CN112946293B - Quantitative detection method for target membrane protein in single cell - Google Patents
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Abstract
The invention provides a method for quantitatively detecting target membrane protein in single cells by adopting a capillary electrophoresis-confocal laser induced fluorescence method. Firstly, measuring fluorescence signals of a plurality of single fluorescent microspheres with known fluorescent molecule number bonded on the surface by a capillary electrophoresis-confocal laser induced fluorescence method, taking a median, and then establishing a standard curve of fluorescence intensity-fluorescent molecule number; secondly, performing immunofluorescence labeling on the membrane protein of the cell, and determining the fluorescence signal of the single-cell membrane protein; finally, the concentration of the target membrane protein in the single cell was calculated by standard curve. The method utilizes the combined action of electrophoresis and electroosmotic flow to drive cells and microspheres to a detection light window, and uses confocal laser to induce fluorescence to detect single-particle fluorescence signals. The method can realize simultaneous detection of multiple target membrane proteins by a multi-channel detector, and has the characteristics of high sensitivity, accurate quantification and low cost.
Description
Technical Field
The invention relates to a quantitative detection method of target membrane protein of single cell, in particular to a quantitative detection method for detecting target membrane protein in single cell by capillary electrophoresis-confocal laser induced fluorescence technology.
Background
The normal progress of various vital activities can not leave protein, and the protein plays an important role in various physiological processes such as information conduction, physiological regulation, material transportation, organism defense and the like. The cells contain tens of thousands of proteins, each of which has its specific structure and function, wherein the membrane proteins are involved in the related processes of substance transport, signal transduction, immune recognition, etc. of the body. Changes in the content of membrane proteins may affect the relevant physiological processes and even lead to diseases. Therefore, quantitative detection of the content of membrane proteins is essential.
In recent years, several single cell membrane protein detection techniques have been developed, such as fluorescence imaging techniques, flow cytometry techniques, and the like. The fluorescence imaging technology can observe the membrane protein content of cells in real time, but can only carry out semi-quantitative analysis; flow cytometry can carry out multi-parameter quantitative detection on cells, can adopt multiple channels to realize simultaneous analysis of multiple membrane proteins, but due to spectral overlap effect, at most dozens of membrane proteins can be detected in one detection process, and due to the limitation of sensitivity, a flow cytometer is only suitable for detecting proteins with high expression content (the expression content needs more than 1000 molecules) and is not suitable for detecting proteins with low expression content. Meanwhile, in the flow cytometry, cells reach a detection optical window in a sheath flow mode, and the sheath flow control mode mainly comprises two modes, wherein the first mode is that a syringe is used for pushing a cell suspension liquid, the periphery of the cell suspension liquid is surrounded by sheath flow gas, so that the cells form a single cell flow which is arranged in the middle and then reach the detection optical window, and an air flow pressure stabilizing device is required to be installed in a system for stabilizing air pressure. The second is that sheath flow liquid and cells need independent control devices, and two sets of motors and two injection pumps are needed. The two driving modes of the cells adopt complicated mechanical structures to enable the cells to stably flow through the detection optical window, are not easy to control and have higher cost.
The confocal Laser Induced Fluorescence Detector (Laser Induced Fluorescence Detector) has high sensitivity and very small detection focusing light spot (5 mu m), is suitable for ultra-trace detection, and is particularly suitable for detecting low-expression protein in single cells. Compared with a flow cytometer, the Capillary Electrophoresis (Capillary Electrophoresis) technology is relatively mature in development, the instrument is relatively simple in structure and easy to control, electroosmotic flow exists in the Capillary, cells can be driven, the cells can rapidly pass through a detection optical window, and high-flux detection is realized. Capillary electrophoresis is combined with confocal laser induced fluorescence detection, and quantitative detection of low-expression membrane protein on single cells is expected to be realized. In order to increase the detection throughput, the single cells are preferably passed through the detection window in intact individual form (without rupture of the membrane). However, such detection methods cannot be quantified using conventional, standard curves established using homogeneous standard solutions. Meanwhile, the electrophoresis behavior of the single cell in the capillary is different from that of the traditional micromolecule or macromolecular protein, and the traditional electrophoresis method cannot be adopted.
Disclosure of Invention
The invention provides a method for quantitatively detecting a single-cell target membrane protein by adopting a capillary electrophoresis-laser induced fluorescence method, which comprises the following steps of firstly, measuring a fluorescence signal of a fluorescent microsphere with the surface bonded with a known fluorescent molecule number by adopting a capillary electrophoresis-confocal laser induced fluorescence method, and establishing a standard curve of fluorescence intensity-fluorescent molecule number; then, performing immunofluorescence labeling on the membrane protein of the cell, and determining the fluorescence signal of the single-cell membrane protein; finally, the concentration of the target membrane protein in the single cell was calculated by standard curve.
The technical scheme of the invention is as follows:
a quantitative detection method of target membrane protein in single cells comprises the following steps:
(1) establishing a standard curve of fluorescence intensity-number of fluorescent molecules, which specifically comprises the following steps:
a. preparing a standard solution: the standard solution is prepared by bonding fluorescent microspheres with known fluorescent molecule number on the surface and preparing the fluorescent microspheres in an electrophoresis buffer solution with a certain volume, wherein more than 3 different standard solutions are prepared, the fluorescent molecules of the microspheres used in the different standard solutions are the same, and only the number of the bonded fluorescent molecules is different; mixing by ultrasonic, the fluorescent molecules and the microspheres have charges on the surface, and the concentration of the microspheres in the prepared standard solution is 103～106Per mL; the difference between the diameter of the microsphere and the diameter of the cell to be measured is 1-2 mu m; the number of the bonded fluorescent molecules on the fluorescent microsphere is 102-106A plurality of;
b. flushing a capillary tube by using an electrophoresis buffer solution, filling the capillary tube with the electrophoresis buffer solution, putting the standard solution prepared in the step a) into a sample bottle, putting the sample bottle into a sample introduction end of capillary electrophoresis, inserting one end of the capillary tube into the sample bottle, and inserting the other end of the capillary tube into the electrophoresis buffer solution;
c. turning on high voltage power supply, adding to two ends of capillary tubeVoltage, the microspheres enter a capillary tube with a detection optical window in sequence under the action of an electric field, and the microspheres are separated by electrophoresis under the combined action of electrophoresis and electroosmotic flow due to the charge on the surfaces of the microspheres, so that the microspheres are not adhered, and the effective length of the capillary tube is more than 30 cm; diameter d for the capillaryLAnd diameter d of the microspheresbA relationship of db＜dL＜2dbThe microspheres are independently and continuously driven to a detection light window from a sample introduction end; optically detecting d of the focal spotwThe relation with the diameter of the microsphere is db＜dw＜2dbA fluorescence detector having wavelength parameters matching the excitation and emission wavelengths of said fluorescent molecule of step a; ensuring that the fluorescence signal detected by the optical window each time is the fluorescence generated by a single microsphere through electrophoretic separation conditions, and recording the fluorescence signals generated by at least 1000 microspheres;
d. repeating the steps b and c in sequence to obtain the fluorescence signal intensity of other different standard solutions;
e. taking the measured median of the fluorescence intensity of the microspheres as the fluorescence intensity of the standard solution, and establishing a standard curve according to the number of fluorescence molecules of the microspheres in the standard solution and the fluorescence intensity of the standard solution;
(2) the method specifically comprises the following steps:
a. washing the cultured cells, removing the cell culture solution by centrifugation, and resuspending the cells by using a cell diluent after washing;
b. fixing the cells by using a cell fixing solution, then washing the cells, and resuspending the cells by using a cell diluent;
c. sealing the protein on the cell membrane by using a sealing reagent, centrifuging to remove the sealing reagent after sealing is finished, and resuspending the cell by using a cell diluent;
d. adopting an antibody with the same fluorescent group as the fluorescent microspheres to carry out immune labeling on target protein on the surface of a cell membrane, washing cells after the labeling is finished, and then adopting an electrophoresis buffer solution to resuspend the cells to form a cell suspension;
e. washing a capillary tube by using an electrophoresis buffer solution, filling the capillary tube with the electrophoresis buffer solution, then putting a cell suspension into a sample bottle, putting the sample bottle at the sample introduction end of capillary electrophoresis, inserting one end of the capillary tube into the sample bottle, and inserting the other end of the capillary tube into the electrophoresis buffer solution;
f. starting a high-voltage power supply, and applying voltage to the two ends of the capillary tube, wherein the electric field intensity range is 300-; the cells enter the capillary tube in sequence under the action of an electric field, and under the combined action of electrophoresis and electroosmotic flow, the cells are separated by the capillary electrophoresis, so that the cells are driven to the detection light window from the sample introduction end independently and continuously; recording the detected fluorescent signal;
g. calculating the number of molecules of the target membrane protein in the single cell according to the standard curve established in (1) and the fluorescence intensity detected in the step f;
the electrophoresis buffer solution in the step (1) is the same as that in the step (2), wherein the electrophoresis buffer solution does not contain an ionic surfactant or a cell membrane breaking reagent, the buffer solution is 20-50mM sodium borate-boric acid or 20-50mM Tris-HCl buffer solution, the pH value range of the electrophoresis buffer solution is 6-8, and the fluorescence detector is a confocal laser induced fluorescence detector.
The diameter of the fluorescent microspheres in the step (1) is 5-30 microns, the microspheres are made of styrene-divinylbenzene, polystyrene or silicon dioxide, and the RSD of the particle size of the microspheres is less than 5%.
The cell diluent is a phosphate buffered solution.
The cell fixing solution is a formaldehyde solution, the volume fraction is 4-6%, and the fixing time is 10-20 min; the blocking reagent is BSA solution or goat serum with the volume fraction of 5-10%, and the blocking time is 10-60 min.
In the step f of quantitatively detecting the single cell membrane protein, the electric field strength is preferably 200-400V/cm.
The method can also adopt two or more than two fluorescence labeling antibodies to carry out immunological labeling on various target membrane proteins on the surface of the cell membrane, adopt two or more than two microspheres with known fluorescent molecule numbers bonded on the corresponding surfaces to establish corresponding standard curves, and adopt a multichannel laser-induced fluorescence detector to simultaneously detect the two or more than two membrane proteins on the single cell.
The fluorescent groups are FITC, dylight488, BV605 and BV 650;
the antibody is one or more than two of rabbit anti-human anti-CD 3, CD63, CD206 and Her2 IgG antibodies with fluorescent groups, and one or more than two of corresponding intracellular target proteins CD3, CD63, CD206 and Her 2.
Compared with the prior art, the method has the following advantages:
1. the method adopts the combined action of electrophoresis and electroosmotic flow to drive the cells and the fluorescent microspheres to reach the detection optical window, can control the speed of electroosmotic flow by controlling the voltage at two ends of the capillary and the concentration of the buffer solution in the capillary, and is easy to control.
2. The method adopts the fluorescent microspheres to establish a standard curve, so that the surface membrane protein can be quantitatively detected even if the single cells do not break the membrane, and the high flux of the analysis method is realized.
3. The capillary electrophoresis-confocal laser induced fluorescence detector system adopted by the method is simple and reliable, and is easy to use in other laboratories.
4. The method can adopt a multi-channel laser-induced fluorescence detector to realize the simultaneous detection of various target membrane proteins.
5. The method combines the laser-induced fluorescence detector with capillary electrophoresis, and has the advantages of high sensitivity, high detection speed and low cost.
Drawings
FIG. 1 schematically shows a micro-fluorescence photograph of a single cell membrane protein after labeling in example 1.
FIG. 2 schematically shows the results of the determination of Her2 protein on various breast cancer cell membranes in example 1.
Detailed Description
In order that the invention may be better understood, it is illustrated by the following examples. The specific embodiments of the present invention are set forth herein for the purpose of illustration only and are not intended to be limiting of the invention.
Example 1:
a method for quantitatively detecting Her2 protein on the surface of a single breast cancer cell comprises the following steps:
(1) establishing a standard curve of fluorescence intensity-number of fluorescent molecules, which specifically comprises the following steps:
a. preparing standard solution, wherein the standard solution is prepared by preparing 1mL of electrophoresis buffer solution from fluorescent microspheres with known fluorescent molecule number bonded on the surfaces, the fluorescent molecules of the microspheres used in different standard solutions are the same and are FITC, only the number of bonded molecules is different, the diameters of the microspheres are equivalent to the diameters of the cells to be detected, the diameters of the microspheres are 12 mu m, the microspheres are made of styrene-divinylbenzene, the RSD of the particle diameters of the microspheres is less than 5%, uniformly mixing the microspheres by ultrasonic treatment for 10min, the fluorescent molecules and the surfaces of the microspheres are charged, and the concentration of the microspheres in the prepared standard solution is 104Per mL; preparing three fluorescent microspheres with surfaces bonded with different fluorescent molecule numbers in 50mM sodium borate-boric acid electrophoresis buffer solution, and then placing the solution into a brown sample bottle, wherein the FITC molecule numbers on the surfaces of the three microspheres are respectively 350, 1000 and 8000 and respectively marked as No. 1, No. 2 and No. 3 standard samples;
b. washing a capillary tube by using an electrophoresis buffer solution, filling the capillary tube with the electrophoresis buffer solution to fully balance the inner wall of the capillary tube, wherein the inner diameter of the capillary tube is 20 micrometers, the outer diameter of the capillary tube is 365 micrometers, putting a No. 1 standard sample into a brown sample bottle, putting the sample bottle into a sample introduction end of capillary electrophoresis, inserting one end of the capillary tube into the sample bottle, inserting the other end of the capillary tube into the electrophoresis buffer solution, and respectively inserting platinum electrodes into the two bottles;
c. the high-voltage power supply is started, voltage is applied to two ends of the capillary tube, the microspheres sequentially enter the capillary tube with the detection optical window under the action of an electric field, the microspheres are separated by electrophoresis under the combined action of electrophoresis and electroosmotic flow due to the fact that the surfaces of the microspheres are charged, the effective length of the capillary tube is 35cm, and the microspheres are free of adhesion in the separation process; the focal spot width of the confocal laser induced fluorescence detector in the detection light window is 15 microns, so that only one microsphere of fluorescence signals can be detected each time, the wavelength parameters of the fluorescence detector are matched with the excitation and emission wavelengths of FITC fluorescent molecules, the excitation wavelength of the fluorescence detector is 488nm, and the detection wavelength is 530-650 nm; the number of each microsphere is about 3000, and the detected fluorescence signals are recorded;
d. repeating the steps b and c in sequence to obtain the fluorescence signal intensity of the No. 2 and No. 3 standard solutions;
e. taking the median of the measured microsphere fluorescence intensity as the fluorescence intensity of the standard solution, and establishing a standard curve of the microspheres with FITC fluorescent molecules according to the number of the fluorescent molecules of the microspheres in the standard solution and the fluorescence intensity of the standard solution;
(2) the method specifically comprises the following steps:
a. collecting cultured breast cancer cells, and counting the collected cells under microscope to obtain the number of cells of about 1 × 106Washing the cells by Phosphate Buffer Solution (PBS), removing cell culture solution by centrifugation, centrifuging for 5min (the rotating speed is 800rpm/min), and after washing is finished, re-suspending the cells by PBS;
b. fixing cells with 200 μ L of 5% formaldehyde solution, fixing at 37 deg.C for 20min, washing cells, centrifuging for 10min (rotation speed of 800rpm/min), and re-suspending cells with PBS;
c. blocking the breast cancer cells by using BSA with the volume fraction of 5%, wherein the blocking time is 15min, centrifuging to remove BSA solution, and resuspending the cells by using 100 mu L of PBS;
d. adding 5 mu L of rabbit anti-human anti-Her 2 protein IgG antibody with FITC into the cell suspension to carry out immune labeling on Her2 protein on the surface of the breast cancer cell membrane, incubating at 37 ℃ for 1hour, washing the cells three times after the labeling is finished, centrifuging for 5min (the rotating speed is 1000rpm/min) each time, then adopting 1mL of 50mM sodium borate-boric acid buffer solution to resuspend the cells to form cell suspension, taking part of the cells, observing under a fluorescence microscope, and determining whether the labeling is successful;
e. the capillary was rinsed with 50mM sodium borate-boric acid buffer and filled with the capillary, and then 1mL of the cell suspension was added to a brown sample bottle at a cell concentration of about 103one/mL, placing the sample bottle at the sample introduction end of capillary electrophoresis, inserting one end of the capillary into the sampleThe other end of the bottle is inserted into the electrophoresis buffer solution;
f. starting a high-voltage power supply, and applying voltage to two ends of the capillary tube, wherein the electric field intensity is 350V/cm; the cells enter the capillary tube in sequence under the action of an electric field, and under the combined action of electrophoresis and electroosmotic flow, the cells are separated by the capillary electrophoresis, so that the cells are driven to the detection light window from the sample introduction end independently and continuously; recording the detected fluorescent signal;
g. and (f) calculating the number of molecules of Her2 protein in each breast cancer cell according to the standard curve established in (1) and the fluorescence signal detected in step f. The experimental results show that the molecular number of the Her2 protein of 100 breast cancer cells ranges from 455 to 12000, and the average molecular number is 6735.
Example 2
A method for quantitatively detecting the content of CD3 protein on the surface of a single T cell comprises the following steps:
(1) establishing a standard curve of fluorescence intensity-number of fluorescent molecules, which specifically comprises the following steps:
a. preparing a standard solution, wherein the standard solution is prepared by preparing 1mL of electrophoresis buffer solution from fluorescent microspheres with known fluorescent molecule number bonded on the surfaces, the fluorescent molecules of the microspheres used in different standard solutions are the same and are BV605, only the number of the bonded molecules is different, the diameters of the microspheres are equivalent to the diameter of a measured cell, the diameters of the microspheres are 13 mu m, the microspheres are made of styrene-divinylbenzene, the RSD of the particle size of the microspheres is less than 5%, uniformly mixing the microspheres by ultrasonic for 5min, the fluorescent molecules and the surfaces of the microspheres are charged, and the concentration of the microspheres in the prepared standard solution is 2 multiplied by 104Per mL; preparing three fluorescent microspheres with surfaces bonded with different fluorescent molecule numbers in a 50mmol/L Tris-HCl electrophoresis buffer solution, and then placing the three fluorescent microspheres into a brown sample bottle, wherein the FITC molecule numbers on the surfaces of the three microspheres are respectively 300, 900 and 9000 and are respectively marked as No. 1, No. 2 and No. 3 standard samples;
b. washing a capillary tube by using an electrophoresis buffer solution, filling the capillary tube with the electrophoresis buffer solution to fully balance the inner wall of the capillary tube, wherein the inner diameter of the capillary tube is 25 micrometers, the outer diameter of the capillary tube is 365 micrometers, then putting a No. 1 standard sample into a brown sample bottle, putting the sample bottle into a sample introduction end of capillary electrophoresis, inserting one end of the capillary tube into the sample bottle, inserting the other end of the capillary tube into the electrophoresis buffer solution, and respectively inserting platinum electrodes into the two bottles;
c. the high-voltage power supply is started, voltage is applied to two ends of the capillary tube, the microspheres sequentially enter the capillary tube under the action of an electric field, the microspheres are separated and conveyed through the detection optical window by electrophoresis under the combined action of electrophoresis and electroosmotic flow due to the fact that the surfaces of the microspheres are charged, the effective length of the capillary tube is 35cm, and the microspheres are free of adhesion in the separation process. The focal spot width of the confocal laser induced fluorescence detector is 15 mu m, the fluorescent signal of only one microsphere can be detected each time, the number of the detected fluorescent microspheres is about 3000, and the detected fluorescent signals are recorded. The wavelength parameters of the used fluorescence detector are matched with the excitation and emission wavelengths of BV605 fluorescent molecules, the excitation wavelength of the used fluorescence detector is 405nm, and the detection wavelength is 590-720 nm;
d. repeating the steps b and c in sequence to obtain the fluorescence signal intensity of the No. 2 and No. 3 standard solutions;
e. taking the median of the measured microsphere fluorescence intensity as the fluorescence intensity of the standard solution, and establishing a standard curve of the microspheres with BV605 fluorescent molecules according to the number of the fluorescent molecules of the microspheres in the standard solution and the fluorescence intensity of the standard solution;
(2) the method specifically comprises the following steps:
a. collecting cultured T cell, and counting the collected cells with a microscope to obtain about 1X 10 cells6Washing the cells by PBS, removing the cell culture solution by centrifugation, centrifuging for 5min (the rotating speed is 1000rpm/min), and after washing is finished, resuspending the cells by PBS;
b. fixing cells with 200 μ l of 4% formaldehyde solution, fixing at 37 deg.C for 15min, washing cells, centrifuging for 5min (at 1000rpm/min), and resuspending cells with PBS;
c. blocking the breast cancer cells by using BSA with the volume fraction of 5%, wherein the blocking time is 15min, centrifuging to remove BSA solution, and resuspending the cells by using 100 mu l of PBS;
d. adding 5 mu L of mouse anti-human anti-CD 3 IgG antibody with BV605 into the cell suspension to carry out immunolabeling on CD3 protein on the surface of a T cell membrane, incubating for 30min at 37 ℃, washing the cells three times after completing labeling, centrifuging for 5min (the rotating speed is 1000rpm/min) each time, then adopting 1mL of 50mM Tris-HCl electrophoresis buffer solution to resuspend the cells to form cell suspension, taking part of the cells to observe under a fluorescence microscope, and determining whether the labeling is successful;
e. the capillary was rinsed with 50mM Tris-HCl buffer and filled with the capillary, and 1mL of the cell suspension was added to a brown vial with a cell concentration of about 104Each mL, placing a sample bottle at the sample introduction end of capillary electrophoresis, inserting one end of a capillary into the sample bottle, and inserting the other end of the capillary into an electrophoresis buffer solution;
f. starting a high-voltage power supply, and applying voltage to two ends of the capillary tube, wherein the electric field intensity is 400V/cm; the cells enter the capillary tube in sequence under the action of an electric field, and under the combined action of electrophoresis and electroosmotic flow, the cells are separated by the capillary electrophoresis, so that the cells are driven to the detection light window from the sample introduction end independently and continuously; recording the detected fluorescent signal;
g. the number of molecules of CD3 protein in each T cell was calculated based on the standard curve established in (1) and the fluorescence signal detected in step f, and the experimental results showed that the number of molecules of CD3 protein ranged from 565 to 11000 for 100T cells, and the average number of molecules was 8435.
Example 3:
a method for quantitatively detecting the content of CD63 and CD206 proteins on the surface of a single macrophage comprises the following steps:
(1) establishing a standard curve of fluorescence intensity-number of fluorescent molecules, which specifically comprises the following steps:
a. preparing standard solution, wherein the standard solution is prepared by preparing 1mL electrophoresis buffer solution from fluorescent microspheres with known fluorescent molecule number bonded on the surface, the fluorescent molecules of the microspheres used in different standard solutions are the same and are FITC, only the number of bonded molecules is different, and the diameters of the microspheres used and the measured fine particles are differentThe diameter of the cells is equivalent, the diameter of the microsphere is 13 μm, the material of the microsphere is polystyrene, the ultrasound is carried out for 10min, the fluorescent molecules and the surface of the microsphere are uniformly mixed, the concentration of the microsphere in the prepared standard solution is 104Per mL; preparing three fluorescent microspheres with surfaces bonded with different fluorescent molecule numbers in a 50mM sodium borate-boric acid electrophoresis buffer solution, and then placing the solution into a brown sample bottle, wherein in a first standard curve, the BV421 molecular numbers on the surfaces of the three microspheres are respectively 400, 1000 and 8000, and are respectively marked as No. 1, No. 2 and No. 3 standard samples; in the second standard curve, the BV711 molecular numbers on the surfaces of the three microspheres are respectively 400, 1000 and 8000, and are respectively marked as No. 4, No. 5 and No. 6 standard samples;
b. washing a capillary tube by using an electrophoresis buffer solution, filling the capillary tube with the electrophoresis buffer solution to fully balance the inner wall of the capillary tube, wherein the inner diameter of the capillary tube is 25 micrometers, the outer diameter of the capillary tube is 365 micrometers, then putting a No. 1 standard sample into a brown sample bottle, putting the sample bottle into a sample introduction end of capillary electrophoresis, inserting one end of the capillary tube into the sample bottle, inserting the other end of the capillary tube into the electrophoresis buffer solution, and respectively inserting platinum electrodes into the two bottles;
c. the high-voltage power supply is started, voltage is applied to two ends of the capillary tube, the microspheres sequentially enter the capillary tube with the detection optical window under the action of the electric field, the microspheres are separated by electrophoresis under the combined action of electrophoresis and electroosmotic flow due to the fact that the surfaces of the microspheres are charged, the effective length of the capillary tube is 35cm, and the microspheres are free of adhesion in the separation process. The diameter of a focal spot of a confocal laser induced fluorescence detector is 15 mu m, only one microsphere fluorescence signal can be detected each time, the wavelength parameters of the fluorescence detector are matched with the excitation and emission wavelengths of BV421 and BV711 fluorescence molecules, dual-channel detection is adopted, the excitation wavelength of a channel 1 is 405nm, and the detection wavelength is 420-550 nm; the excitation wavelength of the channel 2 is 405nm, the detection wavelength is 650-850 nm, the number of each microsphere is about 4000, and the detected fluorescent signals are recorded;
d. repeating the steps b and c in sequence to obtain the fluorescence signal intensity of the standard solution No. 2 to No. 6;
e. taking the median of the measured microsphere fluorescence intensity as the fluorescence intensity of the standard solution, and establishing a standard curve with two types of fluorescent microspheres, BV421 and BV711, according to the number of fluorescent molecules of the microspheres in the standard solution and the fluorescence intensity of the standard solution;
(2) the method specifically comprises the following steps:
a. collecting cultured macrophages, and counting the collected cells with a microscope to obtain a total cell count of about 2X 106Washing the cells by PBS, removing the cell culture solution by centrifugation, centrifuging for 10min (the rotating speed is 800rpm/min), and after washing is finished, resuspending the cells by PBS;
b. fixing cells with 100 mul of formaldehyde solution with volume fraction of 4%, fixing for 15min at 37 ℃, then washing the cells, centrifuging for 10min (the rotating speed is 800rpm/min), and re-suspending the cells with PBS after centrifugation;
c. blocking the macrophages by BSA with the volume fraction of 5%, wherein the blocking time is 15min, centrifuging to remove the BSA solution, and resuspending the cells by 100 μ l of PBS;
d. adding 5 mu L of mouse anti-CD 63 IgG antibody with BV421 and mouse anti-CD 206 IgG antibody with BV711 into the cell suspension to carry out immunolabeling on CD63 and CD206 proteins on the membrane surface of macrophage, incubating at 37 ℃ for 1hour, washing the cells three times after labeling, centrifuging for 5min (the rotating speed is 1000rpm/min) each time, then re-suspending the cells by using 1mL of 50mM sodium borate-boric acid buffer solution to form cell suspension, taking part of the cells to observe under a fluorescence microscope, and determining whether the labeling is successful or not;
e. the capillary was rinsed with 50mM sodium borate-boric acid buffer and filled with the capillary, and then 1mL of the cell suspension was added to a brown sample bottle at a cell concentration of about 104Each mL, placing a sample bottle at the sample introduction end of capillary electrophoresis, inserting one end of a capillary into the sample bottle, and inserting the other end of the capillary into an electrophoresis buffer solution;
f. starting a high-voltage power supply, and applying voltage to two ends of the capillary tube, wherein the electric field intensity is 350V/cm; the cells enter the capillary tube in sequence under the action of the electric field, and under the combined action of electrophoresis and electroosmotic flow, the cells are separated by the capillary electrophoresis due to the charge on the surfaces of the cells, so that the cells are driven to the detection light window from the sample introduction end independently and continuously. Recording the detected fluorescent signal;
g. calculating the molecular number of CD63 and CD206 membrane proteins in each single cell according to the standard curve established in (1) and the fluorescence signal detected in step f, and the experimental result shows that the molecular number of the CD63 protein in 100 macrophages ranges from 745 to 13000 and the average molecular number is 9435; the number of molecules of the CD206 protein ranged from 935 to 15680, with an average number of molecules of 9338.
Claims (7)
1. A quantitative detection method of target membrane protein in single cell is characterized in that:
(1) establishing a standard curve of fluorescence intensity-number of fluorescent molecules, which specifically comprises the following steps:
a. preparing a standard solution: the standard solution is prepared by bonding fluorescent microspheres with known fluorescent molecule number on the surface and preparing the fluorescent microspheres in an electrophoresis buffer solution with a certain volume, wherein more than 3 different standard solutions are prepared, the fluorescent molecules of the microspheres used in the different standard solutions are the same, and only the number of the bonded fluorescent molecules is different; mixing by ultrasonic, the fluorescent molecules and the microspheres have charges on the surface, and the concentration of the microspheres in the prepared standard solution is 103~106Per mL; the difference between the diameter of the microsphere and the diameter of the cell to be measured is 1-2 mu m; the number of the bonded fluorescent molecules on the fluorescent microsphere is 102-106A plurality of;
b. flushing a capillary tube by using an electrophoresis buffer solution, filling the capillary tube with the electrophoresis buffer solution, putting the standard solution prepared in the step a) into a sample bottle, putting the sample bottle into a sample introduction end of capillary electrophoresis, inserting one end of the capillary tube into the sample bottle, and inserting the other end of the capillary tube into the electrophoresis buffer solution;
c. starting a high-voltage power supply, applying voltage to two ends of a capillary tube, enabling the microspheres to sequentially enter the capillary tube with a detection optical window under the action of an electric field, separating the microspheres by electrophoresis under the combined action of electrophoresis and electroosmotic flow due to the fact that the surfaces of the microspheres are charged, enabling the microspheres to be free of adhesion, and enabling the effective length of the capillary tube to be larger than 30 cm; diameter d for the capillaryLAnd microspheresDiameter dbA relationship of db＜dL＜2dbThe microspheres are independently and continuously driven to a detection light window from a sample introduction end; optically detecting d of the focal spotwThe relationship with the diameter of the microsphere is db＜dw＜2dbThe wavelength parameter of the fluorescence detector is matched with the excitation and emission wavelength of the fluorescence molecule in the step a; ensuring that the fluorescence signal detected by the optical window each time is the fluorescence generated by a single microsphere through electrophoretic separation conditions, and recording the fluorescence signals generated by at least 1000 microspheres;
d. repeating the steps b and c in sequence to obtain the fluorescence signal intensity of other different standard solutions;
e. taking the measured median of the fluorescence intensity of the microspheres as the fluorescence intensity of the standard solution, and establishing a standard curve according to the number of fluorescence molecules of the microspheres in the standard solution and the fluorescence intensity of the standard solution;
(2) the method specifically comprises the following steps:
a. washing the cultured cells, removing the cell culture solution by centrifugation, and resuspending the cells by using a cell diluent after washing;
b. fixing the cells by using a cell fixing solution, then washing the cells, and resuspending the cells by using a cell diluent;
c. sealing the protein on the cell membrane by using a sealing reagent, centrifuging to remove the sealing reagent after sealing is finished, and resuspending the cell by using a cell diluent;
d. adopting an antibody with the same fluorescent group as the fluorescent microspheres to carry out immune labeling on target protein on the surface of a cell membrane, washing cells after the labeling is finished, and then adopting an electrophoresis buffer solution to resuspend the cells to form a cell suspension;
e. washing a capillary tube by using an electrophoresis buffer solution, filling the capillary tube with the electrophoresis buffer solution, then putting a cell suspension into a sample bottle, putting the sample bottle at the sample introduction end of capillary electrophoresis, inserting one end of the capillary tube into the sample bottle, and inserting the other end of the capillary tube into the electrophoresis buffer solution;
f. starting a high-voltage power supply, applying voltage to two ends of the capillary tube, wherein the electric field intensity range is 300-500V/cm; the cells enter the capillary tube in sequence under the action of an electric field, and under the combined action of electrophoresis and electroosmotic flow, the cells are separated by the capillary electrophoresis, so that the cells are driven to the detection light window from the sample introduction end independently and continuously; recording the detected fluorescent signal;
g. calculating the number of molecules of the target membrane protein in the single cell according to the standard curve established in the step (1) and the fluorescence intensity detected in the step f;
the electrophoresis buffer solution in the step (1) is the same as that in the step (2), wherein the electrophoresis buffer solution does not contain an ionic surfactant or a cell membrane breaking reagent, the buffer solution is 20-50mM of sodium borate-boric acid or 20-50mM of Tris-HCl buffer solution, and the pH value range of the electrophoresis buffer solution is 6-8; the fluorescence detector is a confocal laser-induced fluorescence detector.
2. The method of claim 1, wherein: the diameter of the fluorescent microspheres in the step (1) is 5-30 microns, the microspheres are made of styrene-divinylbenzene, polystyrene or silicon dioxide, and the RSD of the particle size of the microspheres is less than 5%.
3. The method of claim 1, wherein: the cell diluent is a phosphate buffered solution.
4. The method of claim 1, wherein: the cell fixing solution is a formaldehyde solution, the volume fraction is 4-6%, and the fixing time is 10-20 min; the blocking reagent is BSA solution or goat serum with the volume fraction of 5% -10%, and the blocking time is 10-60 min.
5. The method of claim 1, wherein: in the step f of quantitatively detecting the single cell membrane protein, the electric field strength is 200-400V/cm.
6. The method of claim 1, wherein: adopting two or more than two fluorescence labeling antibodies to carry out immunological labeling on various target membrane proteins on the surface of a cell membrane, adopting two or more than two microspheres with known fluorescent molecule numbers bonded on corresponding surfaces to establish a corresponding standard curve, and adopting a multichannel laser induced fluorescence detector to simultaneously detect the two or more than two membrane proteins on a single cell.
7. The method of claim 1, wherein:
fluorophores are FITC, dylight488, BV605 and BV 650;
the antibody is one or more than two of rabbit anti-human CD3, CD63, CD206 and Her2 IgG antibodies with fluorescent groups, and one or more than two of corresponding intracellular target proteins CD3, CD63, CD206 and Her 2.
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