A bionic taste sensor and its application

Abstract

The present invention discloses a bionic taste sensor based on taste receptors and its application, belonging to the field of electrochemical sensor technology. The bionic taste sensor includes a working electrode and an antibody complex matched with the working electrode. The working electrode includes a glassy carbon electrode, a sheet material loaded with gold nanoparticles modified on the surface of the glassy carbon electrode, and a taste receptor protein assembled on the sheet material; the antibody complex is a metal organic framework material loaded with gold nanoparticles and an antibody against taste receptors. The present invention can effectively distinguish taste substances by utilizing the specific recognition between taste receptors and taste substances or anti-taste receptor antibodies; the metal organic framework material loaded with gold nanoparticles is modified on the antibody, and the electrochemical response performance is significantly improved through immune amplification, thereby improving the detection sensitivity of the biosensor.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical sensors, and particularly relates to a bionic taste sensor based on taste receptors and its applications. Background Art

[0002] As one of the natural chemical senses, gustatory perception is closely related to human life, as it plays an important role in nutrient recognition, ingestion, and avoidance of harmful and indigestible substances. Traditional taste evaluation and detection methods can be divided into two categories: sensory analysis and chemical analysis. Although human sensory evaluation can directly obtain taste information, it is difficult to establish effective evaluation criteria to quantify sweetness. Chemical analysis based on instrument testing (such as HPLC, GC / LC-MS) has the characteristics of high resolution and high sensitivity, but is limited by complex operations and time-consuming procedures. In addition, electronic tongues based on electrochemical technology and chemically sensitive materials (such as polymers, lipid membranes, etc.) have been widely developed. An electronic tongue is a detection technology that uses a multi-sensor array as the basis to sense the overall characteristic response signal of a sample, and performs analog recognition and quantitative and qualitative analysis on the sample, with the advantages of low cost and rapid measurement. However, the specificity and sensitivity of electronic tongues have not yet reached the level of human taste perception. Therefore, researchers have increasingly focused on electrochemical taste biosensors based on highly selective bioactive materials to improve the performance of electronic tongues.

[0003] Currently, some taste biosensors using taste tissues, taste cells, and taste receptors have been widely designed to simulate human sensory perception. Among them, biosensors based on taste tissues and cells can simply and accurately distinguish and detect taste substances, but are limited by individual differences and short lifespan; biosensors based on taste receptors have a simple structure and composition and have become the most promising choice. For example, the literature reports using the umami ligand-binding domain VFT derived from T1R1, immobilizing it on a graphene-based FET to prepare an umami bioelectronic tongue, which can detect monosodium L-glutamate at a high sensitivity of about 1 nM (High-performance bioelectronic tongue using ligand binding domain T1R1 VFT for umami taste detection. Biosensors and Bioelectronics, 2018.117: 628-636).

[0004] Improving sensitivity is crucial for enhancing the detection performance of taste biosensors. Generally, the methods for improving the sensitivity of taste biosensors can be divided into two categories. The first method is to optimize the immobilization of proteins through various nanomaterials. With the development of nanotechnology, more and more nanomaterials have been used to design biosensors. Another method is to use highly sensitive conductors such as field-effect transistor (FET) devices for effective signal amplification. For example, the literature reports that a T1R2 VFT immobilized carbon nanotube field-effect transistor with a floating electrode is used as an artificial sweet taste sensing system, which can be used to detect sweetener solutions as low as 0.1 fM (Ultrasensitive Bioelectronic Tongue Based on the Venus Flytrap Domain of a Human Sweet Taste Receptor. ACS Applied Materials & Interfaces, 2022, 14(2):2478-2487). Although the application of FET devices can significantly improve the detection sensitivity, its complex manufacturing process leads to low production efficiency and high production cost, limiting the development and practical application of FET amplification.

[0005] Therefore, designing new easy-to-operate and low-cost signal amplification methods is of great significance for taste biosensors. Summary of the Invention

[0006] The object of the present invention is to provide a taste biosensor with simple fabrication and low cost, which uses the specific recognition and competition between taste receptors and ligands or anti-taste receptor antibodies to distinguish taste substances, and at the same time improves the detection sensitivity through the principle of immune amplification.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides a bionic taste sensor, which includes a working electrode and an antibody complex matching with the working electrode. The working electrode includes a glassy carbon electrode, a lamellar material loaded with gold nanoparticles modified on the surface of the glassy carbon electrode, and a taste receptor protein assembled on the lamellar material; the antibody complex is composed of a metal-organic framework material loaded with gold nanoparticles and an antibody against the taste receptor.

[0009] The working principle of the bionic taste sensor in the present invention:

[0010] In the absence of taste substances, the antibody of the antibody complex can smoothly recognize the taste receptor, resulting in a relatively large electrochemical impedance (R et) In the presence of a taste substance, the taste substance specifically recognizes the taste receptor protein, occupies the key amino acid residue sites of the antibody, and simultaneously triggers local conformational changes and surface charge redistribution of the taste receptor protein, hindering the binding between the taste receptor and the antibody, resulting in a decrease in R et The change value of the electrochemical impedance is correlated with the content of the taste substance, and the content of the taste substance is calculated by measuring the change value of the electrochemical impedance.

[0011] Furthermore, the preparation method of the working electrode includes: resuspending the sheet material loaded with gold nanoparticles in an aqueous chitosan solution to obtain a suspension, dropping it on the polished glassy carbon electrode, drying, then dropping the taste receptor protein solution, reacting at 4 °C, and then dropping the bovine serum albumin solution for blocking to obtain the working electrode.

[0012] In the above preparation method, the sheet material is adsorbed on the GCE through chitosan, and the protein is assembled on the electrode through physical embedding of chitosan and Au-S bonds.

[0013] Furthermore, the mass percentage concentration of the aqueous chitosan solution is 0.2%.

[0014] Furthermore, the sheet material includes but is not limited to titanium carbide, nickel phosphorus sulfur, and molybdenum disulfide.

[0015] Furthermore, the concentration of the taste receptor protein solution is 100 μg / mL, and after dropping the protein solution, it reacts at 4 °C for several hours.

[0016] Furthermore, the sheet material loaded with gold nanoparticles is titanium carbide loaded with gold nanoparticles, and its preparation method includes: adding the gold nanoparticle solution to the Ti3C2 MXene aqueous solution under magnetic stirring to obtain the titanium carbide loaded with gold nanoparticles. The research of the present invention shows that modifying the glassy carbon electrode with titanium carbide loaded with gold nanoparticles has excellent conductivity.

[0017] Furthermore, the preparation method of the antibody complex includes: adding the anti-taste receptor antibody solution to the suspension of the metal-organic framework material loaded with gold nanoparticles for incubation, centrifuging and washing, and then adding the bovine serum albumin solution for blocking to obtain the antibody complex.

[0018] In the above preparation method, the protein binds to the nanomaterial through Au-N bonds.

[0019] Furthermore, the antibody is an antibody against the antigenic determinant of the taste receptor. The action site of the antibody targets the binding site between the taste receptor and the taste substance, improving the targeting of specific recognition.

[0020] Further, the metal-organic framework material can be, but is not limited to, MIL series metal-organic framework materials, specifically MIL-101(Fe).

[0021] Further, the metal-organic framework material loaded with gold nanoparticles is the MIL-Fe metal-organic framework material loaded with gold nanoparticles, and its preparation method includes: adding a chloroauric acid solution to the suspension of the MIL-Fe metal-organic framework material, and then adding a reducing agent to react. After the reaction is completed, the precipitate is collected by centrifugation to obtain the MIL-Fe metal-organic framework material loaded with gold nanoparticles.

[0022] Further, the reducing agent can be, but is not limited to, sodium citrate.

[0023] In the present invention, the taste receptor can be, but is not limited to, human taste receptor type 1 (T1Rs). Human taste receptor type 1 is a receptor family that can sense sweet and umami tastes, including three members: T1R1, T1R2, and T1R3. The corresponding taste receptor is selected according to the specific taste substance and modified on the working electrode.

[0024] Further, the taste receptor protein is the Venus flytrap domain protein of human taste receptor type 1 member 2 with the amino acid sequence shown in SEQ ID NO.2; the antibody is the antibody against the amino acid sequence at positions 150-450 shown in SEQ ID NO.1. The above modified bionic taste sensor can be used to detect sweet substances.

[0025] Further, the bionic taste sensor consists of an electrochemical three-electrode system, including a working electrode, a counter electrode, and a reference electrode. The counter electrode is a platinum wire electrode, and the reference electrode is a silver / silver chloride electrode.

[0026] The present invention also provides the application of the bionic taste sensor in detecting taste substances, and the application includes:

[0027] (1) Dilute the sample to be tested in a phosphate buffer solution to obtain a test solution, drop it on the working area of the surface of the working electrode, incubate at 30-37 °C, and wash; then drop the antibody complex solution, perform an immunoreaction at 30-37 °C, and wash to obtain a modified working electrode;

[0028] (2) Use an electrochemical workstation to test the electrochemical impedance spectrum, record the electrochemical impedance before and after dropping the sample to be tested, and calculate the change value of the electrochemical impedance ΔR et = R0 - R, where R0 is the blank impedance and R is the test impedance, and substitute it into the standard curve to calculate the content of the taste substance in the test solution.

[0029] Further, in step (1), the incubation time after dropping the test solution is 10-20 minutes.

[0030] Furthermore, the washing is carried out using a phosphate buffer solution.

[0031] Furthermore, the reaction time after adding the antibody complex solution is 20 - 30 minutes.

[0032] Furthermore, when the taste substance is a sweet substance, the method for drawing the standard curve includes: First, weigh sucrose and completely dissolve it in water to make a sucrose mother liquor; then mix the sucrose mother liquor with a phosphate buffer solution and make up the volume to obtain standard solutions to be measured with different concentrations; then take different concentrations of the standard solutions and drop them onto the working area of the surface of the working electrode, repeat steps (1)-(2), record the electrochemical impedance change values of different concentrations of sucrose; finally, draw a calibration linear curve between the electrochemical impedance change values and the logarithm of the concentration.

[0033] Furthermore, the conditions for the electrochemical impedance spectroscopy test are that the frequency range is from 0.01 Hz to 100 kHz and the signal amplitude is 5 mV.

[0034] Furthermore, the electrolyte for the electrochemical measurement is 5 mM [Fe(CN)6] containing 0.1 M KCl 3- / 4- solution.

[0035] The beneficial effects of the present invention are as follows:

[0036] (1) The present invention provides a novel bionic electrochemical taste biosensor based on a bioactive sensing element, immune amplification, and a nanomaterial carrier. By utilizing the specific recognition between a taste receptor and a taste substance or an anti-taste receptor antibody, the designed biosensor can effectively distinguish taste substances; at the same time, a metal-organic framework material loaded with gold nanoparticles is modified on the antibody, and the electrochemical response performance is significantly improved through immune amplification, thereby enhancing the recognition sensitivity of the biosensor.

[0037] (2) The bionic electrochemical taste biosensor provided by the present invention is simple to manufacture, has a low manufacturing cost, has the advantages of easy operation and rapid detection, and the detection sensitivity meets the detection requirements in the food field. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a principle flow chart of the sensor of the present invention.

[0039] Figure 2 It is a step-by-step characterization diagram by EIS during the preparation process of the sensor.

[0040] Figure 3 It is the electrochemical response of the sensor of the present invention in the presence of sucrose.

[0041] Figure 4The figure shows the comparison of the electrochemical response of the present invention with immunoamplification in the presence of antibody complexes and the response in the absence of antibody complexes.

[0042] Figure 5 This is a linear calibration curve between the electrochemical change value of the sensor and the logarithm of sucrose concentration.

[0043] Figure 6 This is the sensor's detection response to commercial apple juice. DETAILED DESCRIPTION

[0044] The present invention will be further described below with reference to specific examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0046] In the following examples, HAuCl4 and sodium citrate were purchased from Sinopharm; bovine serum albumin was purchased from Aladdin; and Ti3C2Mxene was purchased from Xianfeng Nano.

[0047] Example 1

[0048] Taking titanium carbide loaded with gold nanoparticles (TiCAu) as the sheet nanomaterial and MIL-Fe(Au) as the metal-organic framework material as an example, the biomimetic taste sensor was designed and generated. The specific implementation steps are as follows:

[0049] 1. Prediction of antigenic determinant sites of human taste receptor type 1 member 2 (T1R2) Venus flytrap (VFT) domain protein

[0050] The amino acid sequence of human T1R2 VFT is shown in SEQ ID NO. 1. A combination of a Hidden Markov Model (HMM) and a propensity score method was used to predict the positions of linear B cell epitopes. Residues with scores exceeding a threshold were considered to be part of the antigenic determinant epitope.

[0051] The prediction results showed that the antigenic determinant sites included 134-178, 248-274 and 338-374, most of which overlapped with the T1R2 carbohydrate binding site.

[0052] Based on the above results, we commissioned a biological company to prepare the corresponding antibody targeting the sequence of positions 150 to 450 of T1R2 VFT.

[0053] 2. Preparation of human taste receptor type 1 member 2 (hT1R2) Venus flytrap (VFT) domain protein

[0054] The VFT part was truncated from hT1R2, and the amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3. A gene fragment of T1R2-VFT with an N-terminal 6×His tag was synthesized and cloned into the pET28a vector. The plasmid was then transformed into Escherichia coli BL21(DE3) cells and screened on an LB agarose plate containing kanamycin. The transformed cells were cultured in LB medium containing kanamycin (50 μg / mL) at 37°C. When the optical density (OD 600 ) value reached 0.5, the expression of the T1R2 VFT gene was induced with isopropyl β-D-thiogalactoside (1 mM) at 20°C for 8 hours. After cell culture, the cells were collected by centrifugation (12,000 rpm, 5 minutes, 4°C). The pellet was transferred into lysis buffer and then centrifuged at 12,000 rpm at 4°C for 25 minutes. The insoluble fraction, including the inclusion body protein (IBP) of T1R2 VFT, was resuspended in IBP buffer and disrupted by sonication. Next, the dissolved sample was collected by centrifugation at 30°C for 30 minutes (12,000 rpm), and then dialyzed twice (volume ratio of 1:8): first, dialyzed against dialysis buffer I for 1.5 hours using a dialysis membrane with a molecular weight cut-off of 10 kDa, and then dialyzed overnight against dialysis buffer II. The dialyzed sample was filtered through a 0.45 μm filter and loaded onto a pre-equilibrated His-Trap affinity column (5 mL). The column was gradually washed with washing buffer, and finally, T1R2 VFT was eluted with elution buffer and stored at -80°C for later use.

[0055] 3. Synthesis of the MIL-Fe metal-organic framework material loaded with gold nanoparticles (Au@MIL-101(Fe)) and modification with antibodies

[0056] 3.1 Preparation of MIL-101(Fe)

[0057] First, FeCl3·6H2O (1.62 g) and terephthalic acid (H2BDC, 0.824 g) were uniformly dissolved in 60 mL of N,N-dimethylformamide (DMF) with continuous stirring. Second, the resulting mixture was poured into a 100 mL Teflon stainless steel container, sealed, and then heated to 110°C in an oven. After reacting for 20 hours, the mixture was cooled to room temperature and centrifuged at 1000 revolutions per minute for 10 minutes. The excess reactants and impurities in MIL-101(Fe) were removed by washing twice with DMF and ethanol in sequence. Finally, after drying overnight at 60°C, pure MIL-101(Fe) was obtained.

[0058] 3.2 Preparation of Au@MIL-101(Fe)

[0059] First, MIL-101(Fe) was treated with ultrasonic waves for 30 minutes to obtain a uniform suspension (concentration: 1 mg / mL). 250 μL of HAuCl4 solution (concentration: 40 mM) was added to 10 mL of MIL-101(Fe) solution. After 30 minutes, 1 mL of sodium citrate solution (concentration: 40 mM) was added, and stirring continued for 2 hours. The reaction solution was centrifuged at 11200 rpm for 10 minutes. Then, it was washed with deionized water and centrifuged three times to remove unreacted substances. Finally, the final precipitate was transferred to 4 mL of deionized water to obtain Au@MIL-101(Fe).

[0060] 3.3 Preparation of Au@MIL-101(Fe)-antibody complex

[0061] 500 μL of the antibody (100 μg / mL) obtained in step 1 was added to the above Au@MIL-101(Fe) solution, and it was stirred and incubated at room temperature for 1 hour. Then, 50 μL of bovine serum albumin (BSA 1%) was added to block non-specific adsorption sites. After centrifugation, the Au@MIL-101(Fe)-antibody complex was resuspended in 1 mL of phosphate buffer and stored at 4°C for further use.

[0062] 4. Synthesis of titanium carbide loaded with gold nanoparticles (TiCAu)

[0063] 4.1 AuNPs were prepared according to the citrate reduction method of Turkevich / Frens. In a typical synthesis process, 50 mL of 1 mM HAuCl4 was taken and heated to boiling with strong stirring. 5 mL of 38.8 mM sodium citrate was quickly added to the vortex of the solution, and the color of the solution changed from light yellow to bright red. Boiling continued for 10 minutes, then the heating magnetic sleeve was removed, and stirring continued until the solution cooled to room temperature. After the formation of AuNPs, they were stored at 4°C.

[0064] 4.2 Synthesis of TiCAu

[0065] The Ti3C2MXene was ultrasonically treated to obtain a uniform suspension (concentration 1 mg / mL). Then, under magnetic stirring, 2 mL of the AuNPs solution was slowly added to 10 mL of the Ti3C2MXene aqueous solution. After the reaction lasted for 3 hours, the mixture was centrifuged at 8000 rpm for 10 minutes. The excess reactants were removed by washing twice continuously with water. Finally, the pure Ti3C2MXene / AuNPs (TiCAu, concentration approximately 0.25 mg / mL) was transferred to 3 mL of deionized water containing 0.2% chitosan (CS) to obtain the TiCAu suspension.

[0066] 5. Preparation of the working electrode of the electrochemical sensor

[0067] The glassy carbon electrode (GCE) was polished with alumina polishing powders (particle sizes 1.0, 0.3, and 0.05 μm respectively), ultrasonically treated with distilled water and ethanol, and dried with nitrogen for subsequent use.

[0068] Modification of the working electrode: First, 6 μL of the TiCAu suspension prepared in step 4 was dropped onto the clean GCE. After drying, 6 μL of the T1R2 VFT protein solution (100 μg / mL) prepared in step 2 was dropped and reacted for 6 hours, with the temperature maintained at 4 °C. Then, 6 μL of BSA (1%) was dropped and reacted for 1 hour.

[0069] 6. Using an electrochemical workstation to test the electrochemical impedance spectroscopy (EIS)

[0070] The electrochemical experiment was carried out at 25 °C using a Chenhua CHI 660E instrument in China, adopting a three-electrode system. The electrochemical electrode system consisted of the working electrode, counter electrode, and reference electrode prepared in step 5. The counter electrode was a platinum wire electrode (Pt), and the reference electrode was a silver / silver chloride (Ag / AgCl) electrode.

[0071] The frequency range of the electrochemical impedance spectroscopy (EIS) was from 0.01 Hz to 100 kHz, and the signal amplitude was 5 mV; the electrolyte for the electrochemical measurement was 5 mM [Fe(CN)6] 3- / 4- solution containing 0.1 M KCl.

[0072] 6.1 Step-by-step characterization with EIS during the preparation (without condition optimization) of the sensor

[0073] The preparation process of the working electrode of the sensor is as Figure 1 shown. The EIS characterization results are as Figure 2As shown, the bare glassy carbon electrode (GCE) has a small resistance (curve a). After immobilizing TiCAu on the GCE surface by chitosan adsorption, a significant decrease in resistance was observed (curve b). When the biosensitive material T1R2 VFT (curve c) and the blocker BSA (curve d) were successively assembled onto the electrode with the help of physical embedding of chitosan and Au-S bonds, the resistance showed a gradually increasing trend, which was due to the non-conductive protein hindering the electron transfer at the electrode interface (curve c-d). Subsequently, the specific recognition between T1R2 VFT and ligand molecules caused by the addition of sucrose further led to an increase in resistance (curve e). Finally, when Au@MIL-101(Fe) was immobilized on the electrode surface by immunological recognition, the resistance increased significantly (curve f). This increase was due to the introduction of the Au@MIL-101(Fe) nanocomposite. On the one hand, Au@MIL-101(Fe) would create a large steric hindrance, which was not conducive to the approach of the electrochemical indicator 3- / 4- to the electrode. On the other hand, Au@MIL-101(Fe) carried a negative charge and repelled the negatively charged 3- / 4- [Fe(CN)6], resulting in an increase in the total impedance.

[0074] The results showed that recognition and binding between T1R2 VFT and Ab occurred on the electrode surface, indicating the successful preparation of the sensing interface.

[0075] 6.2 After the above sensor was assembled, 100 μL of 10 μM sucrose solution or phosphate buffer (blank control) was dropped onto the working area of the working electrode surface, incubated at 37 °C for 10 minutes, and after the incubation, the electrode was washed with three volumes of PBST. Then, 6 μL of the Au@MIL-101(Fe)-antibody complex prepared in step 3 was added, and an immunological reaction was carried out at 37 °C for 30 minutes. Finally, the modified electrode was washed with three volumes of PBST.

[0076] The electrochemical impedance was measured, and the results were as Figure 3 shown. In the absence of sweet substances (blank control), the antibody could successfully recognize and bind to T1R2 VFT on the working electrode; after adding sucrose, the T1R2 VFT / sucrose complex structure was formed, the conformation of T1R2 VFT changed and hindered the binding between VFT and the antibody, resulting in a decrease in et R.

[0077] 6.3 After the above sensor is assembled, take 100 μL of 1 μM sucrose solution and drop it onto the working area of the working electrode surface. Incubate at 37 °C for 10 minutes. After incubation, wash the electrode with three times the volume of PBST. Then, drop 6 μL of the Au@MIL-101(Fe)-antibody complex prepared in step 3, and carry out an immunoreaction at 37 °C for 30 minutes. Finally, wash the modified electrode with three times the volume of PBST. Test the electrochemical impedance before and after modification with the Au@MIL-101(Fe)-antibody complex.

[0078] As Figure 4 shown, when there is a MIL-Fe metal-organic framework material-antibody complex loaded with gold nanoparticles, the electrochemical response increases by about 3.03 times, highlighting the advantage of designing a bionic sensor through immune amplification.

[0079] 7. Plotting of the standard curve

[0080] Prepare the standard solution: Weigh solid sucrose and dissolve it completely with water to make a sucrose stock solution; mix the sucrose stock solution with 1×PBS buffer solution and make up the volume to obtain standard solutions with different concentrations to be measured.

[0081] Take 100 μL of each of the above-concentration standard solutions and drop them onto the working area of the working electrode surface. Incubate at 37 °C for 10 minutes. After incubation, wash the electrode with three times the volume of PBST. Then, drop 6 μL of the Au@MIL-101(Fe)-antibody complex prepared in step 3, and carry out an immunoreaction at 37 °C for 30 minutes. Finally, wash the modified electrode with three times the volume of PBST.

[0082] Use an electrochemical workstation to test the electrochemical impedance spectrum (EIS). The frequency range of the electrochemical impedance spectrum (EIS) is from 0.01 Hz to 100 kHz, and the signal amplitude is 5 mV; the electrolyte for electrochemical measurement is 5 mM [Fe(CN)6] 3- / 4- solution containing 0.1 M KCl. Record the change value ΔR et of the electrochemical impedance when dropping standard solutions with different concentrations = R0 - R, where R is the measured resistance and R0 is the blank resistance.

[0083] As Figure 5 shown, there is a linear relationship between the change value ΔR et of the electrochemical impedance and the logarithmic concentration (-logC). The sucrose concentration is 50 pM - 50 μM, and the correlation coefficient R 2 is 0.99643.

[0084] 8. Determination of actual samples

[0085] To demonstrate the applicability of our biosensor in real samples, the sweet substances in commercial fruit juices were detected in this experiment.

[0086] The commercial apple juice used in this experiment contains various natural sweet substances such as sucrose, glucose, and fructose, with a total concentration of approximately 100 mg / mL. The commercial apple juice was serially diluted (10 -10 -10 -5 ) with 1×PBS buffer solution.

[0087] As Figure 6 shown, the device started to show a response from a dilution concentration of 10 -10 (V / V) and reached a saturation state at approximately 10 -5 (V / V). The standardized dose-dependent response of the sensor to the diluted apple juice in a complex environment was shown.

[0088] The above is only an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, various changes and modifications can be made to the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the scope of the claims of the present invention.

Claims

1. A bionic taste sensor, characterized in that, It includes a working electrode and an antibody complex that matches the working electrode. The working electrode includes a glassy carbon electrode, a lamellar material loaded with gold nanoparticles modified on the surface of the glassy carbon electrode, and a taste receptor protein assembled on the lamellar material. The lamellar material loaded with gold nanoparticles is titanium carbide loaded with gold nanoparticles, and its preparation method includes: under magnetic stirring, adding a gold nanoparticle solution to an aqueous solution of Ti3C2 MXene, and reacting to obtain the titanium carbide loaded with gold nanoparticles. The antibody complex is composed of a metal-organic framework material loaded with gold nanoparticles and an antibody against a taste receptor. The metal-organic framework material loaded with gold nanoparticles is a MIL-Fe metal-organic framework material loaded with gold nanoparticles, and its preparation method includes: adding a chloroauric acid solution to a suspension of the MIL-Fe metal-organic framework material, then adding a reducing agent to react, and after the reaction is completed, centrifuging to collect the precipitate to obtain the MIL-Fe metal-organic framework material loaded with gold nanoparticles.

2. The bionic taste sensor according to claim 1, wherein, The preparation method of the working electrode includes: resuspending the lamellar material loaded with gold nanoparticles in an aqueous chitosan solution to obtain a suspension, dropping it on the polished glassy carbon electrode, drying, then dropping a taste receptor protein solution, reacting at 4 °C, and then dropping a bovine serum albumin solution to block to obtain the working electrode.

3. The bionic taste sensor according to claim 1, characterized in that, The preparation method of the antibody complex includes: adding an antibody solution against a taste receptor to a suspension of the metal-organic framework material loaded with gold nanoparticles for incubation, centrifuging and washing, and then adding a bovine serum albumin solution to block to obtain the antibody complex.

4. The bionic taste sensor according to claim 1, wherein The antibody is an antibody against the antigenic determinant of a taste receptor. The taste receptor protein is a Venus flytrap domain protein of human taste receptor type 1 member 2 with the amino acid sequence shown in SEQ ID NO.

2. The antibody is an antibody against the sites 150-450 in the amino acid sequence shown in SEQ ID NO.

1.

5. Use of the bionic taste sensor according to any one of claims 1-4 in detecting taste substances, characterized in that, The application includes: (1) Diluting a sample to be tested in a phosphate buffer solution to obtain a test solution, dropping it on the working area of the surface of the working electrode, incubating at 30-37 °C, washing, then dropping an antibody complex solution, performing an immunoreaction at 30-37 °C, and washing to obtain a modified working electrode. (2) Use an electrochemical workstation to test the electrochemical impedance spectrum, record the electrochemical impedance before and after adding the sample to be tested, and calculate the change value of electrochemical impedance ΔR et = R0 - R, where R0 is the blank impedance and R is the test impedance. Substitute it into the standard curve to calculate the content of the taste substance in the sample to be tested.

6. The application according to claim 5, characterized in that, The conditions for electrochemical impedance spectroscopy testing are a frequency range of 0.01 Hz to 100 kHz and a signal amplitude of 5 mV.

7. The application according to claim 5, characterized in that The electrolyte for electrochemical measurement was a 5 mM [Fe(CN)6] solution containing 0.1 M KCl 3- / 4- solution.

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