Application of a mouse taste bud tissue biosensor in screening TRPM8 receptor antagonists

Abstract

The present invention provides an application of a mouse taste bud tissue biosensor in screening TRPM8 antagonists. The mouse taste bud tissue biosensor is constructed by using a screen-printed electrode as a detection device and a mouse taste bud tissue membrane as an identification element. The tissue biosensor method has high sensitivity, strong specificity, small sample volume, and a detection concentration as low as 10 ‑ 22 mol·L ‑1 The invention also achieved the screening of TRPM8 receptor antagonists from candidate natural compounds in traditional Chinese medicine by analogy with enzymatic reaction kinetics. This invention forms a set of methods for screening TRPM8 receptor antagonists based on mouse taste bud tissue sensors.

Description

Technical Field

[0001] The present invention belongs to the field of traditional Chinese medicine, and particularly relates to the application of a mouse taste bud tissue biosensor in screening TRPM8 receptor antagonists. Background Art

[0002] TRP channels are voltage-gated cation channels that sense stimuli such as light, temperature, touch, pain, and taste. Heat-activated channels include TRPV1-4, while cold-activated channels include TRPM8 and TRPA1. TRP channels are closely associated with the pungent-warm and pungent-cool properties of traditional Chinese medicine (TCM). Many natural compounds derived from pungent TCMs can modulate TRP channels. Therefore, research on TRP channels can, to some extent, explain the scientific implications of these properties. TRPM8, one of the most studied TRP channels, is a cold-activated, thermosensitive TRP channel distributed in sensory neurons of the dorsal root ganglion and trigeminal nerve. TRPM8 channels can be activated by a variety of stimuli, including low temperatures (8-26°C) and cooling agents such as WS-3 and menthol. Both TRPM8 agonists and antagonists have been studied for various therapeutic applications, with TRPM8 antagonists being potential treatments for neuropathic pain, inflammation, migraine, and cancer. However, most TRPM8 antagonists lack selectivity for TRPM8 and interact with both TRPV1 and TRPA1. Therefore, specific screening of TRPM8 receptor antagonists is necessary. In addition, research on TRPM8 receptor agonists and antagonists can further expand the exploration of TRPM8 receptor function.

[0003] Biosensors are valuable tools for rapidly screening biological targets with high sensitivity and specificity. Tissue biosensors provide technical support for identifying and screening antagonists, enabling the identification of agonist-antagonist interactions. Mouse taste bud tissue biosensors utilize a biofilm composed of mouse taste bud tissue modified on a screen-printed electrode. Taste bud tissue originates from the tongue, a key organ for collecting information about the taste of substances. The basic unit of taste perception is the taste bud, composed of taste receptor cells, basal cells, and supporting cells. Taste receptors are primarily associated with the perception of sweet, umami, bitter, and pungent flavors. When taste receptors on taste buds bind to ligands, they generate taste signals that are transmitted to the taste center in the brain. This allows for highly specific signaling between taste bud cells and the brain's taste center. Chemical, thermal, and tactile stimuli on the tongue trigger reactions in sensory nerve fibers, which interact with taste receptors on taste cells in the taste papillae, activating nerve fibers that form projections to the taste cells. TRPM8 is present in sensory nerve fibers innervating the tongue. Moreover, the taste mucosal epithelial tissue still maintains normal function for a period of time after being peeled off, and the taste receptor cells can still maintain effective activity to taste substances. In addition, screen-printed electrodes (SPE) are disposable sensors based on screen printing technology. Tissues, enzymes, antigens, antibodies, and nucleic acids can be modified on electrodes to make enzymes, antibodies, and gene sensors, etc. Because they have high sensitivity and selectivity, as well as low detection limits, and allow a large number of experiments, the required sample volume is small, and no electrode pretreatment and maintenance are required, they are widely used in pharmaceuticals, clinical trials, and the food industry. The mouse taste bud tissue biosensor signal constructed by the present invention is generated by the combination of TRPM8 receptor agonists and antagonists, so the biosensor based on screen-printed electrodes can provide reliable results. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the existing technology, expand the detection range of tissue biosensors, improve the specific selection of receptors, and sensitively screen TRPM8 receptor antagonists, the primary purpose of the present invention is to provide a method for using a mouse taste bud tissue biosensor in screening TRPM8 receptor antagonists.

[0005] A method for using a mouse taste bud tissue biosensor in screening TRPM8 receptor antagonists comprises the following steps:

[0006] (1) Construction of mouse taste bud tissue sensors:

[0007] Soluble starch was dissolved in a 1%-5% sodium alginate glutaraldehyde solution to prepare a sodium alginate starch glue solution. The sodium alginate starch glue solution was used to fix the mouse taste bud tissue between two microporous filter membranes to form a sandwich-structured biological tissue membrane. The prepared tissue membrane was immersed in a CaCl2 solution and fixed on a screen-printed working electrode. The mouse taste bud tissue sensor was then constructed.

[0008] (2) Parameter optimization for the construction of mouse taste bud tissue sensors:

[0009] The CV method was used to optimize the detection method for the taste bud tissue sensor parameters of mice. The detection voltage range was -0.4-0.7 V. Capsaicin solution was used as the test sample. Water, PBS, K3[Fe(CN)6] aqueous solution, K3[Fe(CN)6]PBS solution, KCl-5mmol·L -1 K3[Fe(CN)6] solution, KCl-10mmol·L -1 K3[Fe(CN)6] solution was used as the working fluid for the test. Figure 1 (A) It can be seen that KCl-10mmol·L -1 When K3[Fe(CN)6] solution was used as the working solution, the response current was the largest; when KCl-10mmol·L - 1 When K3[Fe(CN)6] solution was used as the working solution, the detection was performed at scan rates of 25, 50, 75, 100, 125, 150, and 175 mV. Figure 1 (B) It can be seen that 50mV is selected as the scanning rate; with KCl-10mmol·L -1 K3[Fe(CN)6] solution was used as the working solution, the scanning rate was 50mV, and the tests were carried out at scanning intervals of 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, and 0.10V / s. Figure 1 (C) It can be seen that 0.01 V / s is selected as the scanning interval.

[0010] (3) Determination of the concentration of TRPM8 agonist solution:

[0011] Accurately weigh 1.1 mg of TRPM8 agonist WS-3 standard (molecular weight 211.344) in a 5 mL volumetric flask and use KCl-10 mmol L -1 The K3[Fe(CN)6] mixed solution was fixed to volume and mixed to obtain a concentration of about 1×10 -3 mol·L -1 WS-3 solution; diluted by 10-fold concentration gradient to obtain 1×10 -22 -1×10-4 mol·L -1 WS-3 solution; using the prepared mouse taste bud tissue biosensor as the reaction device, blank solution and WS-3 solutions with varying concentrations from low to high were added, and the current intensity was recorded using an electrochemical workstation, such as Figure 2 (A) shows that the WS-3 solution concentration (C) is used as the horizontal axis and the current change rate (ΔI) is used as the vertical axis for fitting. The results are shown in Figure 2 (B) As shown in the figure, when the concentration range is 1×10 -21 -1×10 -19 mol / L, there is a good linear relationship, and the concentration is divided into 1×10 -21 , 2×10 -21 , 4×10 -21 , 6×10 -21 , 8×10 -21 , 1×10 -20 , 2×10 -20 , 4×10 -20 , 6×10 -20 , 8×10 -20 , 1×10 -19 mol·L -1 ;

[0012] (4) TRPM8 agonists interact with TRPM8 receptors:

[0013] According to the subdivision concentration in step (3) from low to high (1×10 -21 -1×10 -19 mol·L -1 ) WS-3 solution was added to the mouse taste bud tissue biosensor in sequence, and the current intensity was recorded using an electrochemical workstation; the results are shown in FIG. Figure 3 As shown in (A), the response current change rate shows a good hyperbolic relationship with the TRPM8 agonist WS-3 solution, indicating that the interaction between the WS-3 solution and its receptor is similar to the enzymatic reaction kinetics. When the receptor and ligand bind to a certain extent, there is a ligand saturation effect. The reciprocal of the concentration of the TRPM8 agonist WS-3 solution is used as the horizontal axis and the reciprocal of the response current change rate is used as the vertical axis. The double reciprocal curve is drawn, and the linear relationship is good. The results are shown in FIG. Figure 3 As shown in (B), the fitting equation is y = 8.811 × 10 -21 x+7.940(R 2 =0.040); According to the formula

[0014]

[0015] Slope is the slope of the double reciprocal curve, and Intercept is the intercept, from which the Kd value of TRPM8 agonist and TRPM8 can be calculated, Kd = 1.11 × 10 -21 M

[0016] (5) The mixed solution of TRPM8 receptor agonist and antagonist interacts with the TRPM8 receptor:

[0017] Add 1×10 -20 mol·L -1 A mixed solution of magnoliaside, a TRPM8 candidate antagonist, was prepared and added to the mouse taste bud tissue biosensor in descending order of concentration. The current intensity was recorded using an electrochemical workstation. The concentration of the mixed solution (C) was used as the horizontal axis and the current change rate (ΔI) was used as the vertical axis. A hyperbola fitting was performed by analogy with the enzymatic reaction kinetics. The results are shown in the figure. Figure 4 As shown in (A), when the receptor binds to the ligand to a certain extent, there is a ligand saturation effect. When it reaches a certain level, the response current no longer changes. With the reciprocal of the mixed solution concentration as the horizontal axis and the reciprocal of the current change rate as the vertical axis, a double reciprocal curve is drawn and a linear equation is fitted. The results are shown in Figure 4 As shown in (B), it can be seen from the figure that the linear relationship is good, and the equation is y = 2.776 × 10 -20 x+9.625,R 2 =0.983 Calculate the dissociation constant Kd value, Kd = 2.88 × 10 -21 M;

[0018] (6) Similarly, 1×10 -20 mol·L -1 A mixed solution of forsythiaside, ligustilide, and menthol, a candidate TRPM8 antagonist, was prepared. The interaction strength of the mixture of forsythiaside and TRPM8 agonist with TRPM8 receptor was determined and calculated. The Kd was 3.41×10 -21 M; The interaction strength of the mixture of ligustilide and TRPM8 agonist with TRPM8 receptor Kd = 1.54×10 -21 M; The enzymatic reaction kinetics of the mixture of menthol and TRPM8 agonist showed no ligand saturation effect and could not be fitted with a hyperbola, so it was not a TRPM8 receptor antagonist, while forsythiaside and ligustilide were TRPM8 receptor antagonists, and the results were shown in Table 2. Figure 5-Figure 7 .

[0019] (7) After adding TRPM8 antagonist, the main manifestation is the change of dissociation constant Kd and maximum reaction rate Vmax. According to the formula

[0020]

[0021] The Vmax of the forsythin mixture and the ligustilide mixture were 0.132 and 0.124 respectively, and the Vmax of the TRPM8 agonist solution was 0.126. The forsythin mixture and the ligustilide mixture did not change significantly compared with the TRPM8 agonist solution, while the dissociation constant Kd value increased significantly, which is similar to the competitive inhibition of the enzyme. Therefore, forsythin and ligustilide are competitive antagonists of TRPM8; the Vmax of the magnolin mixture was 0.104, which was smaller than that of the TRPM8 agonist, while the dissociation constant Kd value increased significantly, which belongs to mixed inhibition. According to the formula

[0022]

[0023]

[0024] C represents the inhibitor concentration used, Ki is the inhibition constant, and Ki' is the apparent inhibition constant.

[0025] Ki magnolin = 4.65 × 10 -21 , Ki' magnolia oil = 2.46 × 10 -20 , because Ki' magnolin > Ki magnolin, magnolin is a competitive and non-competitive mixed antagonist of TRPM8; Ki forsythiaside = 5.21 × 10 -21 , Ki ligustilide = 2.47 × 10 -20 , Ki ligustilide > Ki forsythiaside, so the antagonistic effect of ligustilide is stronger than that of forsythiaside.

[0026] The beneficial effects of the present invention are:

[0027] The principle of the present invention is that when the TRPM8 antagonist binds to TRPM8, it will cause Ca 2+ The release of action potentials generates conduction, and the electrical signals are transmitted to a computer through tissue biosensors to simulate the biological body, thereby studying the interaction between TRPM8 and antagonists. Sodium alginate starch glue is used to fix the mouse taste bud tissue membrane on a microporous filter membrane, which is then fixed on a screen-printed electrode to produce a tissue biosensor with a detection concentration as low as 1×10 -22 mol·L -1 , with high sensitivity and specificity, and requiring a small sample volume. Furthermore, the mouse taste bud tissue biosensor can reflect the binding of ligands to receptors. Combined with the kinetics of enzymatic reactions, the reaction kinetic parameters can be obtained by plotting a double reciprocal curve, which can then be used to determine the type of antagonism, providing reliable results in antagonist screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1: Parameter optimization for the construction of mouse taste bud tissue sensors, (A) working fluid type optimization diagram; (B) scanning rate optimization diagram; (C) scanning interval optimization diagram;

[0029] Figure 2 :1×10 -22 -1×10 -4 mol·L -1 Interaction between TRPM8 agonist WS-3 and TRPM8, (A) Current changes of TRPM8 agonist WS-3; (B) Concentration determination of TRPM8 agonist WS-3 solution;

[0030] Figure 3 :1×10 -21 -1×10 -19 mol·L -1 Interaction between TRPM8 agonist WS-3 and TRPM8, (A) hyperbola graph of TRPM8 agonist WS-3; (B) linear fitting graph of TRPM8 agonist WS-3;

[0031] Figure 4 : Interaction between magnolia and TRPM8, (A) hyperbola graph of magnolia; (B) linear fitting graph of magnolia;

[0032] Figure 5 : Interaction between forsythiaside and TRPM8, (A) hyperbola graph of forsythiaside; (B) linear fitting graph of forsythiaside;

[0033] Figure 6 : Interaction between ligustilide and TRPM8, (A) hyperbola graph of ligustilide; (B) linear fitting graph of ligustilide;

[0034] Figure 7 :Interaction between menthol and TRPM8 DETAILED DESCRIPTION

[0035] Example 1: Construction of a mouse taste bud tissue biosensor, the specific steps are as follows:

[0036] (1) Construction of mouse taste bud tissue biosensor:

[0037] Soluble starch was dissolved in a 1%-5% sodium alginate glutaraldehyde solution to prepare a 1%-5% sodium alginate starch glue solution; the sodium alginate starch glue solution was used to fix the mouse taste bud tissue between two microporous filter membranes to form a sandwich-structured biological tissue membrane; the prepared tissue membrane was immersed in a 5% CaCl2 solution for 10 seconds to form a stable chelate, and the biological tissue membrane was fixed to a screen-printed working electrode, completing the construction of the mouse taste bud tissue sensor.

[0038] (2) Parameter optimization of mouse taste bud tissue biosensor:

[0039] The optimized detection method of mouse taste bud tissue sensor parameters was CV method, with the detection voltage range of -0.4-0.7V and 10 -21 mmol·L -1 Capsaicin was used as the test sample. First, the types of working solutions were optimized, including water, PBS, K3[Fe(CN)6] aqueous solution, K3[Fe(CN)6]PBS solution, and KCl-5mmol·L -1 K3[Fe(CN)6] solution, KCl-10mmol·L -1 K3[Fe(CN)6] solution was used as the working fluid for the test. Figure 1 (A) It can be seen that KCl-10mmol·L -1 When K3[Fe(CN)6] solution was used as the working solution, the response current was the largest, so in subsequent experiments, KCl-10mmol·L -1 K3[Fe(CN)6] solution was used as the working solution; secondly, the scanning rates were set to 25, 50, 75, 100, 125, 150, and 175 mV for detection. Figure 1 (B) It can be seen that, considering the scanning interval and sampling conditions, 50mV is selected as the scanning rate; finally, KCl-10mmol·L -1 K3[Fe(CN)6] solution was used as the working solution, the scanning rate was 50mV, and the scanning intervals were set to 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, and 0.10V for testing. Figure 1 (C) It can be seen that taking the scanning rate into consideration, 0.01 V / s is selected as the scanning interval.

[0040] (3) Performance evaluation of mouse taste bud tissue biosensor:

[0041] The current of the same mouse taste bud tissue biosensor was measured for six consecutive days when capsaicin solution of the same concentration was applied. Within the first five days, the fluctuation range was small and tended to be stable, indicating that the mouse taste bud tissue biosensor had good stability. Three mouse taste bud tissue biosensors prepared from different batches were used to test capsaicin solution of the same concentration. The relative standard deviation of the response current change rate was 6.47%, indicating that the mouse taste bud tissue biosensor had good repeatability.

[0042] Example 2 Application of a Mouse Taste Bud Tissue Sensor in Screening TRPM8 Antagonists

[0043] (1) Determination of the concentration of the TRPM8 agonist WS-3 solution:

[0044] Accurately weigh 1.1 mg of TRPM8 agonist WS-3 standard (molecular weight 211.344) in a 5 mL volumetric flask and use 0.1 mol·L -1 KCl and 10 mol·L -1 The mixed solution of K3[Fe(CN)6] was fixed to volume and mixed to obtain a concentration of about 1×10 - 3 mol·L -1 WS-3 standard solution; diluted by 10-fold concentration gradient to obtain 1×10 -22 -1×10 -4 mol·L -1 WS-3 solution; blank solution and WS-3 solutions with increasing concentrations were added to the prepared mouse taste bud tissue biosensor as the reaction device, and the current intensity was recorded using a CHI-660E electrochemical workstation, such as Figure 2 (A) shows that the WS-3 solution concentration (C) is used as the horizontal axis and the current change rate (I0-I) / I0(ΔI) is used as the vertical axis for fitting. The results are shown in Figure 2 As shown in (B), it can be seen from the figure that when the concentration range is 1×10 -21 -1×10 -19 mol·L -1 There is a good linear relationship, and the subdivision concentration is 1×10 -21 , 2×10 -21 , 4×10 -21 , 6×10 -21 , 8×10 -21 , 1×10 -20 , 2×10 -20 , 4×10 -20 , 6×10 -20 , 8×10 -20 , 1×10 -19 mol·L -1 ;

[0045] (2) TRPM8 agonist WS-3 solution interacts with TRPM8 receptors:

[0046] According to the subdivision concentration in step (1) from low to high (1×10 -21 -1×10 -19 mol·L -1 ) WS-3 solution was added to the mouse taste bud tissue biosensor in sequence, and the current intensity was recorded using a CHI-660E electrochemical workstation; the results are shown in FIG. Figure 3As shown in (A), the response current change rate shows a good hyperbolic relationship with the WS-3 concentration, indicating that the interaction between WS-3 and its receptor is similar to the enzymatic reaction kinetics. When the receptor binds to a certain extent with the ligand, there is a ligand saturation effect, similar to the enzyme-substrate interaction. The current first increases and then tends to be stable. The reciprocal of the WS-3 solution concentration The horizontal axis is the inverse of the rate of change of the current. As the vertical coordinate, draw a double reciprocal curve, the linear relationship is good, the result is as follows Figure 3 As shown in (B), the fitting equation is y = 8.811 × 10 -21 x+7.940(R 2 =0.040); According to the formula

[0047]

[0048] Slope is the slope of the double reciprocal curve, and Intercept is the intercept. From this, the dissociation constant Kd value of WS-3 and TRPM8 can be calculated, Kd = 1.11×10 -21 M, Kd is analogous to the Km value of the enzymatic reaction parameter, that is, the ligand concentration when half of the maximum biological effect of receptor saturation is reached. The smaller the Kd value, the higher the biological efficiency of the interaction between the receptor and the ligand.

[0049] (3) The mixed solution of TRPM8 receptor agonist and antagonist interacts with the TRPM8 receptor:

[0050] Add 1×10 -20 mol·L -1 A mixed solution of magnoliaside, a TRPM8 candidate antagonist, was prepared and added to the mouse taste bud tissue biosensor in descending order of concentration. The current intensity was recorded using an electrochemical workstation. The concentration of the mixed solution (C) was used as the horizontal axis and the current change rate (ΔI) was used as the vertical axis. A hyperbola fitting was performed by analogy with the enzymatic reaction kinetics. The results are shown in the figure. Figure 4 As shown in (A), when the receptor binds to the ligand to a certain extent, there is a ligand saturation effect. When it reaches a certain level, the response current no longer changes, so the current first increases and then tends to be stable, similar to the interaction between enzymes and substrates. The horizontal axis is the inverse of the current change rate As the vertical axis, draw a double reciprocal curve and fit the linear equation. The result is as follows Figure 4 As shown in (B), it can be seen from the figure that the linear relationship is good, and the equation is y = 2.776 × 10 -20 x+9.625,R 2=0.983 Calculate the dissociation constant Kd value, Kd = 2.88 × 10 -21 M;

[0051] (4) Similarly, 1×10 -20 mol·L -1 A mixed solution of forsythiaside, ligustilide, and menthol, a candidate TRPM8 antagonist, was prepared. The interaction strength of the mixture of forsythiaside and WS-3 with the TRPM8 receptor, Kd, ​​was determined and calculated to be 3.41×10 -21 M; The interaction strength of the mixture of ligustilide and WS-3 with TRPM8 receptor is Kd=1.54×10 -21 M; The kinetics of the enzymatic reaction of the mixture of menthol and WS-3 showed no ligand saturation effect, and the current showed irregular changes. It could not be compared with the interaction between enzyme and substrate, so it was not a TRPM8 receptor antagonist. The results are shown in Figure 5-Figure 7 .

[0052] (5) After adding TRPM8 antagonist, the main manifestation is the change of dissociation constant Kd and maximum reaction rate Vmax. According to the formula

[0053]

[0054] The Vmax of forsythiaside mixture and ligustilide mixture were 0.132 and 0.124 respectively, and the Vmax of WS-3 solution was 0.126. There was no significant change in the forsythiaside mixture and ligustilide mixture compared with WS-3 solution, while the dissociation constant Kd value increased significantly, which is similar to the competitive inhibition of the enzyme. When the antagonist is added, it can competitively bind to the ligand binding center of the TRPM8 receptor, and the ligand is excluded from the binding center and cannot bind to the receptor, causing the receptor to be inhibited. Therefore, forsythiaside and ligustilide are competitive antagonists of TRPM8; the Vmax of magnolin mixture was 0.104, which was smaller than that of WS-3 solution, while the dissociation constant Kd value increased significantly, which belongs to mixed inhibition. According to the formula

[0055]

[0056]

[0057] C represents the inhibitor concentration used, Ki is the inhibition constant, and Ki' is the apparent inhibition constant.

[0058] Ki 木兰脂素 =4.65×10 -21 , Ki' 木兰脂素 =2.46×10 -20 , because Ki' 木兰脂素 >Ki 木兰脂素Magnoliopsin is a competitive and non-competitive mixed antagonist of TRPM8, which can bind to TRPM8 receptor and the complex of TRPM8 receptor and agonist at the same time; Ki 连翘苷 =5.21×10 -21 , Ki 藁本内酯 =2.47×10 -20 , Ki 藁本内酯 >Ki 连翘苷 Therefore, the antagonistic effect of ligustilide is stronger than that of forsythiaside.

Claims

1. Application of a mouse taste bud tissue biosensor in screening TRPM8 receptor antagonists, characterized in that: The specific steps are as follows: Step 1: Mouse taste bud tissue is placed between two microporous filter membranes and fixed with a 1%-5% sodium alginate starch gel solution to form a sandwich-structured biological tissue membrane. The sodium alginate starch gel solution is prepared by mixing soluble starch with a 1%-5% sodium alginate glutaraldehyde solution. Step 2: After the prepared tissue membrane was immersed in CaCl2 solution, it was fixed on the screen-printed working electrode. The tissue sensor was constructed and the detection parameters were optimized. Capsaicin solution was used as the test sample, CV method was used as the detection method, the detection voltage range was -0.4-0.7V, and the optimized working fluid types were water, PBS, K3[Fe(CN)6] aqueous solution, K3[Fe(CN)6]PBS solution, and KCl-5mmol·L -1 K3[Fe(CN)6] solution, KCl-10mmol·L -1 K3[Fe(CN)6] solution; Step 3: Using the mouse taste bud tissue sensor prepared in step 2, blank solvent and TRPM8 agonist WS-3 solutions with increasing concentrations were added sequentially, and the current intensity between the electrodes was recorded using an electrochemical workstation. Step 4: Using the concentration of the TRPM8 agonist WS-3 solution as the abscissa and the current change rate as the ordinate, a fitting was performed to determine the further subdivided concentration range of the WS-3 solution; a fixed concentration of alternative antagonists, magnolin, ligustilide, or menthol, was added to the subdivided TRPM8 agonist solutions of different concentrations, and a hyperbola fitting was performed using the concentration of the mixed solution as the abscissa and the response current change rate as the ordinate. The kinetic process of the TRPM8 antagonist to the TRPM8 agonist was determined by fitting the kinetic equation; Step 5: Draw a double reciprocal curve with the reciprocal of the concentration of the mixed solution of TRPM8 agonist and antagonist as the horizontal axis and the reciprocal of the response current change rate as the vertical axis, calculate the inhibition constant of the antagonist on the TRPM8 agonist, and determine the antagonism type of the antagonist on the TRPM8 receptor based on the principle of enzymatic kinetics.

2. The use according to claim 1, characterized in that Step 2: Scan rate optimization range was 25–175 mV.

3. The use according to claim 1, characterized in that Step 2: The scanning interval is optimized in the range of 0.001–0.10 V.

4. The use according to claim 1, characterized in that The concentration range of the TRPM8 agonist WS-3 solution in step 3 is 10 -22 -10 -4 mol·L -1 .

5. The use according to claim 1, characterized in that The concentration range of WS-3 solution in step 4 is further divided into 10 -21 -10 -19 mol·L -1 .

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