Flue gas cool feeling intensity rapid detection method based on FLIPR

Abstract

The invention belongs to the technical field of tobacco chemistry, and provides a method for rapidly detecting smoke cool feeling intensity based on FLIPR, which is used for quantitatively evaluating the transmission efficiency and physiological response of cool feeling components in the smoking process. The method mainly comprises five steps of cell detection model construction, smoke sample preparation, cell membrane-smoke collection liquid reaction system construction, FLIPR detection and data analysis. The method overcomes the subjective limitation of a traditional artificial sensory smoke panel test method, avoids the tedious sample pretreatment process of a traditional chemical analysis method, has the advantages of high sensitivity, high repeatability and high flux, can realize rapid, objective and accurate determination of the cool feeling intensity of the cigarette, and has a wide application prospect. And powerful technical support is provided for quality control and accurate blending of tobacco products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of tobacco chemistry and biosensing cross technology, and particularly relates to a FLIPR-based rapid detection method for smoke cooling intensity BACKGROUND

[0002] In recent years, with the continuous improvement of the sensory quality requirements of the tobacco product market, cooling components such as menthol and menthone are increasingly valued as important additives for imparting a cool sensation to cigarettes. These components activate the transient receptor potential channel, such as the TRPM8 receptor, which is specifically expressed on the cell membrane of the oral cavity and respiratory tract, thereby producing a cool stimulation and enhancing the smoking experience of consumers.

[0003] At present, the evaluation of cooling intensity mainly relies on two methods: one is sensory evaluation, in which trained evaluators subjectively score the cigarette samples according to a standardized scoring system; the other is the use of gas chromatography-mass spectrometry (GC-MS) and other instrumental methods to accurately determine the content of cooling components. However, the former is limited by individual differences and environmental factors, has strong subjectivity and poor reproducibility, and also cannot directly quantify the dynamic response characteristics between cooling components and human biological membranes (such as oral mucosal cell membranes); while the latter, although it can accurately detect the concentration of components, cannot directly reflect the actual physiological sensation during smoking, and is often accompanied by complex sample pretreatment, long time consumption and high equipment requirements.

[0004] To solve the above problems, the present application is proposed. SUMMARY

[0005] To overcome the strong subjectivity and poor reproducibility of the artificial sensory evaluation method in the prior art, the present application provides a FLIPR-based rapid detection method for smoke cooling intensity, which is used for quantitatively evaluating the transmission efficiency of cooling components during smoking and their physiological response. The method mainly includes five steps of cell detection model construction, smoke sample preparation, cell membrane-smoke collection liquid reaction system construction, FLIPR detection, and data analysis. Specifically, first, a CHO-TRPM8 cell (Chinese hamster ovary cell) line expressing TRPM8 channels is selected for culture; then a simulated saliva buffer containing cooling components is transferred to a FLIPR detection plate, and the change in intracellular fluorescence intensity is used to obtain the kinetic curve of calcium ion concentration over time; a dose-response curve of cooling agent concentration and ΔF / F0 value is established, and the half-effective concentration EC 50The method can overcome subjective limitations of a traditional artificial sensory evaluation method, avoid a cumbersome sample pretreatment process of a traditional chemical analysis method, and has the advantages of high sensitivity, high repeatability and high throughput, and can realize rapid, objective and accurate determination of the cooling intensity of cigarette smoke, thereby providing strong technical support for quality control and precise deployment of tobacco products.

[0006] To achieve the above object, the specific scheme adopted by the present application is:

[0007] The first aspect of the present application provides a FLIPR-based rapid detection method for smoke cooling intensity, which realizes real-time monitoring of calcium ion concentration changes caused by activation of TRPM8 receptors by a Chinese hamster ovary cell (CHO cell) model stably expressing TRPM8 receptors in combination with a FLIPR platform, and determines the cooling intensity of cigarette smoke by using the value of ΔF / F0 and EC 50 Quantitative evaluation of smoke cooling intensity.

[0008] Preferably, the rapid detection method comprises the following steps:

[0009] Step (1), cell detection model construction:

[0010] The Chinese hamster ovary cell stably expressing TRPM8 receptors is inoculated into each well of a flat-bottom microtiter plate and proliferated to a predetermined confluence under culture conditions, and then the cell culture solution in the flat-bottom microtiter plate is removed, and a certain volume of HEPES buffer physiological saline HBPS buffer containing calcium ion fluorescent dye Fluor-8 and trypan red is added for fluorescence staining;

[0011] Step (2), smoke sample preparation:

[0012] Each cigarette is smoked according to the GB / T 16450-2004 standard (smoking volume 35 mL / time, smoking time 2 seconds, interval 60 seconds), and the test cigarette containing a cooling agent is smoked by using an RM20H rotary disc type smoking machine, so as to capture the mainstream smoke. After the smoke passes through a cold trap or an adsorption tube, the condensate is collected and dissolved in a pre-prepared simulated saliva buffer to form an exposure liquid containing a cooling component;

[0013] Step (3), cell membrane-smoke collection liquid reaction system construction:

[0014] The exposure liquid containing the cooling component obtained in step (2) is added into each well of the flat-bottom microtiter plate in step (1), so that the cooling component can fully contact the TRPM8 receptors expressed on the cell membrane, and a cell membrane-smoke collection liquid reaction system is constructed;

[0015] Step (4), FLIPR detection:

[0016] The cell membrane-smoke collection liquid reaction system constructed in step (3) is placed on the FLIPR detection platform, and the fluorescence signal generated by the change in the intracellular calcium ion concentration is recorded in real time at the preset excitation / emission wavelength;

[0017] Step (5), data analysis:

[0018] The FLIPR Screen Works 3.1 software is used to perform background correction and normalization processing on the original fluorescence signal recorded in step (4), the baseline fluorescence F0 of the cells in each well before stimulation is taken as a reference, then the maximum fluorescence intensity change value ΔF / F0 reached in each well during the reaction is extracted as a quantitative index of the cooling intensity, based on the ΔF / F0 values at different concentrations, a dose-response curve can be drawn, and the half effective concentration EC50 can be further calculated. 50 , realizing the relative quantitative analysis of the cooling intensity of the mainstream smoke of cigarettes.

[0019] The main operation process of the FLIPR Screen Works 3.1 software is as follows:

[0020] (1) fluorescence data acquisition;

[0021] (2) the original fluorescence data is exported as a time-fluorescence intensity curve (F(t));

[0022] (3) baseline correction: taking the average fluorescence value in the 10 seconds before stimulation as F0;

[0023] (4) ΔF / F0 calculation: ΔF / F0=(F-F0) / F0, where F is the fluorescence intensity after stimulation;

[0024] (5) the built-in formula ΔF / F0=(F-F0) / F0 automatically generates a dose-response curve and automatically calculates the EC50 value.

[0025] If the Fluo-3 probe is enhanced by 60-100 times, the signal dynamic range ΔF / F0 value meets the expected dynamic range of the probe, which indicates that the receptor is stably expressed and functions normally.

[0026] Preferably, the inoculation concentration of the Chinese hamster ovary cells in step (1) is 2.0×10 4 ~ 4.0×10 4 cells / well, the culture medium is DMEM, and the culture conditions are 37°C, 5% CO2 and 95% relative humidity for 18-24h until the cell confluence is more than 90%.

[0027] Preferably, the mass percentage of calcium ion fluorescent dye Fluor-8 30mmol / L, conchiolin 10mmol / L and HEPES buffer physiological saline in the HBPS buffer in step (1) is 0.5%.

[0028] The HEPES buffer physiological saline comprises: 10g / L HEPES; 16g / L NaCl; 0.74g / L KCl; 0.27g / L Na2HPO4.2H2O; 2.0g / L dextrose.

[0029] Preferably, the exposure solution in step (2) is prepared by dissolving 10-20 cigarette smoke condensates in 10mL simulated saliva buffer, and the pH value of the simulated saliva buffer used is 6.8-7.2; the simulated saliva buffer is a phosphate buffer system, and the specific formula is sodium dihydrogen phosphate 1.74g / L and disodium hydrogen phosphate 2.7g / L.

[0030] Preferably, the exposure solution containing the cooling component is added in a flat-bottom microtiter plate at an amount of 4-10μL / well, and the concentration of the exposure solution of the cooling component is set to 0.1-100μg / mL.

[0031] Preferably, the detection parameter of FLIPR in step (4) is: Fluo-3 calcium ion probe, excitation wavelength 470-495nm, and emission wavelength 515-575nm.

[0032] Preferably, the normalization method in step (5) is a min-max standardization method.

[0033] The calculation formula of the dose ΔF / F0 of the maximum fluorescence intensity change value is:

[0034] ΔF / F0=(F-F0) / F0, wherein F0 is the initial fluorescence intensity, and F is the fluorescence intensity after treatment.

[0035] The formula of the cooling agent concentration-maximum fluorescence intensity change value dose ΔF / F0-reaction curve is:

[0036] ΔF / F0==K·C, wherein K is a proportional constant, and the value range of K is K>0, and C is the cooling agent concentration.

[0037] The second aspect of the present application provides an application of the method in the first aspect in the rapid detection of the cooling intensity of cigarette smoke.

[0038] The present application has the following beneficial effects:

[0039] 1、The application realizes the objective quantification of the effect of the cooling component in the smoke by constructing a cell detection model stably expressing the TRPM8 receptor and using the FLIPR platform to monitor the change in intracellular calcium ion concentration in real time, avoids the strong subjectivity and poor repeatability of the traditional sensory evaluation method, and compared with the traditional complex chemical analysis method such as gas chromatography-mass spectrometry, the sample pretreatment process is simplified, and the detection difficulty and time cost are reduced.

[0040] 2、The detection method of the application can quickly establish the dose-response curve of the cooling agent concentration and the cell response (ΔF / F0), and calculate the EC 50 value, which is suitable for the detection of a large number of samples, and the method has high sensitivity and good repeatability, is suitable for the detection of a large number of samples, and is conducive to the precise management of tobacco product quality control and product blending.

[0041] 3、The application uses the TRPM8 receptor expressed on the cell membrane to simulate the response of the human body to the cooling component, so that the detection result can better reflect the physiological actual feeling in the smoking process, and provides more consumer experience technical support for tobacco product research and improvement. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 For the specific embodiment, the sensory evaluation results columnar chart of the cooling intensity of the mainstream smoke of a single cigarette containing different cooling agents. DETAILED DESCRIPTION

[0043] The application will be further described below by examples, but is not limited to the examples.

[0044] The experimental methods not specified in the examples are usually carried out according to the conventional conditions and the conditions described in the manual, or the general equipment, materials, reagents, etc. used according to the conditions suggested by the manufacturer, unless otherwise specified.

[0045] Example 1

[0046] (1) Cell detection model construction: the CHO cells expressing the TRPM-8 receptor clone were inoculated in a 384-well flat-bottom microtiter plate at a concentration of 2.0 x 10 4 cells / well, and were cultured in DMEM medium at 37℃, 5% CO2 and 95% relative humidity to a confluence of 95% overnight;

[0047] The culture medium was removed, 30 μL of HEPES buffer physiological saline (HBPS) solution containing 5 μL of Fluor-8 and 5 μL of trypan red (10 mmol / L) was added to replace 10 min, and then fluorescence staining was carried out for 30 min.

[0048] The HBPS buffer formula is as follows: 10 g / L HEPES; 16 g / L NaCl; 0.74 g / L KCl; 0.27 g / L Na2HPO4.2H2O; 2.0 g / L dextrose, and incubation is performed at 37°C in the dark for 20 min.

[0049] (2) Preparation of flue gas sample:

[0050] According to GB / T 16450-2004, the test cigarettes containing L-menthol are smoked by using RM20H smoking machine at a smoking volume of 35 mL / time, 2 seconds of smoking, and 60 seconds of interval, and then the mainstream smoke condensate is collected by a cold trap. A total of 10 cigarettes are smoked, and the 10 cigarette condensates are dissolved in 10 mL of a simulated saliva buffer with a pH of 6.8. After mixing, the mixture is filtered by a 0.22 μm filter membrane to obtain an exposure solution. The exposure solution is simultaneously measured by GC-MS to obtain a measured value of 12.5 μg / mL.

[0051] (3) Construction of cell membrane-flue gas collection liquid reaction system: the exposure solution containing the cooling component collected in (2) is added to each well of a flat-bottom microtiter plate to construct a cell membrane-flue gas collection liquid reaction system.

[0052] (4) FLIPR detection: the cell membrane-flue gas collection liquid reaction system prepared in step (3) at a concentration of 10 cigarettes / 10 mL of buffer is diluted in gradient to prepare a series of concentrations of 0.1-100 μg / mL (wherein the dilution solvent is anhydrous ethanol), and 5 μL of each solution is added to each well of a flat-bottom microtiter plate. After being balanced at 37°C for 5 min, the fluorescence change value is recorded by FLIPR at an excitation / emission wavelength of 470 / 515 nm, and the FLIPR Screen Works 3.1 software is used for analysis.

[0053] (5) Data analysis: the original fluorescence signal recorded in (4) is subjected to background correction and min-max standardization normalization processing, and then the maximum fluorescence intensity change value (ΔF / F0) is extracted as a quantitative index of cooling intensity, so as to calculate the relative quantitative analysis of cooling intensity.

[0054] The concentration EC50 of the mainstream smoke sample containing L-menthol is calculated by the change in fluorescence intensity, and the value is 8.9 μg / mL. The detection limit of the method for the mainstream smoke sample containing L-menthol is 0.08 μg / mL. 50

[0055] In this embodiment, the preparation method of the test cigarette containing L-menthol is a laboratory method. Specifically, 6.5 g of L-menthol is diluted with anhydrous ethanol to 10 mL, and then added to tobacco according to the perfuming mode. Then, the tobacco is further prepared into a cigarette for testing.​

[0056] The same detection method was used to test the cigarette test samples containing WS-5, menthone, WS-3, WS-23, and menthyl acetate, and the following Table 1 test results were obtained:

[0057] Table 1 Test results of test cigarettes containing different cooling agents

[0058]

[0059] Example 2

[0060] (1) Cell detection model construction: CHO cells expressing cloned TRPM-8 receptors were seeded at a concentration of 4.0 x 10 4 The cells were seeded in 384-well flat-bottom microtiter plates at a concentration of 4.0 x 10

[0061] The culture medium was removed, and 30 μL of a HEPES buffer physiological saline (HBPS) solution containing 5 μL of Fluor-8 and 5 μL of trypan red (10 mmol / L) was added to replace it for 10 min, followed by fluorescence staining for 30 min.

[0062] The HBPS buffer formula is: 10 g / L HEPES; 16 g / L NaCl; 0.74 g / L KCl; 0.27 g / L Na2HPO4.2H2O; 2.0 g / L dextrose, and incubated at 37°C for 20 min in the dark.

[0063] (2) Preparation of smoke samples:

[0064] According to the GB / T 16450-2004 standard, RM20H smoking machine was used to smoke test cigarettes containing L-menthol according to the smoking volume of 35 mL / time, 2 seconds of smoking, and 60 seconds of interval, and then the mainstream smoke condensate was collected by a cold trap. A total of 5 cigarettes were smoked, and the 5 cigarette condensates were dissolved in 10 mL of pH 7.0 simulated saliva buffer, mixed, and then filtered through a 0.22 μm filter membrane to obtain an exposure solution. The exposure solution was simultaneously measured by GC-MS, and the actual measured value by GC-MS was 6.5 μg / mL.

[0065] (3) Cell membrane-smoke collection liquid reaction system construction: the exposure liquid containing cooling components collected in (2) was added to each well of the flat-bottom microtiter plate to construct a cell membrane-smoke collection liquid reaction system.

[0066] (4) FLIPR detection: the concentration of 10 cells / 10 mL buffer cell membrane-smoke collection solution reaction system prepared in step (3) was prepared into a series of concentrations of 0.1-100 μg / mL (wherein the dilution solvent was anhydrous ethanol) by gradient dilution method, 5 μL was added to each well of a flat-bottom microtiter plate, 5 min was balanced at 37°C, then the fluorescence change value was recorded by FLIPR at 480 / 540 nm excitation / emission wavelength, and FLIPR Screen Works 3.1 software was used for analysis.

[0067] (5) Data analysis: the original fluorescence signal recorded in (4) was subjected to background correction and min-max standardization normalization processing, then the maximum fluorescence intensity change value (ΔF / F0) was extracted as the quantitative index of cooling intensity, so as to calculate the relative quantitative analysis of cooling intensity.

[0068] By calculating the fluorescence intensity change, the concentration EC50 value of the L-menthol-containing mainstream smoke sample when the fluorescence intensity response was 50% was 8.5 μg / mL, and the detection limit of the L-menthol-containing mainstream smoke sample by the method was 0.09 μg / mL.

[0069] The preparation method of the test cigarette containing L-menthol in the embodiment adopts a laboratory method, specifically, 6.5 g of L-menthol is diluted with anhydrous ethanol to 10 mL, then added to the tobacco according to the flavoring method, and then further prepared into cigarettes for testing.

[0070] At the same time, the same detection method is used to test the cigarette test samples containing WS-5, menthone, WS-3, WS-23, and menthyl acetate, and the following Table 2 test results can be obtained:

[0071] Table 2 Test results of test cigarettes containing different cooling agents

[0072]

[0073] Example 3

[0074] (1) Construction of cell detection model: CHO cells expressing cloned TRPM-8 receptors were seeded at a concentration of 3.0×10 4 The cells were seeded in a 384-well flat-bottom microtiter plate at a concentration of 3.0×10

[0075] The culture medium was removed, 30 μL of HEPES buffer physiological saline (HBPS) solution containing 5 μL of Fluor-8 and 5 μL of trypan red (10 mmol / L) was added to replace 10 min, and then fluorescence staining was performed for 30 min.

[0076] The HBPS buffer formula is as follows: 10 g / L HEPES; 16 g / L NaCl; 0.74 g / L KCl; 0.27 g / L Na2HPO4.2H2O; 2.0 g / L dextrose, and incubation is performed at 37°C in the dark for 20 min.

[0077] (2) Preparation of smoke samples:

[0078] According to GB / T 16450-2004, the cigarettes are smoked by using an RM20H smoking machine at a puff volume of 35 mL / puff, 2 seconds of puffing, and 60 seconds of interval, and then the mainstream smoke condensate is collected by a cold trap. A total of 10 cigarettes are smoked, and the 10 cigarette condensates are dissolved in 10 mL of pH 7.2 simulated saliva buffer. After mixing, the mixture is filtered by a 0.22 μm filter membrane to obtain an exposure solution. The exposure solution is simultaneously measured by GC-MS to obtain a GC-MS measured value of 7.2 μg / mL.

[0079] (3) Construction of a cell membrane-smoke collection liquid reaction system: The exposure solution containing the cooling components collected in (2) is added to each well of a flat-bottom microtiter plate to construct a cell membrane-smoke collection liquid reaction system.

[0080] (4) FLIPR detection: The cell membrane-smoke collection liquid reaction system prepared in step (3) at a concentration of 10 cigarettes / 10 mL of buffer is diluted in gradient to prepare a series of concentrations of 0.1-100 μg / mL (wherein the dilution solvent is anhydrous ethanol). 5 μL of each solution is added to each well of a flat-bottom microtiter plate, and the plate is equilibrated at 37°C for 5 min. Then, the fluorescence change value is recorded by FLIPR at an excitation / emission wavelength of 480 / 540 nm, and the FLIPR Screen Works 3.1 software is used for analysis.

[0081] (5) Data analysis: The raw fluorescence signal recorded in (4) is subjected to background correction and min-max standardization normalization processing, and then the maximum fluorescence intensity change value (ΔF / F0) is extracted as a quantitative index of the cooling intensity, so as to calculate the relative quantitative analysis of the cooling intensity.

[0082] Through the fluorescence intensity change, the concentration EC50 value of the mainstream smoke sample containing L-menthol at which the fluorescence intensity response is 50% is calculated to be 9.1 μg / mL. The detection limit of the method for the mainstream smoke sample containing L-menthol is 0.08 μg / mL.

[0083] In this embodiment, the preparation method of the test cigarette containing L-menthol uses a laboratory method. Specifically, 6.5 g of L-menthol is diluted with anhydrous ethanol to 10 mL, and then added to the tobacco according to the perfuming method. Then, the cigarette is further prepared into a cigarette for testing.

[0084] The same test method was used to test cigarette samples containing WS-5, menthone, WS-3, WS-23, and menthyl acetate, and the results are shown in Table 3 below.

[0085] Table 3 Test results of test cigarettes containing different cooling agents

[0086]

[0087]

[0088] Test Example 1

[0089] In this test example, 8 volunteers were recruited by the Jiangnan University Food Resources and Mining State Key Laboratory as a taste panel, and the taste test was performed on the cigarette samples of Examples 1 to 3 simultaneously according to the Q Grader standard.

[0090] The taste test scheme included each volunteer gargling with 10 mL of 1% (fructose, glucose, or sucrose) aqueous solution containing different concentrations of cooling agents for 10 s, then spitting out, recording their sensory results on a sensory evaluation form, and taking a sufficient rest between each cooling agent test.

[0091] The cooling intensity score of the cooling agent was divided into 0-9 levels. 0 was the least intense feeling, and 9 was the most intense feeling immediately after gargling with the cooling agent for 10 s and spitting it out and 2 min after spitting out the cooling agent.

[0092] Before evaluating the test cooling agent and the calibration panel, each panel member gargled with 10 mL of 1% (fructose, glucose, or sucrose) solution containing 10 μg / mL of L-menthol for 10 s, spit it out, and set the cooling intensity of the solution to 5. All subsequent sensory analyses of the cooling agents were rated as values equivalent to the 10 μg / mL L-menthol calibration value. The volunteers rested for 10 min after each evaluation. During the rest period, the volunteers ate unsalted biscuits and water to remove residual cooling agents. All volunteers evaluated different samples randomly to avoid any order effect.

[0093] As can be seen from the above, the order of cooling intensity evaluated by artificial sensory evaluation and the cell membrane method using the TRPM-8 channel was consistent, and was: WS-5 (5-point control value) > WS-3 > L-menthol > menthone > WS-23 > menthyl acetate. The results of the two methods were highly consistent, verifying the reliability and effectiveness of the TRPM-8 channel cell membrane method in cooling intensity evaluation.

[0094] As can be seen from the above, the order of the cooling intensity is consistent between the artificial sensory evaluation and the cell membrane method using TRPM-8 channel, and is WS-5 (5 points control value) > WS-3 > L-menthol > menthone > WS-23 > menthyl acetate, the results of the two methods are highly consistent, verifying the reliability and effectiveness of the TRPM-8 channel cell membrane method in the evaluation of cooling intensity.

[0095] Meanwhile, the test results of examples 1-3 show that EC 50 The trend of the corresponding GC-MS test value results is also highly close, but the detection limit can be as low as 0.05 μg / mL, and it can be seen that the test method of the present application has high sensitivity.

[0096] It should be understood that, after reading the above content of the present application, those skilled in the art can make various modifications or modifications to the present application, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. A FLIPR-based rapid detection method for the cooling intensity of smoke, characterized in that: The rapid detection method uses a Chinese hamster ovary cell model that stably expresses TRPM8 receptors and combines the FLIPR platform to monitor in real time the changes in calcium ion concentration caused by the activation of TRPM8 receptors by cooling ingredients, and measures the changes in calcium ion concentration by ΔF / F0 value and EC 50 Quantitatively evaluate the cooling intensity of smoke.

2. The method for rapid detection of cooling intensity of smoke based on FLIPR according to claim 1, characterized in that: The rapid detection method comprises the following steps: Step (1), construction of cell detection model: Chinese hamster ovary cells stably expressing the TRPM8 receptor were seeded into each well of a flat-bottom microtiter plate and proliferated under culture conditions to a predetermined degree of confluency. The cell culture medium in the flat-bottom microtiter plate was then removed and a certain volume of HEPES-buffered saline (HBPS) buffer containing the calcium ion fluorescent dye Fluor-8 and trypan red was added for fluorescent staining. Step (2), flue gas sample preparation: Each cigarette was smoked according to GB / T 16450-2004. The test cigarettes containing cooling components were smoked using an RM20H rotary smoking machine to capture mainstream smoke. After the smoke passed through a cold trap or adsorption tube, the condensate was collected and dissolved in a pre-prepared simulated saliva buffer solution to form an exposure solution containing cooling components. Step (3), construction of cell membrane-smoke collection liquid reaction system: The exposure solution containing the cooling component obtained in step (2) is added to each well of the flat-bottom microtiter plate in step (1), so that the cooling component can fully contact the TRPM8 receptor expressed on the cell membrane, thereby constructing a cell membrane-smoke collection solution reaction system; Step (4), FLIPR detection: The cell membrane-smoke collection liquid reaction system constructed in step (3) is placed on a FLIPR detection platform, and the fluorescence signal generated by the change of intracellular calcium ion concentration is recorded in real time at a preset excitation / emission wavelength; Step (5), data analysis: The raw fluorescence signals recorded in step (4) were background corrected and normalized using FLIPR Screen Works 3.1 software. The baseline fluorescence F0 of the cells in each well before stimulation was used as a reference. Subsequently, the maximum fluorescence intensity change value ΔF / F0 achieved in each well during the reaction was extracted as a quantitative indicator of the cooling intensity. Based on the ΔF / F0 values ​​at different concentrations, a dose-response curve was drawn, and the half-effective concentration EC was further calculated. 50 , to achieve relative quantitative analysis of the cooling intensity of mainstream cigarette smoke.

3. The method for rapid detection of cooling intensity of smoke based on FLIPR according to claim 1, characterized in that: The inoculation concentration of Chinese hamster ovary cells in step (1) is 2.0×10 4 ~4.0×10 4 cells / well, the culture medium is DMEM, and the culture conditions are 37°C, 5% CO2 and 95% relative humidity for 18-24 hours until the cell confluence reaches more than 90%.

4. The method for rapid detection of cooling intensity of smoke based on FLIPR according to claim 1, characterized in that: In the step (1), the mass percentage of the calcium ion fluorescent dye Fluor-8 30 μg / ml, trypan red 10 μg / ml, and HEPES buffered saline in the HBPS buffer is 0.5%; The HEPES buffered saline contains: 10 g / L HEPES; 16 g / L NaCl; 0.74 g / L KCl; 0.27 g / L Na2HPO4.2H2O; and 2.0 g / L dextrose.

5. The method for rapid detection of cooling intensity of smoke based on FLIPR according to claim 1, characterized in that: In step (2), the exposure solution is prepared at a ratio of 10-20 cigarette smoke condensates dissolved in every 10 mL of simulated saliva buffer, and the pH value of the simulated saliva buffer used is 6.8-7.2; the simulated saliva buffer formula is a phosphate buffer system, specifically 1.74 g / L sodium dihydrogen phosphate and 2.7 g / L disodium hydrogen phosphate.

6. The method for rapid detection of cooling intensity of smoke based on FLIPR according to claim 1, characterized in that: The exposure solution containing the cooling component is added to a flat-bottom microtiter plate at an amount of 4 to 10 μL / well, and the concentration of the exposure solution containing the cooling component is set to 0.1 to 100 μg / mL.

7. The method for rapid detection of cooling intensity of smoke based on FLIPR according to claim 1, characterized in that: The detection parameters of FLIPR in step (4) are: Fluo-3 calcium ion probe, excitation wavelength 470-495 nm, emission wavelength 515-575 nm.

8. The method for rapid detection of cooling intensity of smoke based on FLIPR according to claim 1, characterized in that: The normalization method in step (5) is a min-max normalization method; The formula for calculating the dose of the maximum fluorescence intensity change value ΔF / F0 is: ΔF / F0=(F-F0) / F0, where F0 is the initial fluorescence intensity and F is the fluorescence intensity after treatment The linear equation formula of the cooling agent concentration-maximum fluorescence intensity change value dose ΔF / F0-response is: ΔF / F0==K·C, where K is a proportional constant, the value range is K>0, and C is the concentration of the cooling agent.

9. Use of the method according to any one of claims 1 to 8 in the rapid detection of the cooling intensity of cigarette smoke.

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