CN109142298B - Quantitative determination method for sweetness at cell level - Google Patents
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Abstract
The invention provides a method for quantitatively measuring sweetness at a cell level, which comprises the steps of intracellular calcium ion fluorescent staining, laser confocal scanning, microscopic measurement of intracellular calcium ion concentration change and the like. The method establishes a method for researching the change of the concentration of the calcium ions in the sweet taste receptor cells by using a fluorescence detection technology, can quantify the perception degree of human to the sweet taste of other unknown samples under the condition of in vitro, and effectively avoids the influence of human subjective factors. The method can be used for rapidly, efficiently and sensitively detecting the sweetness of the compound, and has a good application prospect.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a quantitative determination method for sweetness at a cell level.
Background
Human taste can be classified into five main tastes, namely sour taste, sweet taste, bitter taste, delicate flavor and salty flavor. Most mammals prefer sweetness, which is generally predictive of more energy, while bitter foods are predictive of possible harm to the body. Mammalian sweet receptor cells are distributed primarily on taste bud cells of the tongue and palate epidermis. The taste signaling pathways differ in each cell that senses a different taste.
The T1R gene family can code three conserved GPCRs, namely T1R1, T1R2 and T1R3, and T1R2 and T1R3 are combined to form a heterodimer which can be used as a sweet taste receptor; T1R1 and T1R3 combine to form a heterodimer, which can act as an umami receptor. These taste ligands interact with taste receptors, activating a cascade of signals that lead to neurotransmitter release, followed by neural fibers in the cranial nerve center that transmit the signal from the hypothalamus into the center of the taste cortex, which processes and integrates the collected signal to produce the corresponding taste sensation.
Sweet taste compounds are diverse, including sugars, artificial sweeteners, thaumatin, amino acids, sweet taste signals are mainly sensed by T1R2/T1R3 dimer receptors on type II taste bud cells, sweet taste signal molecules include simple carbohydrates (mono and polysaccharides), D-type amino acids (majority) and L-type amino acids (portion), artificial sweeteners, etc. Related researches prove that signal molecules involved in sweet taste signal transduction comprise alpha-gustducin, PLC beta 2, IP3R, TRPM5 and the like, and the current researches confirm that molecular signal paths caused by stimulation of sweet taste receptors by natural sweet substances and artificial sweeteners are different.
Binding of sweet compounds to sweet receptors activates mainly two signaling pathways. After the natural sweet substance is combined with a sweet receptor, a downstream G protein is activated, Adenylate Cyclase (AC) is further activated to hydrolyze ATP, cAMP is generated, a cNMP-gated channel is opened, calcium ion inflow is caused, the concentration of calcium ions in cells is increased, cAMP can also activate PKA, potassium ion channel phosphorylation outside a small base is caused, and therefore the ion channel is closed, potassium ion outflow is inhibited, cell membrane depolarization is caused, voltage-dependent calcium ion inflow is caused, and the concentration of calcium ions is increased. After the artificial sweet substance is combined with the sweet taste receptor, the downstream G protein is activated, the phospholipase C (PLC) is activated, IP3 and DAG are generated, IP3 can be combined with IP3R on the endoplasmic reticulum membrane, and the calcium ion in the endoplasmic reticulum is released, and the calcium ion concentration in cells is increased. Activation of both pathways results in an increase in intracellular calcium ion concentration.
Calcium ion, as a major intracellular messenger, participates in and regulates a variety of physiological activities. Cells have a tight mechanism to control the balance of intracellular calcium ions, one is dependent on the calcium pump and calcium channels of the cytoplasmic membrane, and the other is dependent on intracellular calcium stores such as endoplasmic reticulum and mitochondria. In animal and plant cells, calcium ions exist mainly in three forms: 1. binding with cell structural components or macromolecular substances to form bound calcium; 2. storing in intracellular calcium stores such as endoplasmic reticulum and mitochondria to store calcium; 3. in the form of ions, i.e. free calcium. The intracellular storage calcium and free calcium can be converted into each other and in static stateThe concentration of free calcium in the cells is very low, about 10-8-10-7And M. When stimulated, the free calcium in the cell cytoplasm rises rapidly, up to 10-5M。
Fluo-4AM is an acetyl methyl ester derivative of Fluo-4, a fluorescent dye capable of penetrating cell membranes and having the molecular formula C51H50F2N2O23The molecular weight is: 1096.95, the structural formula is:
fluo-4AM has very weak fluorescence, and the fluorescence thereof does not increase with the increase of the intracellular calcium ion concentration. Fluo-4AM can be cleaved by intracellular esterase to form Fluo-4 after entering cells, so that the Fluo-4AM is retained in the cells. Fluo-4 can be combined with free calcium ions, and can generate stronger fluorescence after being combined with the calcium ions, the maximum excitation wavelength is 494nm, and the maximum emission wavelength is 516 nm. The recommended laser excitation wavelength for practical application of laser scanning confocal microscope detection is about 488nm, and the emission wavelength detection range is 512-520 nm.
Sweetness, in a sense, is an index reflecting the evaluation of human senses on food taste, and in short, is characterized by the degree of sweetness of a compound or a food. In daily life, people usually evaluate sweetness by the sense of the tongue. In scientific research, various chemical reagents, analytical instruments and the like are mainly used for detecting the sweetness of a sample, but the instruments can only detect the content of sweet soluble solids in food, but not the actual sweetness of the sample. We therefore generally quantify the sweetness of these foods or complex compounds based on the amount of these individual sweet substances and the sweetness of the individual sweet substances.
The existing methods for detecting sweetness mainly comprise two main types, namely an analytical chemical detection method and a biological detection method. Wherein the analytical chemical detection method mainly comprises high performance liquid chromatography, electronic tongue detection method, saccharimeter detection method, and spectrophotometer detection method; the biological method comprises artificial evaluation and the like. The content of sugar can be accurately detected by an analytical chemical method, but the sweetness cannot be quantified at a physiological level; although the biological detection method can quantify the sweetness at the physiological level, the artificial evaluation has strong subjectivity and different human tastes, and the sweetness cannot be quantified accurately. At present, a standard method for objectively quantifying the sweetness of a substance at a physiological level does not exist, and the establishment of a method for quantifying the sweetness of a compound at a physiological level is very important for the development work of foods and sweeteners.
Disclosure of Invention
The invention provides a method for detecting the change of the calcium ion concentration in a single cell based on a laser scanning confocal microscope, thereby quantifying the sweetness on a physiological level.
The cell lines used by the invention are two, namely an HEK293 cell line and an HEK293-T1R2/T1R3 cell line, so that the measurement difference caused by cell background reaction is fully eliminated; please refer to patent 201810425704.1 for the HEK293-T1R2/T1R3 cell line and the preparation method thereof.
In order to achieve the above object, the present invention provides a method for detecting the concentration of calcium ions in the cytoplasm of a cell stably expressing a sweet taste receptor.
A method for the quantitative determination of sweetness at the cellular level comprising the steps of:
(1) preparing suspension cells of HEK293 cells and HEK293-T1R2/T1R3 cells which are cultured in an adherent manner according to a normal passage step, and respectively planting the suspension cells in a confocal culture dish;
(2) when the confluence degree of HEK293 cells and HEK293-T1R2/T1R3 cells subjected to adherent culture in the confocal culture dish in the step (1) reaches 40% -60%, incubating the cells planted in the confocal culture dish by using a calcium ion fluorescent probe solution;
(3) continuously observing and recording the cell fluorescence image after incubation in the step (2) to 120s by using a laser scanning confocal microscope, and respectively obtaining the fluorescence intensity value of each time point of single cells of the HEK293 cell and the HEK293-T1R2/T1R3 cell within 120 s; setting the minimum fluorescence value within the first 16s as Fo and setting the maximum fluorescence value within the range from 16s to 120s as Fm; wherein at 16s, a sweet sample solution to be tested is added;
(4) calculating fluorescence intensity change ratio values of HEK293 cells and HEK293-T1R2/T1R3 cells by using delta F/Fo, wherein the delta F is Fm-Fo;
(5) the value obtained by subtracting the change ratio delta F/Fo of the fluorescence intensity of HEK293 cells from the change ratio delta F/Fo of the fluorescence intensity of HEK293-T1R2/T1R3 cells is used as delta R;
(6) setting the sweetness of a sucrose solution under a certain known concentration to be 1, and setting the value of F/Fo to be Delta Rs; and (4) the ratio of the Delta R value to the Delta Rs obtained in the step (5) is the relative sweetness of the sweet sample solution to be detected.
Preferably, the confocal culture dish in the step (1) is a cell culture dish suitable for oil-lens observation on a laser confocal microscope.
Preferably, the specification of the confocal culture dish is 35mm in outer diameter and 10mm in inner diameter; the confocal dish was incubated with 25. mu.g/ml polylysine solution at 37 ℃ for 30min before use.
Preferably, the calcium ion fluorescent probe in the step (2) is Fluo-4AM, and the concentration thereof in Flex buffer is 10 μ M.
Preferably, the Flex buffer comprises the components as shown in the following table:
| reagent | Volume (mL) |
| HBSS solution | 47.5 |
| HEPES solution | 1 |
| 1M MgSO4 | 0.05 |
| 1M Na2CO3 | 0.165 |
| 1M CaCl2 | 0.065 |
| 10% BSA bovine serum albumin | 0.5 |
| 250mM Probenecid | 0.5 |
| Total of | 50 |
The HBSS solution, namely Hanks balanced salt solution, is a commercial Thermo company product, and the specific components are shown in the following table:
| composition (I) | Concentration (mg/L) | Concentration (mM) |
| CaCl2(Anhydrous) | 140 | 1.261261 |
| MgCl2·6H2O | 100 | 0.492611 |
| MgSO4·7H2O | 100 | 0.406504 |
| KCl | 400 | 5.333334 |
| KH2PO4 | 60 | 0.441176 |
| NaHCO3 | 350 | 4.166667 |
| NaCl | 8000 | 137.931 |
| Na2HPO4(Anhydrous) | 48 | 0.338028 |
| D-Glucose | 1000 | 5.555555 |
HEPES is N-2-hydroxyethyl piperazine-N-2-ethane sulfonic acid, the HEPES solution is a commercial Thermo company product, the HEPES concentration is 1M, and the pH value is 7.2-7.5; the Probenecid is Probenecid.
Preferably, the pH of the Flex buffer is 7.4.
Preferably, the incubation conditions of the cells planted in the confocal culture dish with the calcium ion fluorescent probe solution in the step (2) are as follows: incubating for 45-60min at 37 ℃ in a dark place, so that the area of adherent cells covering the bottom of the culture dish reaches 40-60%.
Preferably, the laser scanning confocal microscope in the step (3) is FV1000 type; the shooting parameters are set as follows: an objective lens is a 60-time oil lens, a laser tube is a neon ion laser, the excitation wavelength is 488nm, the emission wavelength range is 500nm-600nm, the scanning speed is 8 mus/Pixel, the Pixel is 640x640, the cell is subjected to nondestructive continuous scanning photographing by adopting an XYT scanning program, scanning is carried out for 4min at room temperature under the condition of light shielding, and at least 30 fluorescence pictures with the total frame number are obtained; then, the obtained continuous observation fluorescence images are used for carrying out fluorescence quantification on single cells on the fluorescence images by using ImageJ software, and fluorescence values of the single cells within 120s are obtained.
Preferably, the concentration of sucrose solution having a sweetness of 1 at a certain known concentration in step (6) is 150 mM.
A method for quantitatively measuring sweetness at a cellular level specifically comprises the following steps:
(1) preparing suspension cells from the HEK293 cells and the HEK293-T1R2/T1R3 cells which are cultured in an adherent manner according to a normal passage step; removing culture solution from HEK293 cells and HEK293-T1R2/T1R3 cells which are fully paved on a culture dish, washing the cells twice by using 5mL phosphate buffer solution (10mM, pH 7.4), digesting the cells by using 1mL of 0.25% Trypsin (Trypsin) solution for 2min, adding fresh culture solution to stop the cell digestion, transferring the cell digestion solution into a 15mL centrifuge tube, centrifuging the cell digestion solution for 5min by 180g, and removing supernatant; adding 1ml of fresh culture solution into the lower-layer sediment to prepare suspension cells, and uniformly mixing to obtain a cell suspension;
(2) adding 990 μ l of fresh culture solution into another 1.5ml EP centrifuge tube, adding 10 μ l of cell suspension obtained in step (1), and mixing;
(3) taking out 10 mu l of the cell suspension prepared in the step (2), adding the cell suspension into a blood counting plate with 25 grids multiplied by 16 grids, counting under an optical microscope, and determining the cell concentration; diluting the cell suspension obtained in the step (1) according to the measured cell concentration to prepare a working concentration of 105cell/ml cell suspension; taking 1ml of the extract with a concentration of about 105Adding the cell suspension of cells/ml into a 35mm confocal culture dish, and placing the culture dish in a constant-temperature cell culture box at 37 ℃ for culture;
the invention uses a blood counting chamber with 25 grids multiplied by 16 grids, and the calculation formula of the cell concentration is as follows: the number of cells/ml was 4 large square lattice cells/4 × 10000 × 10000. The above formula is referred to relevant literature (Zhou Yiming, calculation related to the use of blood count plate, < New college entrance examination (three physics of higher generation) > 61-64 in 2013)
(4) Inoculating the cell suspension liquid obtained in the step (2) to a confocal culture dish for culturing for 24 hours, observing under an optical microscope, removing culture liquid in the culture dish when the area of the cell covering the bottom of the culture dish reaches 40% -60%, adding 1mL of buffer solution containing a Fluo-4AM (Thermo Fisher, F14202) fluorescent probe (10 mu M) into the culture dish, and continuously culturing in a constant-temperature cell culture box at 37 ℃ in a dark place for 45-60min to obtain cells marked by the Fluo-4 fluorescent probe;
(5) measuring the emitted fluorescence intensity of the cells marked by the Fluo-4 fluorescent probe in the step (4) under different conditions after the Fluo-4 is combined with calcium ions in the cells by using an FV1000 laser scanning confocal microscope; adding the sweet sample solution to be detected into the culture dish at 16s after the FV1000 scanning laser confocal microscope starts scanning, and continuing to scan and photograph the cells to obtain a fluorescence picture; the parameters of the confocal laser scanning microscope are set as follows: an objective lens is a 60-time oil lens, a laser tube is a neon ion laser, the excitation wavelength is 488nm, the emission wavelength range is 500nm-600nm, the scanning speed is 8 mus/Pixel, the Pixel is 640x640, the cell is subjected to nondestructive continuous scanning photographing by adopting an XYT scanning program, the cell is scanned and photographed for 4min at room temperature under the condition of light shielding, and the total frame number is not less than 30 fluorescence pictures;
(6) analyzing the fluorescence intensity of the single cell continuously changing in the first 2min by using ImageJ software according to the fluorescence picture obtained in the step (5); selecting the lowest fluorescence value in the first 16s as Fo, selecting the maximum fluorescence value in the first 120s as Fm, and calculating the single cell fluorescence intensity change ratio by using a formula of delta F/Fo ═ Fm-Fo/Fo; the intracellular fluorescence change ratio DeltaF/Fo of the HEK293 cells was subtracted from the calculated fluorescence change ratio DeltaF/Fo of the HEK293-T1R2/T1R3 cells to obtain the intracellular fluorescence intensity change ratio difference DeltaR between the HEK293-T1R2/T1R3 cells and the HEK293 cells. We define the sweetness of a sucrose solution at a known concentration as 1 (e.g. defining the sweetness of 150mM sucrose as 1), and the difference in the ratio of the change in intracellular fluorescence intensity between HEK293-T1R2/T1R3 cells and HEK293 cells as Δ R reflects the sweetness of the sugar solution to be tested; the greater the difference Δ R in the rate of change in fluorescence, the greater the sweetness of the sugar solution detected.
Wherein the Fluo-4AM fluorescent probe solution obtained in the step (3) contains 10 mu M of Flex buffer solution for dilution, and the composition of the Flex buffer solution is as follows:
| reagent | Volume (mL) |
| HBSS solution | 47.5 |
| HEPES solution | 1 |
| 1M MgSO4 | 0.05 |
| 1M Na2CO3 | 0.165 |
| 1M CaCl2 | 0.065 |
| 10% BSA bovine serum albumin | 0.5 |
| 250mM Probenecid | 0.5 |
| Total of | 50 |
The HBSS solution, namely Hanks balanced salt solution, is a commercial Thermo company product (Cat No.14025092), and has the following specific components:
| composition (I) | Concentration (mg/L) | Concentration (mM) |
| CaCl2(Anhydrous) | 140 | 1.261261 |
| MgCl2·6H2O | 100 | 0.492611 |
| MgSO4·7H2O | 100 | 0.406504 |
| KCl | 400 | 5.333334 |
| KH2PO4 | 60 | 0.441176 |
| NaHCO3 | 350 | 4.166667 |
| NaCl | 8000 | 137.931 |
| Na2HPO4(Anhydrous) | 48 | 0.338028 |
| D-Glucose | 1000 | 5.555555 |
HEPES represents N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, and the HEPES solution is a commercial product of Thermo (Cat No.15630080), has a concentration of 1M and a pH of 7.2 to 7.5; the Probenecid is Probenecid.
Compared with the existing technology for detecting sweet taste, the determination method of the invention has the advantages that:
1. the sweetness of the compound is evaluated by detecting the change of the concentration of calcium ions in living cells stably expressing the human sweet taste receptor, and the sweetness can be reflected on a physiological level. Rather than detecting the amount of sweet soluble solids in the food product as in the prior analytical chemical and biological methods, and then quantifying the sweetness of the food or complex compounds based on the amount of these individual sweet substances. These measurements are not the sweetness of the authentic compound.
2. The measuring method simulates the perception degree of the human sweet taste receptor to the sweet taste substance on the cell level, quantifies the perception degree of the human sweet taste receptor to the sweet taste substance on the cell level, and quantifies the perception degree of the human to the sweet taste of other unknown samples under the condition of vitro. The invention establishes a stable sweetness detection platform in vitro, and can effectively avoid the influence of human subjective factors. The method can be used for rapidly, efficiently and sensitively detecting the sweetness of the compound, and has a good application prospect.
Detailed Description
Example 1
Changes in intracellular calcium ion concentrations in HEK293 cells and HEK293-T1R2/T1R3 cells at 150mM sucrose solution:
(1) planting cells in a confocal culture dish:
a confocal culture dish (outer diameter 35mM, inner diameter 10mM) was incubated with 1ml of polylysine (25. mu.g/ml) at 37 ℃ for 30min, washed three times with phosphate buffer (10mM, pH 7.4), and the cells were then passaged to the confocal culture dish in a certain proportion. When the cells in the culture dish grow for 24-48 h in an adherent way, the area of the bottom of the culture dish covered by the cells is observed to reach 40-60% under an optical microscope.
(2) Prepared Flex buffer (pH 7.4)
| Reagent | Volume (mL) |
| HBSS solution | 47.5 |
| HEPES solution | 1 |
| 1M MgSO4 | 0.05 |
| 1M Na2CO3 | 0.165 |
| 1M CaCl2 | 0.065 |
| 10% BSA bovine serum albumin | 0.5 |
| 250mM Probenecid | 0.5 |
| Total of | 50 |
The HBSS solution, namely Hanks balanced salt solution, is a commercial Thermo company product, and the specific components are shown in the following table:
| composition (I) | Concentration (mg/L) | Concentration (mM) |
| CaCl2(Anhydrous) | 140 | 1.261261 |
| MgCl2·6H2O | 100 | 0.492611 |
| MgSO4·7H2O | 100 | 0.406504 |
| KCl | 400 | 5.333334 |
| KH2PO4 | 60 | 0.441176 |
| NaHCO3 | 350 | 4.166667 |
| NaCl | 8000 | 137.931 |
| Na2HPO4(Anhydrous) | 48 | 0.338028 |
| D-Glucose | 1000 | 5.555555 |
HEPES represents N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; the HEPES solution is a commercial product (Cat No.15630080) of Thermo company, the concentration is 1M, and the pH value is 7.2-7.5; the Probenecid is Probenecid.
(3) Labeling cells with fluorescent probes:
the prepared Flex buffer solution is put in a water bath kettle at 37 ℃ for warm bath for 30 min. Mu.l Fluo-4AM with a concentration of 10mM was diluted to 10ml Flex buffer to obtain a Fluo-4AM (10. mu.M) dilution, protected from light. The culture solution in the confocal culture dish was removed, 1ml of Fluo-4AM (10. mu.M) diluent was added to each dish, and the mixture was incubated at 37 ℃ for 45 to 60min in the dark. After the incubation, the Fluo-4AM dilution (10. mu.M) was removed and washed once with 1ml of Flex buffer. 1ml of Flex buffer solution was added to each dish for subsequent detection by FV1000 laser scanning confocal microscope.
(4) Method for obtaining XYT axis fluorescence picture by laser confocal scanning microscope
Sequentially opening a laser scanning confocal microscope and opening a 488nm excitation light laser;
opening software, adjusting the scanning speed to be 8 mus/Pixel, setting the Pixel to be 640x640, opening a time axis scanning mode, setting excitation light to be 488nm, and setting a light receiving channel to be 500nm-600 nm;
adjusting an objective lens to be a 60-fold lens, dripping a drop of cedar oil on the lens, and fixing a culture dish above an objective table;
the lens is lifted to enable cedar oil to be in contact with the lower surface of the culture dish, and then the fine focusing helix is adjusted to search cells until the cell morphology can be clearly observed under the ocular lens;
finding a visual field with good cell morphology and uniform dispersion (facilitating subsequent fluorescence quantification);
rapidly scanning a fluorescence image, and obtaining a calcium ion fluorescence image which can be clearly scanned out of cells by adjusting the intensity of excitation light, HV (enhanced image signal), Gain (enhanced signal), Offset (background subtraction) and a focal plane;
removing Flex buffer solution in the culture dish by using a pipette, and adjusting the focal plane again;
starting to scan the fluorescent image, wherein the scanning time is 4min, and the total frame number is 60;
at 16s after the start of the scan, 200. mu.l of 150mM sucrose solution was dropped in suspension around the cells to be detected (without touching the petri dish, preventing the visual field from moving);
and (5) after the scanning time is over, storing the image data, and subsequently collecting and processing the data.
(5) Quantifying changes in intracellular calcium ion concentration in single cells
Opening the Image file in Image J software, randomly selecting three fields in the fluorescence picture shot in the step (4), wherein each field has 2-3 cells, and respectively extracting the fluorescence value of the cell in each frame of Image.
The minimum fluorescence value at 16s before is Fo, and the maximum fluorescence value at 2min before is Fm, and the ratio of the fluorescence change of the individual cells is obtained according to the following formula: Δ F/Fo ═ (Fm-Fo)/Fo; and averaging the fluorescence change ratios of a plurality of cells in the image file, and subtracting the value of the HEK293-T1R2/T1R3 cell fluorescence intensity change ratio delta F/Fo from the value of the HEK293 cell fluorescence intensity change ratio delta F/Fo to obtain the fluorescence change ratio delta Rs, wherein the value of the fluorescence change ratio delta Rs is 0.284.
Example 2
The intracellular calcium ion concentrations of HEK293 cells and HEK293-T1R2/T1R3 cells were varied under 50mM sucrose stimulation, using the same procedures and conditions as in example 1.
The fluorescence change ratio Δ R was found to be 0.105. When the sweetness of the 150mM sucrose solution of example 1 was set to 1, the value of Δ R/. DELTA.Rs was 0.370, and the relative sweetness of the 50mM sucrose solution of this example was 0.370.
Example 3
The intracellular calcium ion concentrations of HEK293 cells and HEK293-T1R2/T1R3 cells were varied under the stimulation of 250mM sucrose, according to the same procedures and conditions as in example 1.
The fluorescence change ratio DeltaR is 0.487. When the sweetness of the 150mM sucrose solution of example 1 was set to 1, the value of Δ R/. DELTA.Rs was 1.71, and the relative sweetness of the 250mM sucrose solution of this example was 1.71.
Example 4
The intracellular calcium ion concentrations of HEK293 cells and HEK293-T1R2/T1R3 cells were varied under 150mM glucose stimulation, using the same procedures and conditions as in example 1.
The fluorescence change ratio Δ R was found to be 0.224. When the sweetness of the 150mM sucrose solution of example 1 was set to 1, the value of Δ R/Δ Rs was 0.789, and the relative sweetness of the 150mM glucose solution of this example was 0.789.
Example 5
The intracellular calcium ion concentrations of HEK293 cells and HEK293-T1R2/T1R3 cells were varied under 50mM sucralose stimulation, using the same procedures and conditions as in example 1.
The fluorescence change ratio DeltaR was found to be 5.9. Assuming that the sweetness of the 150mM sucrose solution of example 1 is 1, the value of Δ R/. DELTA.Rs is 20.7, and the relative sweetness of 50mM sucralose of this example is 20.7.
Claims (5)
1. A method for the quantitative determination of sweetness at the cellular level, comprising the steps of:
(1) preparing suspension cells of HEK293 cells and HEK293-T1R2/T1R3 cells which are cultured in an adherent manner according to a normal passage step, and respectively planting the suspension cells in a confocal culture dish;
(2) when the confluence degree of HEK293 cells and HEK293-T1R2/T1R3 cells subjected to adherent culture in the confocal culture dish in the step (1) reaches 40% -60%, incubating the cells planted in the confocal culture dish by using a calcium ion fluorescent probe solution;
(3) continuously observing and recording the cell fluorescence image after incubation in the step (2) to 120s by using a laser scanning confocal microscope, and respectively obtaining the fluorescence intensity value of each time point of single cells of the HEK293 cell and the HEK293-T1R2/T1R3 cell within 120 s; setting the minimum fluorescence value within the first 16s as Fo and setting the maximum fluorescence value within the range from 16s to 120s as Fm; wherein at 16s, a sweet sample solution to be tested is added;
(4) calculating fluorescence intensity change ratio values of HEK293 cells and HEK293-T1R2/T1R3 cells by using delta F/Fo, wherein the delta F is Fm-Fo;
(5) the value obtained by subtracting the change ratio delta F/Fo of the fluorescence intensity of HEK293 cells from the change ratio delta F/Fo of the fluorescence intensity of HEK293-T1R2/T1R3 cells is used as delta R;
(6) setting the sweetness of a sucrose solution under a certain known concentration to be 1, and setting the value of F/Fo to be Delta Rs; the ratio of the Delta R value to the Delta Rs obtained in the step (5) is the relative sweetness of the sweet sample solution to be detected;
the calcium ion fluorescent probe in the step (2) is Fluo-4AM, and the concentration of the calcium ion fluorescent probe in a Flex buffer solution is 10 mu M; the Flex buffer included the components shown in the following table:
The HBSS solution, namely Hanks balanced salt solution, has the following specific components:
HEPES is N-2-hydroxyethyl piperazine-N-2-ethane sulfonic acid, the concentration of the HEPES solution is 1M, and the pH value is 7.2-7.5; the Probenecid is Probenecid;
the model of the laser scanning confocal microscope in the step (3) is FV 1000; the shooting parameters are set as follows: an objective lens is a 60-time oil lens, a laser tube is a neon ion laser, the excitation wavelength is 488nm, the emission wavelength range is 500nm-600nm, the scanning speed is 8 mus/Pixel, the Pixel is 640x640, the cell is subjected to nondestructive continuous scanning photographing by adopting an XYT scanning program, scanning is carried out for 4min at room temperature under the condition of light shielding, and the total frame number is not less than 30 fluorescence pictures; then, carrying out fluorescence quantification on single cells on the obtained continuous observation fluorescence image by using ImageJ software to obtain a fluorescence value of the single cells within 120 s;
and (4) the concentration of the sucrose solution with the sweetness of 1 under a certain known concentration in the step (6) is 150 mM.
2. The assay of claim 1, wherein the confocal culture dish in step (1) is a cell culture dish suitable for oil-lens observation on a laser confocal microscope.
3. The assay of claim 2, wherein the confocal culture dish has a specification of 35mm outer diameter and 10mm inner diameter; the confocal dish was incubated with 25. mu.g/ml polylysine solution at 37 ℃ for 30min before use.
4. The assay method according to claim 1, wherein the pH of the Flex buffer is 7.4.
5. The method according to claim 1, wherein the incubation conditions of the cells planted in the confocal culture dish with the calcium ion fluorescent probe solution in the step (2) are as follows: incubating for 45-60min at 37 ℃ in a dark place, so that the area of adherent cells covering the bottom of the culture dish reaches 40-60%.
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