CN113866425A - Single cell protein digital imaging detection method - Google Patents
Single cell protein digital imaging detection method Download PDFInfo
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- CN113866425A CN113866425A CN202111120430.3A CN202111120430A CN113866425A CN 113866425 A CN113866425 A CN 113866425A CN 202111120430 A CN202111120430 A CN 202111120430A CN 113866425 A CN113866425 A CN 113866425A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
Abstract
The invention discloses a digital imaging detection method of single-cell protein, which relates to the field of protein detection. The invention is a single-cell protein detection means which has high throughput, short time, stable data storage and digital detection result processing, portability, small size, stable function and low cost.
Description
Technical Field
The invention relates to the field of protein detection, in particular to a digital imaging detection method for single-cell protein.
Background
Protein analysis of rare cells has become an increasingly important issue. However, the method is limited by sampling technology, enrichment technology or the organism itself with extremely low content of the cells, and the prior art means are difficult to carry out systematic protein detection on the cells. Rare cells are mostly studied in circulating tumor cells, fertilized eggs and neuronal cells, which have important effects on diseases, development and early cognitive development, respectively, and if protein levels in dozens or even hundreds of rare cells can be depicted, the cells have important effects on promoting developmental biology, tumor immunity and other fields.
The existing protein detection technology can be divided into two categories, namely non-labeled detection and labeled detection. The detection of non-labeled proteins has the important point of finding unknown proteins and defining new protein functions. The non-labeling detection is mainly mass spectrometry, and the analysis sample is a peptide fragment. The mass spectrum technology has better sensitivity and accuracy and can accurately measure the protein. At present, the mass spectrum mainly determines the primary structure of protein, including molecular weight, amino acid sequence of peptide chain and number and position of polypeptide or disulfide bond, and plays an important role in the research of protein structure analysis. The traditional mass spectrum is only used for analyzing small-molecule volatile substances, but with the appearance of new ionization technologies, such as matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization mass spectrometry (ESI-MS), and the like, the appearance of various mass spectrum technologies provides a new, accurate and rapid way for protein analysis. At present, the combined application of enzymolysis, liquid chromatography separation, tandem mass spectrometry and computer algorithm has become a development trend for identifying proteins. Although mass spectrometry of proteins has great advantages, mass spectrometry can only analyze a minimum of 1000 cells at present, and the complicated processing steps of mass spectrometry often result in a large loss of samples, so mass spectrometry is not suitable for rare samples.
Labeled protein detection is usually performed by means of antibodies, which specifically bind to proteins and are used to study the functions and pathways of action of known proteins. The antibody is usually labeled with fluorescein, biotin, quantum dots, metal elements and the like, and is used for data reading at the detection end. Fluorescein, quantum dot, etc. are limited by the overlap of spectra, and less than 6 channels can be detected simultaneously, and if more than ten proteins need to be detected in the same sample, repeated washing and staining are required. This leads to severe loss of antigen during this process; on the other hand, the overlap of fluorescence channels poses a serious cross-color problem, and the signals of many channels require complex compensation calculations. The metal element can perfectly solve the problems and has the following four advantages: the number of I channels increases to hundreds. The inductively coupled plasma mass spectrometry (ICP-MS) device suitable for metal elements has a very wide atomic weight detection range (88-210 Da), so hundreds of different parameters can be detected simultaneously. And the channels II have no interference, and the calculation and compensation are not needed. ICP mass spectrometry has an ultra-high resolving power and can completely distinguish between the various elements used for labeling. Experimental data show that the interference between adjacent channels is less than 0.3%, and the interference can be basically ignored without calculating compensation. Therefore, the experimental process is simplified, and the samples and the reagents are saved. III metal tags are numerous and have a very low background. More than 30 kinds of metal labels are used for labeling the antibody, more elements can be used as the labels with the technical progress, the types of the elements are further increased, the content of the metal elements used as the labels in cells is extremely low, and the nonspecific binding capacity of the metal labels and cell components is extremely low, so the background of signals is extremely low. IV diversified data processing mode, realize the deep analysis to the sample. The increase in the number of channels brings about a multiple increase in the amount of information. The traditional analysis method can not completely meet the requirement, so that various dimensionality reduction processing needs to be carried out on data to extract useful biological information contained in the data, and the common analysis methods comprise the following steps: SPADE, PCA, visNE, and Gemstone, among others.
The high-throughput, high-sensitivity and high-resolution analysis means such as fluorescence flow cytometry, mass flow imaging technology, single-cell secretory protein determination technology, single-cell western blotting technology (western blot) and the like provide powerful tools for the research of single-cell proteomics.
Fluorescence flow cytometry is widely used for the analysis of cellular protein expression levels. Irish et al used multiparameter flow cytometry to examine the phosphorylation state of signaling proteins in acute myelogenous leukemia cells stimulated by various cytokines. Sachs et al have examined the phosphorylation levels of 11 proteins and lipids in human CD4+ T cells using flow cytofluorescent sorting.
Bendall and Nolan et al, combined with flow cytometry and Mass spectrometry, propose a new Mass cytometry (Mass cytometry). The metal element label is used for labeling antibody dye, the mass spectrum principle is utilized to carry out multi-parameter quantitative detection on the single cells, and the application in the aspect of single cell proteomics analysis is good. Traditional flow cytometry techniques are mainly based on the detection of fluorescence emission spectra, so that there are limitations on the number of detection channels and complicated compensation processes for spectral overlap cross-color. The mass flow cytometry is the fusion of two experimental platforms of the mass flow cytometry and the flow cytometry, not only inherits the characteristic of high-speed analysis of the traditional flow cytometer, but also has the high resolution capability of mass detection. Compared with the traditional flow technology, the ICP mass spectrometer in the mass flow has a very wide atomic weight detection range (88-210 Da), the resolution is extremely high, the interference between adjacent channels is less than 0.3%, the number of the channels is increased, and compensation calculation is avoided. Therefore, the experimental process is simplified, and the samples and the reagents are saved. Evan W.Newell et al used mass spectrometry flow cytometry to analyze the expression of 25 surface markers and functional proteins of human CD8T cells and the binding of three different viral epitopes, and showed the continuous process of T cell maturation, revealing that there are complex functional polymorphisms in T cell populations.
In order to obtain spatial information of protein expression, a scholars such as Giesen C and the like organically combines immunohistochemistry and mass flow spectrometry by using a laser ablation technology to obtain subcellular localization images of 32 proteins in the same visual field (the image resolution can reach 1 um). The imaging mass spectrometry flow technology is utilized to research a human breast cancer slice sample, the cell is subjected to subgroup typing and the polymorphism of tumor tissue cells is researched according to the analysis of the obtained image information, the tissue function and polymorphism are promoted to be researched actively, and a technical basis is provided for personalized molecular targeted therapy. The IsoLight single cell function information multiple detection system of IsoPleexis is the most advanced and indispensable solution in the single cell analysis field at present, and can provide complete immune cell function response analysis under the resolution and sensitivity of single cells to help predict and understand the complex patient reaction of cancer immunotherapy, so that the immune function response is directly associated with CAR-T or other important immunotherapy clinical results, thereby promoting the development of high-demand fields such as cellular immunotherapy research and development, product characterization analysis, immune biomarker discovery, patient efficacy prediction and late-stage monitoring.
Western blotting (Western Blot) is a commonly used protein assay in cell and molecular biology and immunogenetics. A specific procedure is to detect proteins in a sample by separating the proteins in the sample using gel electrophoresis, followed by transfer of the proteins to a membrane (typically nitrocellulose or PVDF), followed by probing with an antibody specific for the target protein. Since the protein is subjected to electrophoretic separation and then to antibody binding reaction, the protein is less affected by the cross-reactivity of the antibody. Thus, even in complex samples such as cell lysates, on-target and off-target signals can be clearly distinguished. However, the results determined in the conventional western blotting method are based on the average expression level of proteins in a large number of cell samples, and the results mask the specificity and diversity of the expression amount of proteins in individual cells.
Hughes and Herr developed single cell immunoblotting (scWB), a single cell protein detection technique that combines microchip-based sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with in-gel protein light trapping and immunodetection. scWB used light-induced blotting technique to immobilize proteins in situ and could detect 11 protein targets in a single cell by detecting fluorescent antibodies. Based on this technique, Herr et al monitored neural stem cell differentiation and response to mitogen stimulation in a single rat. At the same time, the technology is also used for analyzing the expression of eight proteins from single CTC in breast cancer patients, and has important significance for understanding cancer metastasis. Undoubtedly, the development of scWB has provided a strong impetus for proteomic analysis of rare cells. However, scWB has high background fluorescence in detecting target proteins due to limitation of photosensitizer in gel, which is not satisfactory for detecting low abundance proteins.
In recent years, microfluidic technology has developed rapidly and a series of microchip-based technologies have emerged, providing a more efficient platform for single-celled omics analysis. Recent developments in microchip technology have provided a variety of new and feasible solutions for single cell research. By creating a micro-laboratory of about one unit size, sample consumption and reaction time can be reduced while significantly increasing throughput and analytical scale. Furthermore, operating under laminar flow conditions can provide a well-controlled environment, allowing for more accurate operation and more sensitive measurements at the single cell level. These techniques also provide a platform for automation, integration, and parallelization that can further reduce labor and cost requirements, thereby pushing single cell-based research to a higher level.
The above techniques have proved to be very important for analyzing the significant differences in protein expression levels among single cells, but all suffer from drawbacks.
1. Fluorescence flow cytometry, mass flow imaging technology and the like do not really achieve single cell sampling and single cell detection. These techniques are performed after a minimum of 105 cells are loaded, and thus these methods cannot be applied to rare cells such as embryos, circulating tumor cells, or cells with low transfection efficiency.
2. There is a degree of specificity in the binding between antigen and antibody. Therefore, protein immunoassay methods which rely only on the binding of antigen and antibody to recognize target protein molecules for detection, such as microfluidic technology, fluorescence flow cytometry, mass spectrometry flow imaging technology, single cell secreted protein isoplex technology and the like, have low specificity and may have high false positive. And also limited by the variety of specific probes, making such methods limited in their application.
3. Considering the detection of cell surface proteins, transmembrane proteins, secretory proteins and intracellular and even nuclear proteins, direct detection of intracellular and nuclear proteins in flow cytometry is difficult to operate. The single cell secretory protein detection technology can only detect secretory proteins, but cannot detect cell surface, transmembrane proteins, intracellular and nuclear proteins.
4. The microfluidic technology has the advantages of less sample reagent consumption, diversified structural functions, high integration degree and the like. However, the preparation process of the chip is complex, and the single cell separation and detection are difficult to control. The biocompatibility of the material used for preparing the chip is not good enough, and the cell function representation can be changed, so that the measured result can not reflect the real situation in the real human body life activity state.
5. The single cell western blot combines the molecular sieve effect of SDS-PAGE, and through the double verification of the molecular weight of the protein and the recognition of antigen and antibody, the specificity of detection is ensured, and meanwhile, the single cell western blot can detect cell surface protein, transmembrane protein, intracellular and nuclear protein and distinguish epigenetic modified protein. Based on BPMAC, however, the photosensitive immobilization efficiency is low, the lower detection limit is 27000 single-cell protein molecules, and the method is difficult to be applied to the detection of low-abundance proteins and has a great promotion space. And the background fluorescence is higher, and the method sensitivity is low. The excitation wavelength is shorter (UVB, 280-320 nm), and the inactivation of protein antigen sites can be caused, so that false negative is caused. Furthermore, it is difficult to apply to single cell protein analysis of rare cell populations. For example, circulating tumor cells (1-10 cells/ml blood). The single cell western blot detection uses the traditional fluorescent label, the channel is few, the signals are easy to overlap, the detection needs multiple washing and dyeing, and the antigen loss is large.
Therefore, those skilled in the art have made efforts to develop a single-cell protein detection means which can store data stably and process detection results digitally with high throughput and short time, and which is portable, compact, functionally stable and low cost.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention is directed to a single-cell protein detection device with high throughput, short time, stable data storage and digital detection, portability, small size, stable function, and low cost.
In order to achieve the above object, the present invention provides a single-cell protein digital imaging detection method, which is characterized in that the method comprises the following steps:
and 6, carrying out target protein immunoblotting detection by using the labeled antibody, and observing an analysis result.
Preferably, the step 1 further comprises:
step 1.1, baking a silicon wafer with one side coated with SU8-2050 gel on a hot plate at 65 ℃ for 1 minute, and then baking at 95 ℃ for 8 minutes;
step 1.2, the silicon wafer is placed on an exposure machine for exposure for 4 minutes of UV exposure, the UV wavelength is 365nm, and the intensity is 30mW/cm2；
Step 1.3, baking the silicon wafer subjected to exposure in the step 1.2 on a hot plate at 65 ℃ for 2 minutes, and then baking at 95 ℃ for 7 minutes;
step 1.4, coating a second layer of SU8-2050 gel on the first layer of SU8-2050 gel layer of the silicon wafer for homogenization, then baking the mixture on a hot plate at 65 ℃ for 1 minute, and then baking the mixture at 95 ℃ for 2 minutes;
step 1.5, sticking the designed film mask on a silicon wafer, and then placing the silicon wafer into an exposure machine for UV exposure for 4 minutes, wherein the UV wavelength is 365nm, and the intensity is 30mW/cm2；
Step 1.6, baking the silicon wafer subjected to exposure in the step 1.5 on a hot plate at 65 ℃ for 2 minutes, and then baking at 95 ℃ for 7 minutes to prepare a chip template;
step 1.7, fixing the chip template by using tweezers, and washing for 2-3 minutes by using a developing solution;
step 1.8, the chip template is cleaned with isopropanol, acetone, ethanol and deionized water and then dried at 65 ℃.
Preferably, the photosensitizer for protein-immobilized gel in step 2 is N- (3-methacrylamidoylpropyl) -2- (1-methyl-1H-pyrrol-2-yl) -2H-tetrazole-5-carboxamide, MAP-mPyTC, which is prepared as follows:
step 2.1, preparation of ethyl 2- (1-methyl-1H-pyrrol-2-yl) -2H-tetrazole-5-carboxylate:
step 2.1.1, dissolve 5g, i.e. 12.5mmol, of methyl-1 hydro-pyrrole in 30mL of trifluoroethanol;
step 2.1.2, adding 20g of 62.5mmol of iodobenzene diacetate into the solution obtained in the step 2.1.1 at the temperature of minus 40 ℃, and stirring for 2 hours at the temperature of minus 40 ℃ under the protection of nitrogen;
step 2.1.3, concentrating the product of the step 2.1.2 to be black oil, and dissolving the black oil in dichloromethane;
step 2.1.4, adding 3g of 21.6mmol of 5-ethyl formate tetrazole, 3.1g of 8.6mmol of copper (II) trifluoromethanesulfonate and 16ml of 108mmol of triethylamine into the solution in the step 2.1.3, and stirring for 24 hours at room temperature under the protection of nitrogen to react;
step 2.1.5, after the reaction is finished, washing the product by using saturated ammonium chloride, drying by using anhydrous magnesium sulfate, and then filtering and drying;
and 2.1.6, further purifying the product by silica gel flash chromatography, wherein an eluent of the silica gel flash chromatography is PE: EA ═ 5:1 in volume ratio to obtain a brown oily product I, and the product I is 2- (1-methyl-1H-pyrrole-2-yl) -2H-tetrazole-5-carboxylic acid ethyl ester, and the structural formula is shown as the formula I:
step 2.2, preparing 2- (1-methyl-1H-pyrrole-2-yl) -2H-tetrazole-5-carboxylic acid;
step 2.2.1, dissolving 450mg, i.e. 2mmol, of product I in step 2.1 in a volume ratio of MeOH: in a total of 20ml of the mixed solution of H2O-1: 1;
step 2.2.2, adding 850mg of lithium hydroxide, namely 10mmol of lithium hydroxide into the solution obtained in the step 2.2.1 at the temperature of 0 ℃;
2.2.3, placing the mixture obtained in the step 2.2.2 at room temperature, and stirring and reacting for 2 hours under the protection of nitrogen;
step 2.2.4, after the reaction is finished, adding 2N HCL at the temperature of 0 ℃, and adjusting the pH to 4-5;
step 2.2.5, extracting with 98% ethyl acetate solution, washing the mixed organic phase with saturated ammonium chloride, drying with anhydrous sodium sulfate, filtering and concentrating to obtain brown solid product II, namely 2- (1-methyl-1H-pyrrole-2-yl) -2H-tetrazole-5-carboxylic acid, the structural formula is shown as formula II:
step 2.3, preparing N- (3-methacrylamidoylpropyl) -2- (1-methyl-1H-pyrrol-2-yl) -2H-tetrazole-5-carboxamide;
step 2.3.1, dissolve 370mg, i.e. 1.67mmol, of N- (3-aminopropyl) methacrylate/hydrochloride in 20mL of tetrahydrofuran;
step 2.3.2, 650mg, i.e. 3.34mmol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide-HCl, i.e. EDCl. HCl, 220mg, i.e. 1.67mmol of 1-hydroxybenzotriazole, i.e. HOBt, are added to the solution from step 2.3.1 at 0 ℃ and reacted for 30 minutes;
step 2.3.3, adding 300mg of 1.67mmol of product II and 850mg of 8.35mmol of triethylamine in the step 2.2 into the mixed solution in the step 2.3.2 at 0 ℃, and stirring, refluxing and reacting overnight;
step 2.3.4, after the reaction is finished, purifying the product by preparative HPLC to obtain a white powder product III, namely N- (3-methacrylamidopropyl) -2- (1-methyl-1H-pyrrol-2-yl) -2H-tetrazole-5-formamide, namely MAP-mPyTC, the structural formula of which is shown in formula III:
the MAP-mPyTC prepared in the step 3 is copolymerized with acrylamide and N, N' -methylene bisacrylamide to form MPyTC Modified Polyacrylamide (MMP) light-sensitive protein immobilized gel.
In another preferred embodiment, the structural formula of the protein immobilized gel photosensitizer in step 2 is shown as formula IV, wherein R group is an electron-withdrawing group:
the R group is selected from one of the following groups:
preferably, the step 3 further comprises:
step 3.1, treating a standard glass slide with 98 percent of 3- (trimethoxysilyl) propyl methacrylate serving as a silylation reagent for 30 minutes, washing the standard glass slide twice by using methanol and deionized water, and drying the standard glass slide by using nitrogen;
step 3.2, dissolving the gel photosensitizer prepared in the step 2 in dimethyl sulfoxide (DMSO) to prepare a storage solution S1, wherein the concentration of S1 is 100 mM;
step 3.3, in a 1.5ml Ep tube, 25uL of 1.5M Tris-HCl with pH8.8, 166.7uL of 30% acrylamide/methylene bisacrylamide mixture with 29:1, 265 were added.3uL ddH2O, 3.75-15uL 100mM S1, and preparing a colloid precursor (precursor);
step 3.4, adding 10ul of 5% SDS, 4ul of APS and 4ul of TEMED into the gel precursor in the step 3.3, and shaking up lightly to prepare the light-sensitive protein immobilized gel solution;
step 3.5, spreading the protein immobilized gel solution prepared in step 3.4 on the chip template prepared in step 1, and then covering with the silanized glass slide in step 3.1;
step 3.6, standing for 20 minutes, lifting the glass slide, stripping the chip template to prepare a western blot chip, and storing the western blot chip in a refrigerator at 4 ℃;
step 3.7, preparing RIPA-like lysate: preparing a mixed solution of SDS with the final concentration of 0.5%, Triton X-100 with the final concentration of 0.1%, sodium deoxycholate with the final concentration of 0.25%, Tris with the final concentration of 12.5mM and glycine with the pH value of 8.3 to obtain a lysate, wherein the lysate is also an electrophoresis buffer and is stored at the temperature of 4 ℃.
Preferably, the western blot chip prepared in step 3 is a light-sensitive protein immobilized gel attached to one side of the slide, the chip template prepared in step 1 has a plurality of protruding cylinders to form a cylinder array, so that the gel part on the western blot chip prepared in step 3 has a plurality of micropores to form a porous microarray, in addition, the chip template has a plurality of hydrophobic regions, the hydrophobic regions are created by drawing lines with hydrophobic isolation pens, so that the gel part of the western blot chip forms a plurality of isolation regions without gel, the diameter of the cylinders is 30 μm, the width of the isolation regions is 2mm, and the isolation regions divide the western blot chip into three isolation regions.
Preferably, the sample to be tested in step 4 is a cell sample transfected with a reporter (reporter) gene in advance, and step 4 further includes loading the cell sample into the western blot chip prepared in step 3, allowing the cell to sink into a micropore in the chip, allowing the diameter of the micropore to ensure that one cell falls into one micropore, assigning a cell trap coordinate to each cell based on the position of the micropore, washing the excess cells with 0.01M, pH ═ 7 phosphate buffer, observing and recording transfected cells expressing the reporter (reporter) gene by a confocal fluorescence microscope, and recording the coordinates.
Preferably, the step 5 further comprises:
step 5.1, heating the lysis solution/electrophoresis buffer solution prepared in the step 3 to 50-55 ℃ in a water bath, and turning on ultraviolet in advance to stabilize a light source;
step 5.2, placing the Western blot chip prepared in the step 3 in a new dish with the glue surface facing upwards, dropwise adding 200ul of BSA solution with the concentration of 5.12mg/mL, slightly shaking the slide to ensure that the BSA solution is uniformly distributed, and standing for 3 minutes;
step 5.3, placing the Western blot chip in an electrophoresis tank, and pouring 10ml of RIPA-like lysate preheated in a water bath from one corner of the electrophoresis tank gently as soon as possible;
step 5.4, immediately turning on a voltage power supply, wherein the voltage is 200V, and E is 40V/cm2Protein is separated for 30 seconds by electrophoresis;
step 5.5, immediately stopping the voltage, and exposing for 15 seconds to 10 minutes under the ultraviolet of 340-;
step 5.6, after the exposure is finished, taking out the Western blot chip, and placing the Western blot chip in Coomassie brilliant blue dye solution for dyeing for 5 minutes, wherein the Coomassie brilliant blue dye solution is prepared by dissolving 0.1g of Coomassie brilliant blue powder in mixed solution of 20mL of methanol, 16mL of water and 4mL of acetic acid;
step 5.7, taking a picture, and recording the protein fixation condition;
5.8, shaking and washing by using 0.01M TBST, changing the liquid every 15 minutes for 2 hours, and then shaking and washing overnight;
and 5.9, taking a picture and recording the protein fixation condition.
Preferably, the step 6 further comprises:
step 6.1, metal-labeled antibody: selecting antibody solution without BSA, carrying out metal labeling, carrying out antibody titration after the labeling is finished and determining the use concentration of the metal labeled antibody, wherein the antibody solution labeled with the metal has at least 30 metal elements;
step 6.2, incubation of the protein with metal antibody: mixing antibody solutions of various target proteins according to the use concentration of the metal-labeled antibody determined in the step 6.1, dropwise adding the mixed antibody solutions to the surface of a western blotting chip, and incubating for 4 hours at 37 ℃;
and 6.3, after the incubation is finished, washing the protein blot chip by using 0.01M TBST, freezing the marked protein blot chip in a refrigerator at the temperature of minus 80 ℃, and freezing the protein blot chip again by using liquid nitrogen before loading.
Step 6.4, detecting by a mass spectrometer: ablating the coordinate region recorded in the step 4, setting the ablation energy as 12, and detecting by using a mass spectrometer;
and 6.5, carrying out digital processing and analysis on data detected by a mass spectrometry imager by using image J and ilastik software and a statistical analysis method so as to realize specific quantitative or semi-quantitative detection on the target protein and construct a single-cell protein map.
In other embodiments of the present invention, the sample to be tested in step 4 is selected from one of single cell, multi-cell, extracted protein, purified protein solution, DNA-protein sample or RNA-protein sample, and the antibody in step 6 is selected from one of fluorescence, enzyme, colloidal gold or superparamagnetic microsphere labeled antibody.
Through the technical scheme of the invention, the following technical effects can be achieved:
1. and (5) detecting single cells. Realize unicellular loading in the true sense, solve the difficult problem of rare cell protein analysis. High throughput, multiple species loading.
2. The primary antibody is marked by metal, and a secondary antibody is not needed for incubation, so that the experiment time is saved.
3. Multiple proteins can be detected at the single cell level at the same time, which is convenient for constructing a protein interaction network in the same cell.
4. The data can be processed by an algorithm to deeply mine the interaction relationship between the proteins.
5. The device is small and portable. Compared with a fluorescence flow cytometer and a mass spectrometer, the chip of the mass spectrometer has the characteristics of small size and portability, and is more beneficial to developing clinical application.
6. The chip data can be stably stored. In order to effectively compare data for many times, the protein data in the chip should not be attenuated within a certain time so as to facilitate the check and detection for many times.
7. The price is lower. Compared with the traditional fluorescence flow cytometry, the mass spectrometry flow cytometry is required to realize detection with lower price.
The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.
Drawings
FIG. 1 is a schematic diagram of a film mask according to a preferred embodiment of the present invention.
FIG. 2 is a synthesis route diagram of the novel light-sensitive protein-immobilized gel photosensitizer in accordance with a preferred embodiment of the present invention.
FIG. 3 is a NMR spectrum of ethyl 2- (1-methyl-1H-pyrrol-2-yl) -2H-tetrazole-5-carboxylate, a product of a photosensitizer synthesis scheme in accordance with a preferred embodiment of the present invention.
FIG. 4 is a NMR spectrum of the photosensitizer MAP-mPyTC in accordance with a preferred embodiment of the present invention.
FIG. 5 is a mass spectrometric identification of the photosensitizer MAP-mPyTC in accordance with a preferred embodiment of the present invention.
FIG. 6 is a UV-VIS absorption spectrum of a light-sensitive protein immobilized gel using MAP-mPyTC according to a preferred embodiment of the present invention.
FIG. 7 is a schematic diagram of a chip template and a Western blot chip according to a preferred embodiment of the invention.
FIG. 8 is a graph showing the results of comparing the capture efficiency of proteins by UV irradiation at different concentrations of photosensitizer and for different durations in accordance with a preferred embodiment of the present invention.
FIG. 9 is a graph showing the results of a comparison of protein separations for different concentrations of the novel photosensitive gel of the present invention.
FIG. 10 is a schematic diagram of a method for detecting a protein by using a metal-labeled antibody according to a preferred embodiment of the present invention.
FIG. 11 is a diagram of a mass spectrometer imager detection software interface in accordance with a preferred embodiment of the present invention.
Detailed Description
The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
In the drawings, structurally identical elements are represented by like reference numerals, and structurally or functionally similar elements are represented by like reference numerals throughout the several views. The size and thickness of each component shown in the drawings are arbitrarily illustrated, and the present invention is not limited to the size and thickness of each component. The thickness of the components may be exaggerated where appropriate in the figures to improve clarity.
The innovation point of the method provided by the invention is that the variant chemistry of the scWB method combining in-situ immunoblotting and light-click bioorthogonal chemistry can detect hundreds of target proteins in one cell simultaneously in theory to construct a single-cell protein map. The specific method comprises the following steps:
and 6, carrying out target protein immunoblotting detection by using the labeled antibody, and observing an analysis result.
In step 1, a schematic diagram of a film mask is shown in fig. 1, a mask pattern adopted in a preferred embodiment of the present invention is designed by CAD software, a film mask is produced by shenzhen nanochip electronics, and in the preferred embodiment, the space between each row of cell traps is 500 μm, the radius of the cell traps is 15 μm, and the space between each row of cell traps is 2mm, that is, the electrophoretic distance is 2mm at the corresponding cell trap positions on the film mask.
step 1.1, baking a silicon wafer with one side coated with SU8-2050 gel on a hot plate at 65 ℃ for 1 minute, and then baking at 95 ℃ for 8 minutes;
step 1.2, the silicon wafer is placed on an exposure machine for exposure for 4 minutes of UV exposure, the UV wavelength is 365nm, and the intensity is 30mW/cm2；
Step 1.3, baking the silicon wafer subjected to exposure in the step 1.2 on a hot plate at 65 ℃ for 2 minutes, and then baking at 95 ℃ for 7 minutes;
step 1.4, coating a second layer of SU8-2050 gel on the first layer of SU8-2050 gel layer of the silicon wafer for homogenization, then baking the mixture on a hot plate at 65 ℃ for 1 minute, and then baking the mixture at 95 ℃ for 2 minutes;
step 1.5, sticking the designed film mask on a silicon wafer, and then placing the silicon wafer into an exposure machine for UV exposure for 4 minutes, wherein the UV wavelength is 365nm, and the intensity is 30mW/cm2；
The film mask is adhered to the transparent glass in the same size, the glued silicon wafer is set below the transparent glass, and the ultraviolet exposure irradiates the glued silicon wafer through the glass with the film mask.
Step 1.6, baking the silicon wafer subjected to exposure in the step 1.5 on a hot plate at 65 ℃ for 2 minutes, and then baking at 95 ℃ for 7 minutes to prepare a chip template;
step 1.7, fixing the chip template by using tweezers, and washing for 2-3 minutes by using a developing solution;
step 1.8, the chip template is cleaned with isopropanol, acetone, ethanol and deionized water and then dried at 65 ℃.
Example 1:
in a preferred embodiment of the present invention, N- (3-methacrylamidopropyl) -2- (1-methyl-1H-pyrrol-2-yl) -2H-tetrazole-5-carboxamide, MAP-mPyTC, is used as an additive in a protein immobilization gel, and the synthesis steps of MAP-mPyTC are as follows, referring to FIG. 2:
step 2.1, preparation of ethyl 2- (1-methyl-1H-pyrrol-2-yl) -2H-tetrazole-5-carboxylate:
step 2.1.1, dissolve 5g, i.e. 12.5mmol, of methyl-1 hydro-pyrrole in 30mL of trifluoroethanol;
step 2.1.2, adding 20g of 62.5mmol of iodobenzene diacetate into the solution obtained in the step 2.1.1 at the temperature of minus 40 ℃, and stirring for 2 hours at the temperature of minus 40 ℃ under the protection of nitrogen;
step 2.1.3, concentrating the product of the step 2.1.2 to be black oil, and dissolving the black oil in dichloromethane;
step 2.1.4, adding 3g of 21.6mmol of 5-ethyl formate tetrazole, 3.1g of 8.6mmol of copper (II) trifluoromethanesulfonate and 16ml of 108mmol of triethylamine into the solution in the step 2.1.3, and stirring for 24 hours at room temperature under the protection of nitrogen to react;
step 2.1.5, after the reaction is finished, washing the product by using saturated ammonium chloride, drying by using anhydrous magnesium sulfate, and then filtering and drying;
and 2.1.6, further purifying the product by silica gel flash chromatography, wherein an eluent of the silica gel flash chromatography is PE: EA ═ 5:1 in volume ratio to obtain a brown oily product I, and the product I is 2- (1-methyl-1H-pyrrole-2-yl) -2H-tetrazole-5-carboxylic acid ethyl ester, and the structural formula is shown as the formula I:
yield: 450mg (9.6%). 1H NMR (400MHz, DMSO) δ 7.13-7.00(M,1H),6.65(dd, J ═ 3.9,1.9Hz,1H),6.24(dd, J ═ 3.9,3.0Hz,1H),4.47(q, J ═ 7.1Hz,2H),3.65(s,3H),1.37(t, J ═ 7.1Hz,3H) [ M + H + ],222.0, and a nuclear magnetic resonance hydrogen spectrum is shown in fig. 3.
Step 2.2, preparing 2- (1-methyl-1H-pyrrole-2-yl) -2H-tetrazole-5-carboxylic acid;
step 2.2.1, dissolving 450mg, i.e. 2mmol, of product I in step 2.1 in a volume ratio of MeOH: in a total of 20ml of the mixed solution of H2O-1: 1;
step 2.2.2, adding 850mg of lithium hydroxide, namely 10mmol of lithium hydroxide into the solution obtained in the step 2.2.1 at the temperature of 0 ℃;
2.2.3, placing the mixture obtained in the step 2.2.2 at room temperature, and stirring and reacting for 2 hours under the protection of nitrogen;
step 2.2.4, after the reaction is finished, adding 2N HCL at the temperature of 0 ℃, and adjusting the pH to 4-5;
step 2.2.5, extracting with 98% ethyl acetate solution, washing the mixed organic phase with saturated ammonium chloride, drying with anhydrous sodium sulfate, filtering and concentrating to obtain brown solid product II, namely 2- (1-methyl-1H-pyrrole-2-yl) -2H-tetrazole-5-carboxylic acid, the structural formula is shown as formula II:
step 2.3, preparing N- (3-methacrylamidoylpropyl) -2- (1-methyl-1H-pyrrol-2-yl) -2H-tetrazole-5-carboxamide;
step 2.3.1, dissolve 370mg, i.e. 1.67mmol, of N- (3-aminopropyl) methacrylate/hydrochloride in 20mL of tetrahydrofuran;
step 2.3.2, 650mg, i.e. 3.34mmol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide-HCl, i.e. EDCl. HCl, 220mg, i.e. 1.67mmol of 1-hydroxybenzotriazole, i.e. HOBt, are added to the solution from step 2.3.1 at 0 ℃ and reacted for 30 minutes;
step 2.3.3, adding 300mg of 1.67mmol of product II and 850mg of 8.35mmol of triethylamine in the step 2.2 into the mixed solution in the step 2.3.2 at 0 ℃, and stirring, refluxing and reacting overnight;
step 2.3.4, after the reaction is finished, purifying the product by preparative HPLC to obtain a white powder product III, namely N- (3-methacrylamidopropyl) -2- (1-methyl-1H-pyrrol-2-yl) -2H-tetrazole-5-formamide, namely MAP-mPyTC, the structural formula of which is shown in formula III:
white powder III (41mg, 6.7% yield) was obtained.
1H NMR (400MHz, MeOD) δ 6.97-6.82 (M,1H),6.60(dd, J ═ 4.0,1.9Hz,1H), 6.23(dd, J ═ 3.9,3.0Hz,1H), 5.78-5.64 (M,1H), 5.50-5.23 (M,1H),3.71(s,3H),3.50(t, J ═ 6.8Hz,2H),3.36(t, J ═ 6.7Hz,2H), 2.03-1.92 (M,3H),1.87(p, J ═ 6.7Hz,2H) [ M + H + ], 318.1. As shown in formula III, the NMR spectrum and the mass spectrum identification spectrum are respectively shown in FIGS. 4 and 5.
MAP-mPyTC prepared in the step is copolymerized with acrylamide and N, N' -methylene bisacrylamide in the step 3 at room temperature to form MPyTC Modified Polyacrylamide (MMP) light-sensitive protein immobilized gel.
Light absorption characteristics:
the ultraviolet-visible absorption spectrum of the compound synthesized in the embodied example 1 was measured with an ultraviolet-visible spectrophotometer manufactured by agilent technologies (china) ltd. The instrument model is Evolution 220UV-Vis, and the spectrum scanning range is from 300nm to 800 nm. The UV-VIS absorption spectrum shown in FIG. 6 was obtained, thereby obtaining the light absorption characteristics of the light-sensitive protein-immobilized gel of the present invention.
Example 2:
in addition, acrylamide gel based on tetrazole as a photosensitive group can replace acrylamide gel, and other analogues can be adopted as the photosensitizer in the photosensitive protein fixing gel, wherein the structural formula is shown as formula IV, and R groups are electron-pulling groups:
preferably, the R group is selected from one of the following groups.
In step 3-4, a light-sensitive protein immobilized gel needs to be prepared, and a single-cell western blot chip is prepared by matching the chip template in step 1, the schematic diagram of the chip template and the chip in one embodiment of the invention is shown in fig. 7, and a protruding structure on the surface of the chip template is formed due to the special design of the film mask in step 1, so that the chip prepared in step 3 forms a complementary recessed structure, namely a micropore, under the action of the template. When the sample is loaded as a cell, the microwell can be used to capture cells, often referred to as a "cell trap," since the diameter of the microwell is comparable to the diameter of the cell.
Example 3:
as shown in FIG. 7, in a preferred embodiment of the present invention, the Western blot chip prepared in step 3 is a light-sensitive protein immobilized gel attached to one side of the slide glass, the chip template prepared in step 1 has a plurality of protruding cylinders constituting a cylinder array, such that the gel portion of the Western blot chip prepared in step 3 has a plurality of micropores, forming a porous microarray, and further, the chip template has a plurality of hydrophobic regions, which are created by hydrophobic isolation stroke lines, the hydrophobic pen is a common tool in immunohistochemistry, and can draw a thick line on the glass, the line is a hydrophobic chemical substance, the aqueous solution is obstructed by the line and will not cross the border, such that the gel portion of the Western blot chip forms a plurality of isolated regions without gel, the diameter of the cylinders is 30 μm, the width of the isolation region is 2mm, and the isolation region divides the Western blot chip into three isolation regions.
The method for carrying out silanization treatment on the glass slide in advance comprises the following steps:
standard glass slides were treated with 98% 3- (trimethoxysilyl) propyl methacrylate in silanizing reagent for 30 minutes, then washed twice with methanol, deionized water, and dried with nitrogen.
In step 4, when the sample is a cell, the sample to be tested needs to be transfected with a cell sample of a reporter (reporter) gene in advance, and the step 4 further comprises loading the cell sample into the western blot chip prepared in step 3, allowing the cell to settle into a micropore in the chip, allowing the diameter of the micropore to ensure that one cell falls into one micropore, assigning a cell trap coordinate to each cell based on the position of the micropore, washing the excess cells with 0.01M, pH ═ 7 phosphate buffer, observing and recording the transfected cell expressing the reporter (reporter) gene by using a confocal fluorescence microscope, and recording the coordinate. And 5, during electrophoresis, the electrophoresis solution is also a lysis solution, the cells are cracked into a protein solution, the protein solution enters the gel under the action of an electric field force, the separation of the proteins is realized under the action of the electrophoresis due to different molecular weights of different proteins, and after the proteins are separated in the gel, photosensitive groups are excited under the action of ultraviolet illumination, react with but are not limited to the proteins, and the proteins are fixed in the gel in a crosslinking manner.
The sample to be tested can be directly dripped on the chip or can be loaded in other modes, and in a preferred embodiment of the invention, the sample to be tested can be loaded on the chip through a piece of glass and added by capillary action, so that the cells are captured in the flowing process.
To further investigate the effect of different photosensitizer concentrations and different UV exposure times on the fixation efficiency of proteins, different control experiments were performed in accordance with the present invention, with specific reference to examples 4-5.
Example 4:
fixing efficiency of different-concentration light-sensitive protein fixing gel on bovine serum albumin BSA (bovine serum albumin)
1. The product III from example 1 was dissolved in dimethyl sulfoxide DMSO and prepared as a 100mM stock solution S1.
2. In a 1.5ml Ep tube, 25uL of 1.5M Tris-HCl, pH8.8, 166.7uL 30%29:1 acrylamide/methylene bisacrylamide, 265.3uL ddH2O, and 15uL (3%), 7.5uL (1.5%), 3.75uL (0.75%), 0uL (0%) S1 were added to prepare a gel precursor.
3. And 10ul of 5% SDS, 4ul of APS, 4ul of TEMED were added and shaken gently.
And dropwise adding and spreading the solution on a porous microarray chip template, slightly covering the slide subjected to silanization treatment to avoid air bubbles, standing for 20min, and stripping the chip template after the glue is solidified to prepare the western blotting chip with the porous microarray gel.
4. A RIPA-like lysate (also known as running buffer) was prepared. 0.5% SDS, 0.1% v/v Triton X-100, 0.25% sodium deoxycholate, 12.5mM Tris, 96mM glycine, pH 8.3. Stored at 4 degrees.
5. Heating lysis solution/electrophoresis solution in water bath to 50-55 deg.C. The ultraviolet is turned on in advance to stabilize the light source.
6. The chip was placed in a new dish with the glue side up, 200ul of BSA solution (5.12mg/mL) was added dropwise, and the slide was gently shaken to distribute the BSA solution evenly. Standing for 3 min.
7. The gel is placed in an electrophoresis tank, and is gently poured into 10ml of RIPA-like cracking solution preheated in a water bath from one corner of the electrophoresis tank as soon as possible.
8. Immediately turn on the voltage supply, 200V (E ═ 40V/cm2), and the protein was electrophoretically separated for 30 s.
9. The voltage was immediately terminated and UV exposure at 346nm was carried out for 10 min.
10. After the exposure, the chip was removed and stained in Coomassie Brilliant blue stain (0.1g Coomassie Brilliant blue powder in 20mL methanol +16mL water +4mL acetic acid) for 5 min.
11. Pictures were taken and protein fixation was recorded.
12. Shaking with 0.01M TBST, changing solution every 15min for 2h, and then washing overnight with shaking.
13. Pictures were taken and protein fixation was recorded.
14. As shown in fig. 8. The result shows that the S1 light-sensitive protein fixed gel with low concentration level has strong and higher fixing efficiency on the protein in the gel after being excited by ultraviolet.
Example 5:
fixing efficiency of light-sensitive protein fixing gel on bovine serum albumin BSA (bovine serum albumin) at different ultraviolet irradiation times
1. The product III from example 1 was dissolved in dimethyl sulfoxide DMSO and prepared as a 100mM stock solution S1.
2. In a 1.5ml Ep tube, 25uL of 1.5M Tris-HCl pH8.8, 166.7uL of 30% 29:1 acrylamide/methylene bisacrylamide, 265.3uL of ddH were added2O, 3.75uL (0.75%) of S1, formulated as a gel precursor.
3. And 10ul of 5% SDS, 4ul of APS, 4ul of TEMED were added and shaken gently. And dropwise adding and spreading the solution on a porous microarray chip template, slightly covering the silanized slide to avoid bubbles, standing for 20min, and stripping the chip template after the gel is solidified to prepare the protein blotting chip with the porous microarray gel.
4. A RIPA-like lysate (also known as running buffer) was prepared. 0.5% SDS, 0.1% v/v Triton X-100, 0.25% sodium deoxycholate, 12.5mM Tris, 96mM glycine, pH 8.3. Stored at 4 degrees.
5. Heating lysis solution/electrophoresis solution in water bath to 50-55 deg.C. The ultraviolet is turned on in advance to stabilize the light source.
6. The chip was placed in a new dish with the glue side up, 200ul of BSA solution (5.12mg/mL) was added dropwise, and the slide was gently shaken to distribute the BSA solution evenly. Standing for 3 min.
7. The gel is placed in an electrophoresis tank, and is gently poured into 10ml of RIPA-like cracking solution preheated in a water bath from one corner of the electrophoresis tank as soon as possible.
8. Immediately turn on the voltage supply, 200V (E ═ 40V/cm2), and the protein was electrophoretically separated for 30 s.
9. The voltage was immediately terminated, and UV exposure at 346nm was carried out. The exposure time is set to 10min, 6min, 4min, 2min, 1min, 30s, 15s, respectively.
10. After the exposure, the chip was removed and stained in Coomassie Brilliant blue stain (0.1g Coomassie Brilliant blue powder in 20mL methanol +16mL water +4mL acetic acid) for 5 min.
11. Pictures were taken and protein fixation was recorded.
12. Shaking with 0.01M TBST, changing solution every 15min for 2h, and then washing overnight with shaking.
13. Pictures were taken and protein fixation was recorded.
14. As shown in fig. 8. The light-sensitive protein fixing gel has stronger and higher fixing efficiency on the protein in the gel after being excited by ultraviolet within a shorter time (within 30 s).
As shown in the above two examples, in the preparation of mPyTC-modified polyacrylamide (MMP) gels, Triton X-100 was removed from the gel contents, otherwise the cell sample would fall into the microwell and start to lyse, which affects the cell status during subsequent observation using a confocal fluorescence microscope, since when using metal-labeled antibodies it is necessary to ensure that the cell status is intact during observation using a confocal fluorescence microscope.
In addition, when the protein is immobilized, the wavelength of ultraviolet light is preferably 340-350nm, and the irradiation time is preferably 30 seconds to 10 minutes.
Example 6:
the invention also carries out a comparison experiment of common gel, common photosensitive gel and novel photosensitive gel added with the photosensitizer synthesized in the invention under different acrylamide mixture gel concentrations, and the result is shown in figure 9, and the figure shows that the novel photosensitive gel added with the photosensitizer used in the embodiment of the invention can better separate different proteins.
Example 7:
in step 6, as shown in fig. 10, in one embodiment of the present invention, a metal-labeled antibody is used, and the hydrogel is incubated with the metal-labeled antibody, so that the hydrogel has no obstacle to the gold-labeled antibody, and thus, good labeling can be achieved. The process of metal-labeled antibodies is currently the only standard process in the industry.
The detection method comprises the following steps:
step 6.1, metal-labeled antibody: selecting antibody solution without BSA, performing metal labeling by using a standard experimental process of Fuluda (Shanghai) instruments and science and technology Limited, wherein the antibody solution labeled with the metal has at least 30 metal elements, and performing antibody titration after the labeling is finished to determine the use concentration of the metal-labeled antibody;
step 6.2, incubation of the protein with metal antibody: mixing antibody solutions of various target proteins according to the use concentration of the metal-labeled antibody determined in the step 6.1, dropwise adding the mixed antibody solutions to the surface of a western blotting chip, and incubating for 4 hours at 37 ℃;
and 6.3, after the incubation is finished, washing the protein blot chip by using 0.01M TBST, freezing the marked protein blot chip in a refrigerator at the temperature of minus 80 ℃, and freezing the protein blot chip again by using liquid nitrogen before loading.
Step 6.4, detecting by a mass spectrometer: ablating the coordinate region recorded in the step 4, setting the ablation energy as 12, and detecting by using a mass spectrometer;
and 6.5, carrying out digital processing and analysis on data detected by a mass spectrometry imager by using image J and ilastik software and a statistical analysis method so as to realize specific quantitative or semi-quantitative detection on the target protein and construct a single-cell protein map.
Wherein, the mode of dripping the antibody solution in the step 6.2 can adopt the micromanipulation to directly add into the micropore, and can also drip on the surface of the chip in a large scale.
The antibody is selected according to the target protein, for example, if the reference proteins Actin and GAPDH are detected, then the metal-labeled Actin antibody and GAPDH are selected for incubation. The metals currently available for labeling antibodies are lanthanide metals, 40 of which are commercially available, and are commercially available for attachment to antibodies, and the metals are not specific and can be attached to either antibody.
After the antibody is labeled and incubated with protein, detection and data processing of a mass spectrometer are carried out, and the method specifically comprises the following steps:
taking out the chip at minus 80 ℃, and after the temperature of the chip is recovered to normal temperature, setting the centrifugal force of 200g by using a chip centrifugal machine, and centrifuging for 10 s. The mass spectrometry imaging detector is started up in advance and preheated for 20 minutes, the chip is placed into a sample loading groove of the mass spectrometry imaging detector, the glue surface is upward, the ablation laser intensity is set to be 12, a metal antibody used in protein incubation is selected on a software interface, and a region to be detected is selected, as shown in fig. 11. Click start and start the scan, every 1mm x 1mm size area for 1 hour.
And after the scanning is finished, downloading the original data. The method comprises the steps of performing background processing by using software Image J, performing machine learning by using ilastik software, highlighting a signal of data, analyzing the difference of the data by using conventional methods such as t test and the like to realize specific quantitative or semi-quantitative detection of target protein and construct a single-cell protein map.
In addition, the sample to be tested in step 4 of the present invention is selected from one of single cell, multiple cell, extracted protein, purified protein solution, DNA-protein sample or RNA-protein sample, and the antibody in step 6 is selected from one of fluorescence, enzyme, colloidal gold or superparamagnetic microsphere labeled antibody. When used in a complex, for example, a nucleic acid and protein complex, DNA and protein can bind to each other, and RNA and protein can bind to each other, and the molecular weight of the bound substance is different from that of the protein alone. Therefore, substances with different molecular weights are separated by electrophoresis, and the co-detection of three substances, namely DNA-protein, RNA-protein and protein, can be realized.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (10)
1. A single cell protein digital imaging detection method is characterized by comprising the following steps:
step 1, preparing a chip template by using a designed film mask;
step 2, preparing a protein immobilized gel photosensitizer;
step 3, preparing light-sensitive protein fixing gel liquid by using the protein fixing gel photosensitizer in the step 2, and preparing a single-cell western blotting chip by matching with the chip template in the step 1, wherein the western blotting chip forms a plurality of micropores due to the shape of the chip template; preparing a lysis solution which is also an electrophoresis buffer solution;
step 4, loading a sample to be tested into the Western blot chip prepared in the step 3;
step 5, carrying out electrophoresis on the Western blot chip loaded with the sample to be detected according to a single cell immunoblotting (scWB) program, immediately fixing the protein through UV crosslinking after electrophoresis, and enabling the wavelength to be 300-inch and the 370nm intensity to be 40J/cm2The crosslinking time is 15 seconds to 10 minutes;
and 6, carrying out target protein immunoblotting detection by using the labeled antibody, and observing an analysis result.
2. The digital imaging detection method of single-cell protein according to claim 1, wherein the step 1 further comprises:
step 1.1, baking a silicon wafer with one side coated with SU8-2050 gel on a hot plate at 65 ℃ for 1 minute, and then baking at 95 ℃ for 8 minutes;
step 1.2, the silicon wafer is placed on an exposure machine for exposure for 4 minutes of UV exposure, the UV wavelength is 365nm, and the intensity is 30mW/cm2；
Step 1.3, baking the silicon wafer subjected to exposure in the step 1.2 on a hot plate at 65 ℃ for 2 minutes, and then baking at 95 ℃ for 7 minutes;
step 1.4, coating a second layer of SU8-2050 gel on the first layer of SU8-2050 gel layer of the silicon wafer for homogenization, then baking the mixture on a hot plate at 65 ℃ for 1 minute, and then baking the mixture at 95 ℃ for 2 minutes;
step 1.5, the designed film mask is pasted on a silicon wafer and then is exposedUV exposure is carried out in the machine for 4 minutes, the UV wavelength is 365nm, and the intensity is 30mW/cm2；
Step 1.6, baking the silicon wafer subjected to exposure in the step 1.5 on a hot plate at 65 ℃ for 2 minutes, and then baking at 95 ℃ for 7 minutes to prepare a chip template;
step 1.7, fixing the chip template by using tweezers, and washing for 2-3 minutes by using a developing solution;
step 1.8, the chip template is cleaned with isopropanol, acetone, ethanol and deionized water and then dried at 65 ℃.
3. The digital imaging detection method of single-cell protein as claimed in claim 1, wherein the photosensitizer for protein-immobilized gel in step 2 is N- (3-methacrylamidopropyl) -2- (1-methyl-1H-pyrrol-2-yl) -2H-tetrazole-5-carboxamide, MAP-mPyTC, which is prepared as follows:
step 2.1, preparation of ethyl 2- (1-methyl-1H-pyrrol-2-yl) -2H-tetrazole-5-carboxylate:
step 2.1.1, dissolve 5g, i.e. 12.5mmol, of methyl-1 hydro-pyrrole in 30mL of trifluoroethanol;
step 2.1.2, adding 20g of 62.5mmol of iodobenzene diacetate into the solution obtained in the step 2.1.1 at the temperature of minus 40 ℃, and stirring for 2 hours at the temperature of minus 40 ℃ under the protection of nitrogen;
step 2.1.3, concentrating the product of the step 2.1.2 to be black oil, and dissolving the black oil in dichloromethane;
step 2.1.4, adding 3g of 21.6mmol of 5-ethyl formate tetrazole, 3.1g of 8.6mmol of copper (II) trifluoromethanesulfonate and 16ml of 108mmol of triethylamine into the solution in the step 2.1.3, and stirring for 24 hours at room temperature under the protection of nitrogen to react;
step 2.1.5, after the reaction is finished, washing the product by using saturated ammonium chloride, drying by using anhydrous magnesium sulfate, and filtering and drying;
and 2.1.6, further purifying the product by silica gel flash chromatography, wherein an eluent of the silica gel flash chromatography is PE: EA ═ 5:1 in volume ratio to obtain a brown oily product I, and the product I is 2- (1-methyl-1H-pyrrole-2-yl) -2H-tetrazole-5-carboxylic acid ethyl ester, and the structural formula is shown as the formula I:
step 2.2, preparing 2- (1-methyl-1H-pyrrole-2-yl) -2H-tetrazole-5-carboxylic acid;
step 2.2.1, dissolving 450mg, i.e. 2mmol, of product I in step 2.1 in a volume ratio of MeOH: in a total of 20ml of the mixed solution of H2O-1: 1;
step 2.2.2, adding 850mg of lithium hydroxide, namely 10mmol of lithium hydroxide into the solution obtained in the step 2.2.1 at the temperature of 0 ℃;
2.2.3, placing the mixture obtained in the step 2.2.2 at room temperature, and stirring and reacting for 2 hours under the protection of nitrogen;
step 2.2.4, after the reaction is finished, adding 2N HCL at the temperature of 0 ℃, and adjusting the pH to 4-5;
step 2.2.5, extracting with 98% ethyl acetate solution, washing the mixed organic phase with saturated ammonium chloride, drying with anhydrous sodium sulfate, filtering and concentrating to obtain brown solid product II, namely 2- (1-methyl-1H-pyrrole-2-yl) -2H-tetrazole-5-carboxylic acid, the structural formula is shown as formula II:
step 2.3, preparing N- (3-methacrylamidoylpropyl) -2- (1-methyl-1H-pyrrol-2-yl) -2H-tetrazole-5-carboxamide;
step 2.3.1, dissolve 370mg, i.e. 1.67mmol, of N- (3-aminopropyl) methacrylate/hydrochloride in 20mL of tetrahydrofuran;
step 2.3.2, 650mg, i.e. 3.34mmol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide-HCl, i.e. EDCl. HCl, 220mg, i.e. 1.67mmol of 1-hydroxybenzotriazole, i.e. HOBt, are added to the solution from step 2.3.1 at 0 ℃ and reacted for 30 minutes;
step 2.3.3, adding 300mg of 1.67mmol of product II and 850mg of 8.35mmol of triethylamine in the step 2.2 into the mixed solution in the step 2.3.2 at 0 ℃, and stirring, refluxing and reacting overnight;
step 2.3.4, after the reaction is finished, purifying the product by preparative HPLC to obtain a white powder product III, namely N- (3-methacrylamidopropyl) -2- (1-methyl-1H-pyrrol-2-yl) -2H-tetrazole-5-formamide, namely MAP-mPyTC, the structural formula of which is shown in formula III:
the MAP-mPyTC prepared in the step 3 is copolymerized with acrylamide and N, N' -methylene bisacrylamide to form MPyTC Modified Polyacrylamide (MMP) light-sensitive protein immobilized gel.
4. The digital imaging detection method of single-cell protein as claimed in claim 1, wherein the structural general formula of the protein-immobilized gel photosensitizer in step 2 is shown as formula IV, wherein R group is an electron-withdrawing group:
the R group is selected from one of the following groups:
5. the digital imaging detection method of single-cell protein according to claim 1, wherein said step 3 further comprises:
step 3.1, treating a standard glass slide with 98 percent of 3- (trimethoxysilyl) propyl methacrylate serving as a silylation reagent for 30 minutes, washing the standard glass slide twice by using methanol and deionized water, and drying the standard glass slide by using nitrogen;
step 3.2, dissolving the gel photosensitizer prepared in the step 2 in dimethyl sulfoxide (DMSO) to prepare a storage solution S1, wherein the concentration of S1 is 100 mM;
step 3.3, in a 1.5ml Ep tube, 25uL of 1.5M Tris-HCl pH8.8, 166.7uL of 30% 29:1 acrylamide/methylene bisacrylamide mixture, 265.3uL ddH2O, 3.75-15uL 100mM S1, prepared into colloidal precursor (precursor);
step 3.4, adding 10ul of 5% SDS, 4ul of APS and 4ul of TEMED into the gel precursor in the step 3.3, and shaking up lightly to prepare the light-sensitive protein immobilized gel solution;
step 3.5, spreading the protein immobilized gel solution prepared in step 3.4 on the chip template prepared in step 1, and then covering with the silanized glass slide in step 3.1;
step 3.6, standing for 20 minutes, lifting the glass slide, stripping the chip template to prepare a western blot chip, and storing the western blot chip in a refrigerator at 4 ℃;
step 3.7, preparing RIPA-like lysate: preparing a mixed solution of SDS with the final concentration of 0.5%, Triton X-100 with the final concentration of 0.1%, sodium deoxycholate with the final concentration of 0.25%, Tris with the final concentration of 12.5mM and glycine with the pH value of 8.3 to obtain a lysate, wherein the lysate is also an electrophoresis buffer and is stored at the temperature of 4 ℃.
6. The digital imaging detection method of single-cell protein according to claim 1, wherein the western blot chip prepared in step 3 is a light-sensitive protein-immobilized gel attached to one side of a glass slide, the chip template prepared in step 1 has a plurality of protruding cylinders constituting a cylinder array, such that the gel portion of the western blot chip prepared in step 3 has a plurality of micropores, forming a porous microarray, and further, the chip template has a plurality of hydrophobic regions, which are created by drawing lines with hydrophobic isolation pens, such that the gel portion of the western blot chip forms a plurality of isolated regions without gel, the cylinders have a diameter of 30 μm, the width of the isolated regions is 2mm, and the isolated regions divide the western blot chip into three isolated regions.
7. The digital imaging detection method of single-cell protein according to claim 1, wherein the sample to be tested in step 4 is a cell sample transfected with a reporter gene in advance, and step 4 further comprises loading the cell sample into the western blot chip prepared in step 3, allowing the cells to settle into the microwells of the chip, allowing the diameter of each microwell to ensure that one cell falls into one microwell, assigning coordinates of cell traps to each cell based on the position of the microwell, washing the excess cells with 0.01M, pH ═ 7 phosphate buffer, observing and recording the transfected cells expressing the reporter gene by a confocal fluorescence microscope, and recording the coordinates.
8. The digital imaging detection method of single-cell protein according to claim 1, wherein said step 5 further comprises:
step 5.1, heating the lysis solution/electrophoresis buffer solution prepared in the step 3 to 50-55 ℃ in a water bath, and starting ultraviolet in advance to stabilize a light source;
step 5.2, placing the Western blot chip prepared in the step 3 in a new dish with the glue surface facing upwards, dropwise adding 200ul of BSA solution with the concentration of 5.12mg/mL, slightly shaking the slide to ensure that the BSA solution is uniformly distributed, and standing for 3 minutes;
step 5.3, placing the Western blot chip in an electrophoresis tank, and pouring 10ml of RIPA-like lysate preheated in a water bath from one corner of the electrophoresis tank gently as soon as possible;
step 5.4, immediately turning on a voltage power supply, wherein the voltage is 200V, and E is 40V/cm2Protein is separated for 30 seconds by electrophoresis;
step 5.5, immediately stopping the voltage, and exposing for 15 seconds to 10 minutes under the ultraviolet of 340-;
step 5.6, after the exposure is finished, taking out the Western blot chip, and placing the Western blot chip in Coomassie brilliant blue dye solution for dyeing for 5 minutes, wherein the Coomassie brilliant blue dye solution is prepared by dissolving 0.1g of Coomassie brilliant blue powder in a mixed solution of 20mL of methanol, 16mL of water and 4mL of acetic acid;
step 5.7, taking a picture, and recording the protein fixation condition;
5.8, shaking and washing by using 0.01M TBST, changing the liquid once every 15 minutes for 2 hours, and then shaking and washing overnight;
and 5.9, taking a picture and recording the protein fixation condition.
9. The digital imaging detection method of single-cell protein as claimed in claim 7,
the step 6 further comprises:
step 6.1, metal-labeled antibody: selecting antibody solution without BSA, carrying out metal labeling, carrying out antibody titration after the labeling is finished and determining the use concentration of the metal labeled antibody, wherein the metal labeled antibody solution contains at least 30 metal elements;
step 6.2, incubation of the protein with metal antibody: mixing antibody solutions of various target proteins according to the use concentration of the metal-labeled antibody determined in the step 6.1, dropwise adding the mixed antibody solutions to the surface of a western blotting chip, and incubating for 4 hours at 37 ℃;
and 6.3, after the incubation is finished, washing the protein blot chip by using 0.01M TBST, freezing the marked protein blot chip in a refrigerator at the temperature of minus 80 ℃, and freezing the protein blot chip again by using liquid nitrogen before the machine is operated.
Step 6.4, detecting by a mass spectrometer: ablating the coordinate region recorded in the step 4, setting the ablation energy as 12, and detecting by using a mass spectrometer;
and 6.5, carrying out digital processing and analysis on data detected by a mass spectrometry imager by using image J and ilastik software and a statistical analysis method so as to realize specific quantitative or semi-quantitative detection on the target protein and construct a single-cell protein map.
10. The digital imaging detection method of single-cell protein according to claim 1, wherein the sample to be detected in step 4 is selected from one of single cell, multi-cell, extracted protein, purified protein solution, DNA-protein sample or RNA-protein sample, and the antibody in step 6 is selected from one of fluorescent, enzyme, colloidal gold or superparamagnetic microsphere labeled antibody.
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