Method for detecting bitter substances based on nano-channel bionic taste sensor and application

Abstract

The invention belongs to the technical field of analysis and detection, and particularly discloses a method for detecting bitter substances through a bionic taste sensor based on a nano-channel and application. The bionic taste sensor is characterized in that a nano-pore film body is provided; activating grafting sites of carboxyl groups on the surface of the nanoporous film body by adopting a chemical reaction activator; the bitter receptor protein and the grafting sites are subjected to a chemical bonding amidation reaction, the bitter receptor protein and the grafting sites are arranged between a reference electrode and a working electrode, channels of the nano-channel film body form nano channels for transmitting ions, the bitter protein identifies bitter substances to be detected to change transmembrane current, and the bitter receptor protein and the grafting sites are subjected to a chemical bonding amidation reaction. Therefore, the ionic current signal in the nano channel is obviously changed, so that the specific detection of the bitter substance to be detected is realized. The method has the advantages of high sensitivity, high specificity, small sample dosage and low cost, has important significance for improving the application performance of the method in actual sample analysis, and has wide market application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular, to a method and application for detecting bitter substances using a nanochannel-based bionic taste sensor. Background Art

[0002] Taste is a chemical sensation generated when food stimulates taste buds in the oral cavity. For most mammals such as humans, the taste perception system mainly functions to recognize and distinguish five basic tastes, including sour, sweet, bitter, umami, and salty. These basic tastes together constitute the taste space of organisms, representing different nutritional or physiological needs, and have important impacts on food selection, nutrition, and health. Among the five tastes, bitterness is particularly important as it helps organisms detect and avoid toxic and indigestible substances. Therefore, detecting bitter substances is crucial for the survival of organisms and for improving food safety, flavor, and drug efficacy. However, due to the diversity of the structures of bitter substances, their specificity and sensitivity are poor, which limits the precise detection of bitter substances.

[0003] Sensory evaluation is the main method for testing various tastes. However, this method needs to be completed in a specialized and qualified laboratory and is subjective, being affected by personal preferences and perception abilities. In addition, sensory analysis is not applicable to toxic or health-harmful bitter substances. Electronic tongues use multiple low-selectivity chemical sensors and are widely used, but they have problems of poor selectivity and low sensitivity, which makes it challenging to accurately identify bitter compounds in complex systems. In recent years, bioelectronic tongues using bioactive substances such as taste receptors, cells, and tissues as sensing elements have strong taste detection capabilities and clear detection mechanisms, and can simultaneously detect multiple bitter substances, thus attracting people's attention. However, bioelectronic tongues face challenges such as low detection limits, complex preparation, poor repeatability, and poor stability. Summary of the Invention

[0004] The object of the present invention is to provide, in view of the above deficiencies of the prior art, a method and application for detecting bitter substances using a nanochannel-based bionic taste sensor. After a bitter substance binds to the corresponding bitter receptor, it causes the release of various protein subunits, calcium ions, and sodium ions, and finally transmits taste information to the taste nervous system. Therefore, in the process of bitter perception, biological ion channels play an extremely important role. Inspired by the taste perception in organisms, a suitable bitter protein is selected according to the bitter molecule to be detected and modified on the surface of a nanopore membrane having a structure similar to that of a biological ion channel, so as to accurately identify bitter substances in a complex system.

[0005] The first aspect of the present invention is to provide a method for detecting bitter substances using a nanochannel-based bionic taste sensor, including the following steps:

[0006] S1. Construct a bionic taste sensor,

[0007] Provide a nanochannel thin film body, activate the grafting sites on the surface carboxyl groups of the nanochannel thin film body using a chemical reaction activator, and then bond the bitter receptor protein to the grafting sites through a chemical bonding amidation reaction to obtain the bionic taste sensor. The bitter receptor protein is the receptor protein for the bitter substance to be detected; the nanochannel thin film body is a polyethylene terephthalate nanochannel thin film or a graphene oxide thin film; the bitter substance solution is prepared by dissolving the bitter substance in a phosphate buffer solution.

[0008] S2. Conduct detection,

[0009] Use a detection device for detection. The detection device includes two chambers, and the bionic taste sensor is arranged between the two chambers. Both ends of the bionic taste sensor are communicated with the two chambers respectively. Both of the two chambers are filled with the bitter substance solution to be measured, and a working electrode and a reference electrode are respectively arranged; record the transmembrane ion current.

[0010] S3. Calculate,

[0011] Calculate the current change rate compared with the phosphate buffer solution. The formula is:

[0012] Current change rate % = (I - I0) / I0,

[0013] where I0 is the current value measured with the phosphate buffer solution as the electrolyte, and I is the current value measured with the bitter substance solution to be measured as the electrolyte.

[0014] Further, the bitter substances include allyl isothiocyanate, yohimbine hydrochloride, limonin, sinigrin, soyasaponin I, and / or berberine.

[0015] Further, the bitter receptor protein is T2R38 or T2R16.

[0016] Further, the chemical reaction activator is EDC / NHS, EDC is (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), and NHS is N-hydroxysuccinimide.

[0017] Further, the method for forming the grafting sites includes: dropping EDC / NHS onto the surface of the nanochannel thin film for carboxyl terminal activation; then modifying the receptor protein of the bitter substance to be measured onto the surface of the nanochannel thin film.

[0018] Further, in step S2, the working electrode is an Ag electrode, and the reference electrode is an AgCl electrode.

[0019] The second aspect of the present invention is to provide the application of the above method in food quality assessment and safety monitoring.

[0020] Furthermore, the detection limits of allyl isothiocyanate, yohimbine hydrochloride, limonin, sinigrin, soyasaponin I, and berberine are 0.262 pM, 0.018 pM, 0.231 pM, 1.783 pM, 14.409 pM, and 0.121 pM, respectively.

[0021] Furthermore, the bionic bitter taste sensor is stored at 4 °C for at least 7 days.

[0022] Furthermore, the food includes any one of fresh orange juice, spoiled orange juice, vinegar, cola, coffee, monosodium glutamate, or edible salt.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The present invention provides a method for detecting bitter substances using a bionic taste sensor based on nanochannels. The bionic taste sensor is obtained by providing a nanoporous membrane body; activating the grafting sites of the carboxyl groups on the surface of the nanoporous membrane body using a chemical reaction activator; and then performing a chemical bonding amidation reaction between the bitter receptor protein and the grafting sites. It is arranged between the reference electrode and the working electrode. The pores of the nanoporous membrane body form nanochannels for transmitting ions. The bitter protein in the nanochannels recognizes and binds to the bitter substances to be detected, causing a change in the transmembrane current generated by the transmission of Na+, Cl-, and other ions in the phosphate buffer solution, resulting in a significant change in the ion current signal in the nanochannels to achieve specific detection of the bitter substances to be detected.

[0025] (2) The method for detecting bitter substances using the bionic taste sensor based on nanochannels provided by the present invention selects T2R38 in T2R as a probe, which can effectively recognize allyl isothiocyanate (AI), yohimbine hydrochloride (YH), limonin (L), sinigrin (S), soyasaponin I (SS I), and berberine (B), and the detection limits are as low as 0.262 pM, 0.018 pM, 0.231 pM, 1.783 pM, 14.409 pM, and 0.121 pM, respectively. The bionic bitter taste sensor can be stored at 4 °C for more than one week without obvious loss of activity and can be reused multiple times. In addition, the bionic taste sensor in the present invention has good selectivity and reliability.

[0026] (3) The method for detecting bitter substances provided by the present invention has high sensitivity and high specificity, requires less sample volume, and has low cost, which is of great significance for improving its application performance in actual sample analysis and has a broad market application prospect. Description of the Drawings

[0027] Figure 1 Method for detecting bitterness by the PET-T2R38 bionic taste sensor in Example 1;

[0028] Figure 2 Energy dispersive X-ray spectroscopy (EDS) of PET and PET-T2R38 in Example 1;

[0029] Figures 3a - 3f Curves of the change rate of ion current of AL, YH, L, S, SS I, and B versus bitterness concentration respectively;

[0030] Figure 4 Pore size distribution of PET-T2R38 before and after binding with bitter molecules in Example 1;

[0031] Figure 5 Stability of the sensor during a 7-day storage period in Example 2;

[0032] Figure 6 Detection results of limonin concentration by the bionic taste sensor and high performance liquid chromatography (HPLC) in Example 3;

[0033] Figure 7 Detection of real samples by the PET-T2R38 bionic taste sensor in Example 4;

[0034] Figure 8 Schematic diagram of simultaneously modifying two protein receptors, T2R38 and T2R16, on the outer surface of the graphene oxide film, which expands the detection range of bitter substances and avoids the influence of the change in the inner surface pore size on the change rate of current. Detailed implementation mode

[0035] In order to more clearly and comprehensively explain the technical solution and beneficial effects of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and embodiments. It should be clear that the described reference drawings are only partial embodiments of the present invention, which are only used to explain the present invention and should not be construed as a limitation to the present invention. Unless otherwise specified, the equipment and reagents used in the present invention are conventional commercially available products in the technical field.

[0036] Explanation of English abbreviations in the present invention:

[0037] PET nanoporous film: polyethylene terephthalate nanoporous film; PBS buffer solution: phosphate buffer solution.

[0038] In organisms, the process of bitter taste perception is mainly mediated by bitter taste receptors (T2Rs). T2Rs are G protein-coupled receptors, and different T2Rs can recognize different bitter compounds. After bitter substances bind to their corresponding bitter taste receptors, the release of various protein subunits, calcium ions, and sodium ions is induced, ultimately transmitting taste information to the taste nervous system. Therefore, biological ion channels play an extremely important role in the process of bitter taste perception. Inspired by taste perception in organisms, the present invention aims to provide a method for detecting specific bitter molecules through a nanochannel-based bionic taste sensor.

[0039] It should be noted that the bitter taste receptor protein in the present invention can be selected according to the bitter substance to be detected, and no limitation is made here. To better illustrate the high sensitivity and high specificity of the bitter substance detection method provided by the present invention, a nanochannel-based bionic bitter taste sensor is constructed by taking T2R38 in T2Rs as an example. In organisms, the T2R38 bitter taste protein can recognize various bitter substances such as allyl isothiocyanate (AI), yohimbine hydrochloride (YH), limonin (L), sinigrin (S), soyasaponin I (SSI), and berberine (B).

[0040] As Figure 1 shown, it is a schematic diagram of the principle of the method for detecting bitter substances by the nanochannel-based bionic taste sensor provided by the present invention. The bionic taste sensor of the present invention is applicable to measuring the properties of the liquid to be tested. According to the specific binding of the bitter substance to be tested with the bitter taste protein modified on the PET nanopore membrane, the helical structure of the bitter taste protein becomes loose, changing the pore size of the PET nanopore membrane, and further causing the ionic current in the nanochannels of the PET nanopore membrane to decrease with the increase in the concentration of the bitter substance, and the current change rate to decrease with the increase in the concentration of the bitter substance. The relative current between the working electrode and the reference electrode is measured, and the current difference between the two electrodes is measured. The size range of the PET nanopore membrane is (0.3 - 0.6) × (0.3 - 0.6) cm, the pore size is 30 - 50 nm, and the pore density is 5×10 7 pieces / cm -1 . Ions such as Na+ and Cl- in the PBS buffer solution perform transmembrane transport, and electrochemical analysis is carried out on both sides of the electrode, and ionic signal transmission is carried out through the PET nanopores.

[0041] The working electrode can include a substrate made of platinum, gold, platinum black, etc., and the reference electrode can include a substrate made of silver coated with a silver chloride film.

[0042] Example 1

[0043] 1) Select a columnar pore with a pore diameter of 30 nm, a thickness of 12 μm, and a pore density of 5×10 7 pieces / cm -1PET nanoporous membrane (purchased from IT4IP S.A., Belgium);

[0044] 2) Add 150 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and 75 mg of N-hydroxysuccinimide (NHS) to 3 mL of deionized water respectively. Subsequently, immerse the cut PET nanoporous membrane in a petri dish containing the above EDC / NHS mixed solution and react at room temperature for 3 h;

[0045] 3) After the above reaction is completed, remove the EDC / NHS mixed solution in the petri dish, and rinse the PET with deionized water to remove the residual EDC / NHS on the surface;

[0046] 4) Drop 0.1 mg / mL of T2R38 bitter protein on the surface of the PET nanoporous membrane and react overnight to modify T2R38 on the PET surface through an amide reaction. Name the modified product PET-T2R38;

[0047] 5) After the above reaction is completed, rinse the PET with PBS buffer solution to remove the residual T2R38 on the surface of the PET nanoporous membrane;

[0048] 6) Analyze the elemental composition of PET and PET-T2R38 by energy-dispersive X-ray spectroscopy (EDS). The results are as Figure 2 shown. The appearance of S element and the increase of N element in PET-T2R38 are due to the cysteine and methionine of T2R38.

[0049] 7) Place PET and PET-T2R38 between two chambers, and inject 1 mL of AI, YH, L, S, SSI, B solutions with different concentrations (prepared with PBS buffer solution, the concentration range of AI is 10 -12 M - 10 -4 M, the concentration range of YH is 10 -13 M - 10 -4 M, the concentration range of L is 10 -12 M - 10 -4 M, the concentration range of S is 10 -11 M - 10 -4 M, the concentration range of SSI is 10 -11 M - 10 - 4 M, the concentration range of B is 10 -13 M - 10 -4 M), record the transmembrane ion current respectively, and calculate the current change rate compared with the PBS buffer solution. The results are as Figures 3a - 3fAs shown, after T2R38 binds to bitter molecules, the 7 helical structures of T2R38 become loose, the pore diameter of PET decreases, resulting in a decrease in the transmembrane current of PET-T2R38 with the increase of the concentration of bitter molecules, and the current change rate decreases with the increase of the concentration of bitter molecules. The calculation formula is: Current change rate % = (I - I0) / I0,

[0050] where I0 is the current value measured with PBS as the electrolyte, and I is the current value measured with the solution of the bitter substance to be tested as the electrolyte.

[0051] 8) Observe the pore diameter of PET-T2R38 before and after binding to bitter molecules. The results are as Figure 4 shown. After binding to bitter molecules, the pore diameter decreases from 18.47 ± 6.40 nm to 15.83 ± 3.01 nm.

[0052] Example 2

[0053] Evaluate the stability of the bionic taste sensor.

[0054] The bionic taste sensor prepared in Example 1 was stored at 4 °C for 7 days. The results are as Figure 5 shown. On the 7th day, the initial response currents of the bionic taste sensor to AI, YH, and L were retained at 91.81%, 90.24%, and 83.15% respectively, indicating that the PET-T2R38 bionic taste sensor has good stability.

[0055] Example 3:

[0056] Verify the reliability of the bionic taste sensor for detecting bitter substances.

[0057] A comparative measurement was carried out on limonin (L) at a certain concentration (C0 6×10 -5 M) by two methods: high performance liquid chromatography (HPLC) and the bitter taste sensor.

[0058] The results are as Figure 6 shown. The concentration of L prepared by measuring with the bitter taste sensor platform is C a 6.412×10 -5 M, and the measurement result by the HPLC method is about 5.862×10 -5 M. The errors of the two methods are about 6.87% and 2.30% respectively, indicating that the bionic taste sensor has high accuracy and reliability for detecting bitter molecules.

[0059] Example 4

[0060] Verify that the method for detecting bitter substances provided by the present invention can be used for food quality assessment and safety monitoring.

[0061] Orange juice was selected for the demonstration. During the juice processing, the acidic pH of the juice induces the conversion of a-cyclocitralide in citric acid into bitter limonin, and the d-cyclocitralide hydrolase in citric acid further accelerates this process, resulting in a slight bitterness in the juice. Long-term storage under acidic conditions and in the open air will increase the production of limonin, thus exacerbating the bitterness and damaging the quality of the juice (i.e., spoilage).

[0062] As Figure 7 shown, the current change rates of fresh orange juice (FOJ), spoiled samples (DOJ), and actual samples such as vinegar, cola, coffee, monosodium glutamate, and salt solution are presented. Due to the higher content of limonin in the spoiled orange juice, its current change rate is significantly greater than that of other real samples. The results indicate that the PET-T2R38 bitter sensor has the potential to detect bitter compounds in various real samples, demonstrating its broad application potential in food quality assessment and safety monitoring.

[0063] Example 5

[0064] As Figure 8 shown, the schematic diagram of simultaneously modifying two protein receptors, T2R38 and T2R16, on the outer surface of the graphene oxide film expands the detection range of bitter substances and avoids the influence of the change in the inner surface pore size on the current change rate. The specific operation is as follows: Prepare a graphene oxide dispersion with a concentration of 1 mg / mL using flaky monolayer graphene oxide (flake diameter: 0.5 - 5 μm; thickness 0.8 - 1.2 nm), and perform ultrasonic and suction filtration treatments. After the suction filtration is completed, transfer the graphene oxide film to an oven at 60 °C for drying to obtain the graphene oxide film, and cut it into a size of 2 mm × 8 mm. Two protein receptors, T2R38 and T2R16, are simultaneously modified on the outer surface of the graphene oxide film. Subsequently, the bitter substance solution is dropped on the surface of the graphene oxide film, and the PBS solution is dropped at both ends of the graphene oxide film. Due to the recognition of the bitter protein for the bitter substance to be detected, the Na+ and Cl- ions passing through the interlayer channels of the graphene oxide nanochannels change, resulting in a significant change in the ion current signal within the nanochannels to achieve specific detection of bitter substances within a larger range.

[0065] Where not otherwise involved above, it shall apply to the prior art.

[0066] Although some specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are only for illustration purposes and not for limiting the scope of the present invention. Those skilled in the technical field to which the present invention pertains can make various modifications or supplements to the described specific embodiments or use similar means for substitution, but will not deviate from the direction of the present invention or exceed the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc. made to the above embodiments based on the technical essence of the present invention should all be included within the protection scope of the present invention.

Claims

1. A method for detecting bitter substances by a nanochannel-based bionic taste sensor, characterized in that, It includes the following steps: S1. Construct a bionic taste sensor. Provide a nanoporous membrane body, activate the grafting sites of the carboxyl groups on the surface of the nanoporous membrane body with a chemical reaction activator, and then carry out a chemical bonding amidation reaction between the bitter receptor protein and the grafting sites to obtain the bionic taste sensor. The bitter receptor protein is the receptor protein for the bitter substance to be detected. The nanoporous membrane body is a polyethylene terephthalate nanoporous membrane or a graphene oxide membrane. S2. Conduct detection. Detect with a detection device. The detection device includes two chambers. The bionic taste sensor is arranged between the two chambers. Both ends of the bionic taste sensor are communicated with the two chambers respectively. Both of the two chambers are filled with the solution of the bitter substance to be detected, and a working electrode and a reference electrode are respectively arranged, and record the transmembrane ionic current. The solution of the bitter substance is prepared by dissolving the bitter substance with a phosphate buffer solution. S3. Calculate. Calculate the current change rate compared with the phosphate buffer solution. The formula is: Current change rate % = (I - I0) / I0, where I0 is the current value measured with the phosphate buffer solution as the electrolyte, and I is the current value measured with the solution of the bitter substance to be detected as the electrolyte. The bitter substances include allyl isothiocyanate, yohimbine hydrochloride, limonin, sinigrin, soyasaponin I and / or berberine. The bitter receptor protein is T2R38 and / or T2R16. The chemical reaction activator is EDC / NHS. EDC is (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), and NHS is N-hydroxysuccinimide.

2. The method according to claim 1, wherein The formation method of the grafting sites includes: dropping EDC / NHS onto the surface of the nanoporous membrane for carboxyl terminal activation; and then modifying the receptor protein of the bitter substance to be detected onto the surface of the nanoporous membrane.

3. The method according to claim 2, characterized in that In step S2, the working electrode is an Ag electrode, and the reference electrode is an AgCl electrode.

4. The method according to claim 1, wherein 7. Application of the method according to any one of claims 3-6 in food quality assessment and safety monitoring.

5. The method according to claim 4, wherein The minimum detection limits of allyl isothiocyanate, yohimbine hydrochloride, limonin, sinigrin, soyasaponin I and berberine are 0.262 pM, 0.018 pM, 0.231 pM, 1.783 pM, 14.409 pM and 0.121 pM respectively.

6. The method according to claim 1, characterized in that, The bionic bitter sensor is stored at 4°C for at least 7 days. The food includes any one of fresh orange juice, spoiled orange juice, vinegar, cola, coffee, monosodium glutamate or edible salt.

8. The application according to claim 7, characterized in that ​ 9. The application according to claim 7, wherein ​ 10. The application according to claim 7, characterized in that, ​

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