place the electrode in a mixed eluent with a methanol / acetic acid volume ratio of 9:1 and shake for 35 min to remove the template molecules; (5) Finally, wash thoroughly with ultrapure water and dry at room temperature to obtain the IMP molecularly imprinted modified electrode (MIP / SPCE after elution).

[0041] Comparative Example 1 A non-molecularly imprinted modified electrode is prepared in a manner that is basically the same as that in Example 4 or Example 5, except that template molecules MSG or IMP are not added to the electropolymerization system. The remaining steps are kept the same, and a non-molecularly imprinted modified electrode (NIP / SPCE) is finally obtained.

[0042] Example 6 A dual-recognition sandwich electrochemical sensor based on MSG is fabricated as follows: (1) Take the MSG molecularly imprinted modified electrode obtained in Example 4; (2) Add 10 μL of MSG standard solution of different concentrations to the surface of the electrode and incubate at room temperature for 25 min to allow MSG to specifically bind to the imprint cavity; (3) Gently rinse the electrode surface with ultrapure water to remove unbound molecules; (4) Add 10 μL of the AuNPs-T1R1-VFT / Ni-MOF signal probe solution prepared in Example 3 to the electrode surface and continue to incubate at room temperature for 25 min. (5) After incubation, rinse the electrode surface with ultrapure water and dry it with nitrogen to obtain the MSG-based dual recognition sandwich electrochemical sensor (i.e., MIP / SPCE after target incubation).

[0043] Example 7 A dual-recognition sandwich electrochemical sensor based on IMP is fabricated as follows: (1) Take the IMP molecularly imprinted modified electrode obtained in Example 5; (2) Add 10 μL of IMP standard solution of different concentrations to the surface of the electrode and incubate at room temperature for 30 min to allow IMP to specifically bind to the imprint cavity; (3) Gently rinse the electrode surface with ultrapure water to remove unbound molecules; (4) Add 10 μL of the AuNPs-T1R1-VFT / Ni-MOF signal probe solution prepared in Example 3 to the electrode surface and continue to incubate at room temperature for 30 min. (5) After incubation, rinse the electrode surface with ultrapure water and dry it with nitrogen to obtain the IMP-based dual recognition sandwich electrochemical sensor (i.e., MIP / SPCE after target incubation).

[0044] Example 8 A method for detecting umami substances in fish and shrimp samples, comprising the following steps: (1) Purchase crucian carp and whiteleg shrimp from a local supermarket. Take the back muscle of the crucian carp and remove the head and shell of the whiteleg shrimp to take the shrimp meat. (2) The samples were divided into four groups according to the heat treatment method: raw group, steamed group, boiled group and roasted group. Among them, crucian carp was steamed for 8 min, boiled for 8 min and roasted at 180 °C for 5 min; whiteleg shrimp was steamed for 5 min, boiled for 5 min and roasted at 180 °C for 3 min. (3) After the samples in each group are processed, weigh 5 g of fish or shrimp meat, add 20 mL of 5% trichloroacetic acid solution, homogenize thoroughly, and shake at 4 °C for 20 min. (4) Centrifuge the mixture at 10,000 rpm for 15 min and collect the supernatant; (5) Adjust the pH of the supernatant to 7.0 with 5% NaOH, and then filter it three times through a 0.22 μm microporous membrane; (6) Before testing, dilute the extract with 0.01 M PBS by 10. 4 times; (7) The electrochemical sensors prepared in Example 6 and Example 7 were used to quantitatively detect MSG and IMP in the samples, and high performance liquid chromatography was used for comparison and verification.

[0045] Figure 1 The detection conditions were as follows: a screen-printed carbon electrode was used as the substrate; the electrode system included a carbon working electrode, a carbon counter electrode, and an Ag / AgCl reference electrode; and the detection solution contained 5 mM [Fe(CN)6]. 3- / 4- A mixed solution of 0.1M KCl. During the detection process, the target molecule first binds to the recognition cavity in the molecularly imprinted polymer film, and then assembles with the AuNPs-T1R1-VFT / Ni-MOF signal probe to form a MIP-target-T1R1-VFT dual recognition sandwich structure.

[0046] Figure 2 The detection conditions were as follows: the bare screen-printed electrode, the molecularly imprinted modified screen-printed electrode before elution, and the molecularly imprinted modified screen-printed electrode after elution were naturally dried and then characterized by atomic force microscopy to test the changes in their surface morphology and roughness, which was used to verify the formation of the molecularly imprinted polymer film and the generation of imprint cavities after template molecules were eluted.

[0047] Figure 3 The detection conditions were as follows: AuNPs / Ni-MOF composite material was fully dispersed and dropped onto the surface of a conductive substrate, and then naturally dried before being observed by scanning electron microscopy to analyze the morphology and structure of Ni-MOF and the loading and dispersion of AuNPs on the Ni-MOF surface.

[0048] Figure 4 The detection conditions were as follows: Fourier transform infrared spectroscopy was used to characterize the structures of AuNPs / Ni-MOF and AuNPs-T1R1-VFT / Ni-MOF, with a scanning range of 4000–500 cm⁻¹. -1 By comparing the changes in the characteristic absorption peaks of the material before and after T1R1-VFT fixation, the successful fixation of T1R1-VFT on the surface of AuNPs / Ni-MOF composite material was verified.

[0049] Figure 5 The detection conditions were as follows: differential pulse voltammetry was used to sequentially detect the bare screen-printed electrode (a), the MIP / SPCE corresponding to Example 4 before elution (b), the MIP / SPCE corresponding to Example 4 after elution (c), the MIP / SPCE after target molecule incubation (d), and the sandwich-type sensing interface after AuNPs-T1R1-VFT / Ni-MOF signal probe assembly (e). The detection solution contained 5 mM [Fe(CN)6]. 3- / 4- A mixed solution of 0.1 M KCl was used, with a scan range of -0.2 to 0.6 V and a pulse amplitude of 50 mV. The test was conducted at room temperature.

[0050] Figure 6 The testing conditions are: 10 -6 A 10 μL solution of MSG standard was added dropwise to the surfaces of MSG-MIP / SPCE (Example 4 sample) and NIP-SPCE (Comparative Example 1 sample), and incubated at room temperature for 25 min. After rinsing, 10 μL of AuNPs-T1R1-VFT / Ni-MOF signal probe was added, and incubation continued for another 25 min. The electrodes were then placed in a solution containing 5 mM [Fe(CN)6]. 3- / 4- Differential pulse voltammetry was performed in a detection solution containing 0.1 M KCl, with an MSG concentration of 10. -6 M.

[0051] Figure 7 The detection conditions were as follows: MSG standard solutions of different concentrations were added dropwise to the MSG-MIP / SPCE surface in a volume of 10 μL, and incubated at room temperature for 25 min to allow the MSG to fully bind to the imprint cavity. After rinsing, 10 μL of AuNPs-T1R1-VFT / Ni-MOF signal probe was added, and incubation continued for 25 min to form a MIP-MSG-T1R1-VFT dual-recognition sandwich structure. The electrode was then placed in a solution containing 5 mM [Fe(CN)6]. 3- / 4- Differential pulse voltammetry was performed in a detection solution containing 0.1 M KCl, with MSG concentrations ranging from 10... -9 ~10-2 M.

[0052] Figure 8 The detection conditions were as follows: IMP standard solutions of different concentrations were added dropwise to the IMP-MIP / SPCE surface in a volume of 10 μL, and incubated at room temperature for 30 min to allow IMP to fully bind to the imprint cavity. After rinsing, 10 μL of AuNPs-T1R1-VFT / Ni-MOF signal probe was added, and incubation continued for 30 min to form a MIP-IMP-T1R1-VFT dual-recognition sandwich structure. The electrode was then placed in a solution containing 5 mM [Fe(CN)6]. 3- / 4- Differential pulse voltammetry was performed in a detection solution containing 0.1 M KCl, with IMP concentrations ranging from 10⁻⁶. -10 ~10 -3 M.

[0053] Figure 9 The detection conditions were as follows: selective testing was performed using differential pulse voltammetry. In the MSG system, 10... -6 M MSG and interfering substances at 100-fold concentration, including IMP, sodium succinate, L-glutamic acid, L-aspartic acid, sucrose, citric acid, NaCl, and quinine. In the IMP system, 10... -6 M IMP and interfering substances at 100-fold concentrations, including MSG, sodium succinate, GMP, AMP, sucrose, citric acid, NaCl, and quinine. All samples were treated under the same sandwich assembly conditions before electrochemical detection.

[0054] Figure 10 The detection conditions were as follows: MSG molecularly imprinted modified electrodes, IMP molecularly imprinted modified electrodes, and corresponding non-molecularly imprinted modified electrodes were used for testing. 10 μL of the target or interfering agent solution was added to the electrode surface and incubated at room temperature for 25–30 min to allow for sufficient interaction with the imprinted membrane. After rinsing, 10 μL of AuNPs-T1R1-VFT / Ni-MOF signal probe was added, and incubation continued for 25–30 min to form the corresponding recognition and assembly interface. The electrode was then placed in a solution containing 0.1 M KCl and 5 mM [Fe(CN)6]. 3- / 4- In the mixed detection solution, differential pulse voltammetry was used for detection, with a scanning range of -0.2 to 0.6 V and a pulse amplitude of 50 mV. The imprinting factor was calculated based on the ratio of the difference in response between the molecularly imprinted electrode and the non-molecularly imprinted electrode to the same analyte, and this factor was used to evaluate the specific recognition ability of the molecularly imprinted membrane for the target analyte.

[0055] Figure 11The detection conditions were as follows: crucian carp were harvested from the back muscle, and whiteleg shrimp were decapitated and shelled before being harvested from the meat. Samples were divided into raw, steamed, boiled, and baked groups. Crucian carp were steamed for 8 min, boiled for 8 min, and baked at 180 °C for 5 min; whiteleg shrimp were steamed for 5 min, boiled for 5 min, and baked at 180 °C for 3 min. After processing, 5 g of each sample was weighed, added to 20 mL of 5% trichloroacetic acid solution for homogenization, and extracted by shaking at 4 °C for 20 min. The mixture was then centrifuged at 10000 rpm for 15 min, and the supernatant was collected. The pH of the supernatant was then adjusted to 7.0 with 5% NaOH, filtered through a 0.22 μm microporous membrane, and diluted 10 μL with 0.01 M PBS. 4 The results were measured using MSG and IMP sensors, respectively, and verified by high-performance liquid chromatography.

[0056] Figure 1 This is a schematic diagram of the electrochemical sensor detection system based on a molecularly imprinted polymer-target-umami receptor dual-recognition sandwich assembly provided by the present invention. The system was constructed using an atomic force microscope (AFM). Figure 2 The molecularly imprinted films constructed in Examples 4 and 5 were characterized. Roughness results showed that ( Figure 2 The roughness of the bare SPCE was 29.1, which increased to 34.8 after electrodeposition and further increased to 47.2 after elution, indicating the successful formation of the imprinted cavity.

[0057] Using scanning electron microscopy (SEM) Figure 3 ) and Fourier transform infrared spectroscopy ( Figure 4 The AuNPs-T1R1-VFT / Ni-MOF signal probe in Example 3 was characterized. Figure 3 The results show that Ni-MOF has a loose and porous aggregated structure, and under high magnification, it appears as short rod-shaped crystals with a length of about 100 nm. After introducing PVP-modified gold nanoparticles, a large number of spherical nanoparticles are uniformly attached to the surface of Ni-MOF, and the dispersion is good. Figure 4 The results showed that, after further binding with T1R1-VFT, at 1674 cm⁻¹ -1 The presence of a characteristic peak belonging to the protein amide I band indicates that T1R1-VFT was successfully immobilized on the surface of the AuNPs / Ni-MOF composite material.

[0058] Differential pulse voltammetry (PDV) Figure 5The bare SPCE (a), MIP / SPCE before elution (b), MIP / SPCE after elution (c), MIP / SPCE after target incubation (d), and the sandwich-type sensing interface (e) after assembling the AuNPs-T1R1-VFT / Ni-MOF signal probe were characterized. The test conditions were: scan range -0.2 V to 0.6 V, pulse amplitude 50 mV.

[0059] Figure 5 The results showed that the peak current of the MIP / SPCE before elution was significantly lower than that of the bare SPCE, which is attributed to the dense structure and strong insulation of the molecularly imprinted polymer film. After template elution, the peak current increased, indicating that the formation of the imprinted cavity improved interfacial mass transfer. After the target molecules recombinated, interfacial charge transport was somewhat hindered. Upon further assembly of the AuNPs-T1R1-VFT / Ni-MOF signal probe, the peak current significantly increased, indicating that the target-mediated sandwich structure effectively improved the enrichment of the signal probe at the interface, while the conductive Ni-MOF and AuNPs significantly promoted interfacial electron transport.

[0060] For comparison, a non-imprinted membrane modified electrode (NIP / SPCE) was prepared using the same steps without the addition of template molecules during electropolymerization. Subsequently, the NIP / SPCE and MIP / SPCE were incubated with the target analyte MSG under the same conditions to evaluate their specific recognition ability. The corresponding DPV curves are shown below. Figure 6 As shown, after electrodeposition of the non-imprinted polymer and incubation of the target and signal probe, the peak current at approximately +0.2 V is relatively low (curve b). This is because the NIP film lacks binding sites that match the target, making it difficult to recognize the target and assemble the sandwich structure. Under the same treatment conditions, the peak current of the imprinted electrode increases significantly (curve a). This indicates that the imprinted cavity can effectively capture the target and promote the enrichment of the signal probe, exhibiting superior recognition ability and electrochemical response compared to the non-imprinted polymer film.

[0061] Differential pulse voltammetry (PDV) Figure 7-8 The dual-recognition sandwich electrochemical sensors prepared in Examples 6 and 7 were tested. Figure 7-8 The results showed that as the concentration of MSG or IMP increased, the differential pulse voltammetric response current gradually increased, indicating that the increase in target molecule concentration can promote the assembly of more AuNPs-T1R1-VFT / Ni-MOF signal probes onto the electrode surface, thereby achieving concentration-dependent electrochemical signal amplification.

[0062] Among them, the MSG system ( Figure 7 ) in 10 -9 M~10 -2 It exhibits a good linear relationship within the range of M, and the regression equation is I. MSG(μA) = 29.47 + 1.58lgC (M), correlation coefficient is 0.993, detection limit is 0.33 nM. IMP system ( Figure 8 ) in 10 -10 M~10 -3 It exhibits a good linear relationship within the range of M, and the regression equation is I. IMP (μA) = 34.01 + 1.86lgC (M), the correlation coefficient is 0.992, and the detection limit is 0.03 nM. These results demonstrate that the dual-recognition sandwich electrochemical sensor constructed in this invention can achieve highly sensitive and rapid quantitative detection of MSG and IMP.

[0063] Differential pulse voltammetry (PDV) Figure 9-10 Selective testing was performed on the sensors prepared in Examples 6, 7, and Comparative Example 1. For the MSG system ( Figure 9 A), add 10 drops of each. -6 M MSG and 100-fold concentrations of IMP, sodium succinate, L-glutamic acid, L-aspartic acid, sucrose, citric acid, sodium chloride, and quinine were detected. For the IMP system ( Figure 9 B), add 10 drops of each. -6 MIMP, as well as 100 times the concentration of MSG, sodium succinate, GMP, AMP, sucrose, citric acid, sodium chloride, and quinine were tested.

[0064] Figure 9 The results of A indicate that the MSG system is effective against 10 -6 The response current of MMSG reached 20.47 μA, while the response to various interfering substances at 100 times the concentration was less than 3.5 μA. Figure 9 The results of B indicate that the IMP system is effective against 10 -6 The response of MSG and IMP was 22.35 μA, while the response to 100-fold concentrations of interfering substances was less than 5.5 μA. Further calculations showed that the imprinting factors of MSG and IMP were 4.95 and 5.07, respectively. The selectivity factors of the main interfering substances in both systems were greater than 5, indicating that the sensor constructed in this invention has high selectivity for the target umami molecules and good anti-interference ability.

[0065] Imprinting factor reflects the binding ability of molecular imprints to target substances. Figure 10 The results showed that the imprinting factors of MSG and IMP were 4.95 and 5.07, respectively, which were much larger than those of other interfering substances, indicating that the molecularly imprinted layer has a stronger specific recognition ability than the non-imprinted polymer.

[0066] The method described in Example 8 was used to process crucian carp under different processing methods. Figure 11 A) and whiteleg shrimp ( Figure 11B) MSG and IMP in the sample were detected. Figure 11 The results showed that the MSG detection signals of fish and shrimp samples were generally higher after steaming and baking than those in the raw group, while the boiled group had the lowest. The IMP detection signals showed the same trend, with the steamed group having the highest signal, the baked group slightly higher than the raw group, and the boiled group having the lowest signal. This is because heat treatment can promote the release of intracellular umami molecules, while the boiling process causes MSG and IMP to dissolve in water and decrease their content. Furthermore, IMP may undergo further degradation subsequently. The detection results of the electrochemical sensor of the present invention (sensor of Example 8) were compared with those of high performance liquid chromatography (Table 1). The results in Table 1 show that the trends of the two methods are consistent, indicating that the dual-recognition sandwich electrochemical sensor constructed in this invention can be used for the accurate detection of MSG and IMP in complex food matrices.

[0067] Table 1

[0068] Example 9 Based on Example 1, the solid-liquid ratio of nickel acetate tetrahydrate to DMF / DMA mixed solvent in step (1) was changed (7:(10-15) mg / mL), the reaction temperature in step (2) was changed (65-70 °C), and the reaction time was changed (1.5-2.5 h), while other conditions were the same as in Example 1.

[0069] Table 2

[0070] Table 2 shows that different Ni-MOF preparation conditions have a significant impact on the sensor's detection performance. Specifically, when the solid-liquid ratio of nickel acetate tetrahydrate to DMF / DMA mixed solvent is 7:12 mg / mL, the reaction temperature is 65 °C, and the reaction time is 2 h, the sensor's detection range reaches 10... -10 ~10 -2 M is significantly better than other groups. This indicates that the Ni-MOF structure is formed more fully under these conditions, which is beneficial for AuNPs recombination and T1R1-VFT fixation, thereby improving the conductivity and signal amplification capability of the signal probe.

[0071] When the solid-liquid ratio is too low or the reaction conditions deviate from the optimal range, the detection range narrows significantly. This may be due to insufficient Ni-MOF crystal growth, structural inhomogeneity, or a reduction in active sites, affecting the construction effect of the signal probe. Based on comprehensive comparison, 7:12 mg / mL, 65 °C, and 2 h were determined to be the optimal conditions for Ni-MOF preparation.

[0072] Example 10 Based on Example 2, the volume ratio of 1 wt% tetrachloroauric acid to 1 wt% sodium citrate was changed (1:(2-3)) and the stirring time in step (4) was changed (23-25 ​​h) in the dark. Other conditions were the same as in Example 1.

[0073] Table 3

[0074] Table 3 shows that the volume ratio of tetrachloroauric acid to sodium citrate and the stirring time in the dark have a significant impact on the preparation effect of PVP-AuNPs. When the volume ratio of the two is 1:2.5 and the stirring time in the dark is 24 h, the detection range of the constructed sensor reaches 10. -10 ~10 -2 M was significantly better than other groups, indicating that the conditions were conducive to the formation of PVP-AuNPs with suitable particle size, good dispersibility and high stability.

[0075] When the amount of sodium citrate is too low or too high, or the stirring time in the dark is insufficient or too long, the detection range is significantly narrowed. This may be due to insufficient reduction of AuNPs, uneven particle size distribution, or decreased surface stability, which in turn affects their composite with Ni-MOF and the immobilization effect of T1R1-VFT. Based on comprehensive comparison, a tetrachloroauric acid:sodium citrate volume ratio of 1:2.5 and stirring in the dark for 24 hours are determined to be the optimal conditions for the preparation of PVP-AuNPs.

[0076] Example 11 Based on Example 3, the volume ratio of 5 mg / mL Ni-MOF dispersion to 1 wt% PVP-AuNPs was changed (3:(0.8-1.2)) and the ultrasonic time in step (2) was changed (4-6 min). Other conditions were the same as in Example 3.

[0077] Table 4

[0078] Table 4 shows that the volume ratio of Ni-MOF to PVP-AuNPs and the ultrasonic time have a significant impact on the construction effect of AuNPs / Ni-MOF composite materials. When the volume ratio is 3:1 and the ultrasonic time is 5 min, the detection range of the constructed sensor reaches 10. -10 ~10 -2 M was significantly better than other groups, indicating that this condition is conducive to the uniform dispersion and stable recombination of AuNPs on the Ni-MOF surface, thereby improving the conductivity of the signal probe and the acceptor loading capacity.

[0079] When the amount of AuNPs is too low or the ultrasonic time is insufficient, the composite is incomplete, resulting in limited signal amplification. Conversely, excessive AuNPs or prolonged ultrasonic time may lead to nanoparticle aggregation or damage to the Ni-MOF structure, thereby reducing sensing performance. Based on comprehensive comparison, a Ni-MOF:PVP-AuNPs volume ratio of 3:1 and an ultrasonic time of 5 min were determined to be the optimal conditions for preparing AuNPs / Ni-MOF composite materials.

[0080] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A dual-recognition sandwich electrochemical sensor, characterized in that, It includes molecularly imprinted modified working electrodes and receptor-functionalized signal probes; The molecularly imprinted modified working electrode is a molecularly imprinted polymer film (MIP) modified electrode constructed on the surface of a screen-printed electrode. The molecularly imprinted polymer film uses umami substances as template molecules to form specific recognition cavities. The receptor functionalization signal probe is an AuNPs-T1R1-VFT / Ni-MOF signal probe; During detection, the target umami molecules first specifically bind to the recognition cavity of the molecularly imprinted polymer membrane, and then act as a bridging medium to promote the assembly of the receptor-functionalized signal probe onto the surface of the working electrode, forming a MIP-target-T1R1-VFT dual recognition sandwich structure, thereby achieving electrochemical signal amplification.

2. The dual-recognition sandwich electrochemical sensor according to claim 1, characterized in that, The umami substance is monosodium glutamate or inosine 5'-monophosphate or a combination thereof; The functional monomer of the molecularly imprinted polymer film is o-phenylenediamine; The working electrode is a screen-printed carbon electrode.

3. The dual-recognition sandwich electrochemical sensor according to claim 1, characterized in that, The molecularly imprinted modified working electrode was prepared according to the following steps: Step A1: Dissolve the template molecule and o-phenylenediamine in PBS buffer, and electropolymerize using cyclic voltammetry with a screen-printed electrode as the substrate to form a molecularly imprinted polymer film. Step A2: The obtained electrode is then placed in a methanol / acetic acid mixed elution solution for template molecule elution to obtain a molecularly imprinted modified working electrode with specific recognition cavity.

4. The dual-recognition sandwich electrochemical sensor according to claim 3, characterized in that, The PBS buffer concentration is 0.05-0.3 M, and the pH is 7.1-7.5; the volume ratio of the methanol / acetic acid mixed elution buffer is (8-10):1; Preferably, the PBS buffer concentration is 0.1 M and the pH is 7.4; the volume ratio of the methanol / acetic acid mixed elution buffer is 9:

1. The electropolymerization conditions are as follows: template molecule concentration of 4 mM to 5 mM, o-phenylenediamine concentration of 5 mM, scanning potential range of -0.5 V to +0.8 V, scanning rate of 50 mV / s, and number of cycles of 14 to 16; the template molecule elution time is 30 min to 35 min.

5. The dual-recognition sandwich electrochemical sensor according to claim 1, characterized in that, The AuNPs-T1R1-VFT / Ni-MOF signal probe was prepared according to the following steps: Step B1: Dissolve nickel acetate in a DMF / DMA mixed solvent, add sodium acetate solution and HATP·6HCl solution, and obtain Ni-MOF through a solvothermal reaction; Step B2: Add tetrachloroauric acid to boiling ultrapure water, then add sodium citrate solution for reduction reaction, cool and add polyvinylpyrrolidone for surface modification to obtain PVP-AuNPs; Step B3: Mix Ni-MOF with PVP-AuNPs to obtain AuNPs / Ni-MOF composite material; Step B4: Incubate the AuNPs / Ni-MOF composite material with T1R1-VFT to obtain the AuNPs-T1R1-VFT / Ni-MOF signal probe.

6. The dual-recognition sandwich electrochemical sensor according to claim 5, characterized in that, In step B1, the solid-liquid ratio of nickel acetate tetrahydrate to DMF / DMA mixed solvent is 7:(10-15) mg / mL, the volume ratio of DMF to DMA is (1-2):(1-2), the solid-liquid ratio of nickel acetate tetrahydrate, sodium acetate solution and HATP·6HCl solution is 7:(7-9):(2-5) mg / mL / mL, the molar concentration of sodium acetate solution is 2-4 M, the molar concentration of HATP·6HCl solution is 9.3-9.9 μM, and the reaction conditions are 65-70 °C for 1.5-2.5 h. In step B2, the volume ratio of 1 wt% tetrachloroauric acid solution to boiling ultrapure water is 1:(90-110), the volume ratio of 1 wt% tetrachloroauric acid solution to 1 wt% sodium citrate solution is 1:(2-3), the volume ratio of 1 wt% tetrachloroauric acid solution to 30 mg / mL polyvinylpyrrolidone solution is 1:(4-6), the stirring time is 1-3 min, the reaction time continues for 14-16 min, and the stirring time in the dark is 23-25 ​​h. In step B3, the volume ratio of 5 mg / mL Ni-MOF dispersion to 1 wt% PVP-AuNPs solution is 3:(0.8-1.2), the ultrasonic treatment time is 4-6 min, and the stirring time at room temperature is 10-14 h (overnight). In step B4, the volume ratio of AuNPs / Ni-MOF dispersion to 0.15 mg / mL T1R1-VFT protein solution is 1:(0.25-0.35), the incubation temperature is 3-5 °C, and the incubation time is 10-14 h.

7. The application of the dual-recognition sandwich electrochemical sensor according to any one of claims 1 to 6 in the quantitative detection of umami substances; The umami substance is monosodium glutamate or inosine 5'-monophosphate or a combination thereof, and the application is the rapid quantitative detection of umami substances in fish and shrimp.

8. A method for quantitatively detecting umami substances using the dual-recognition sandwich electrochemical sensor according to any one of claims 1 to 6, characterized in that, Includes the following steps: S1: Add solutions of umami substances of different concentrations to the surface of the molecularly imprinted working electrode and incubate them to allow the umami substances to bind to the imprint cavity; S2: After rinsing, AuNPs-T1R1-VFT / Ni-MOF signal probes are dropped onto the surface of the working electrode and incubated to allow them to bind with the target molecules to form a MIP-target-T1R1-VFT dual recognition sandwich structure. S3: Place the constructed electrode in a detection solution containing electrolyte for electrochemical detection; S4: A standard curve is obtained by linear fitting with the logarithm of the umami substance concentration as the abscissa and the response current value as the ordinate. S5: Detect the response current of the sample to be tested, and calculate the concentration of umami substances in the sample to be tested in combination with the standard curve.

9. The method according to claim 8, characterized in that, The umami substance solution was added in a volume of 10 μL, and the incubation time was 25 min–30 min; the signal probe solution was added in a volume of 10 μL, and the incubation time was 25 min–30 min; the detection solution contained 0.1 M KCl and 5 mM [Fe(CN)6]. 3- / 4- The mixed solution; the electrochemical detection method is cyclic voltammetry, differential pulse voltammetry and electrochemical impedance spectroscopy.

10. The method according to claim 8, characterized in that, The test samples were derived from crucian carp and whiteleg shrimp. The test samples were pretreated as follows: fish or shrimp meat samples were weighed, added to trichloroacetic acid solution for homogenization, extracted by shaking at 4 °C, centrifuged, the supernatant was collected and the pH was adjusted to neutral. After filtration through a microporous membrane, the samples were diluted with PBS buffer and then subjected to electrochemical detection.

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