CN112964881A - High-throughput high-sensitivity single cell transfection protein analysis chip - Google Patents
High-throughput high-sensitivity single cell transfection protein analysis chip Download PDFInfo
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- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The invention discloses a high-flux high-sensitivity single cell transfection protein analysis chip, which relates to the field of microchip analysis platforms and consists of a carrier and a light-sensitive protein fixed gel coating, wherein the coating is divided into more than one detection area by a hydrophobic separation band, and a cell trap is arranged in the detection area. According to the invention, the polyacrylamide gel with tetrazole as a photosensitive group is used as a photosensitive protein fixing gel, the polyacrylamide gel is easy to crosslink with proteins through ultraviolet illumination, has a sensitive and accurate detection effect on low-abundance proteins, and can truly realize gene expression detection on transfected single cells and rare cells; and the design of the regional microchip can realize the simultaneous detection of multiple targets and multiple targets, and has important significance for the analysis of foreign gene expression and the evaluation and research of transfection conditions.
Description
Technical Field
The invention relates to the field of microchip analysis platforms, in particular to a single cell transfection protein analysis chip with high flux and high sensitivity.
Background
Complex signal transduction and protein-modified expression are the basis for many functions of organisms. Biologists often introduce foreign DNA, RNA and proteins into cells to alter their signals and behavior(1). Transfection is the process of introducing foreign nucleic acids into cells to produce transgenic cells, which are powerful analytical tools for studying gene function and regulation, as well as protein function(2). Transfection can be divided into two categories: transient transfection and stable transfection. Stable transfection generally refers to the integration of the introduced genetic material (transgene) with a selectable marker gene into the host genome, and transgene expression is maintained even after replication of the host cell. However, the construction of stably transfected cells requires a long time and is cumbersome(3). Transiently transfected genes are expressed at high levels for only a limited time compared to stably transfected genes and are not integrated into the genome. Transient transfection is simple and rapid, and is a common important tool in signal path research(4)。
The transfection efficiency of transient transfection is influenced by a number of factors. Such as cell type, transfection environment, gene delivery vector, size of target gene, transfection reagent, etc(5). The underlying reason is that these factors affect the integrity of the plasmid and its ability to reach the nucleus(6). Furthermore, in biological studies, target and reporter genes typically form a fused DNA plasmid and are transfected into cells. The marker gene is usually a gene expressing a fluorescent protein, such as EGFP, mCherry and the like, and can be used for fluorescent flow cytometry sorting or fluorescent microscope observation(7). Theoretically, the fusion gene means that the reporter gene and the target gene will be co-expressed, but actually, transfected cells expressing the reporter gene cannot express 100% of the target gene. This may be due to the complex enzymatic environment and translational modification mechanisms in the cell(8). This results in a greatly reduced proportion of cells that can be successfully transfected for study.
For cells with low transfection efficiency, enrichment is usually performed using fluorescence flow sorting, followed by subsequent proteome or transcriptome analysis. However, by fluorescent flow cytometryCells expressing the reporter gene are screened for incorporation of false positive transfected cells expressing only the reporter gene. In subsequent proteomic analysis, these false positives interfere with the analysis results. Furthermore, traditional proteomic analysis methods (e.g. immunoblot analysis, co-immunoprecipitation analysis, fluorescence flow cytometry and mass spectrometry flow cytometry) impose stringent requirements on the number of loaded cells. For example, traditional Western blotting requires that the total protein loading concentration be up to 1. mu.g/. mu.l, and then from at least 106Extraction of proteins from individual cells(9). Mass cytometry also requires at least 10 as the latest tool for high throughput proteomic analysis5Sample size of individual cells to ensure statistical significance(10). For rare cells, such as egg cells, circulating tumor cells and spermatocytes, transfection is difficult and inefficient, and these methods are no longer suitable(11). Furthermore, these analytical methods still perform statistical analysis on a large number of cells, thereby ignoring inter-cell heterogeneity. However, analysis of cellular heterogeneity is of great importance for understanding the particular physiological functions of an organism and the diagnosis and development of disease(12). Therefore, a technique is required: the transfection effect can be easily and clearly determined without screening and enrichment, and single cell protein analysis can be carried out.
In recent years, microfluidics technology has rapidly evolved and a series of microchip-based technologies have emerged, providing a more efficient platform for unicellular omics analysis(13). The recent development of microchip technology provides various new feasible solutions for single cell research(14). 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(15). In addition, operating under laminar flow conditions can provide a well-controlled environment, allowing more accurate operation and more sensitive measurements at the single cell level(16). 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(17). However, chip preparation processThe method is complex and difficult to control single cell separation detection. 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.
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 blot and the like provide a powerful tool 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 detect the phosphorylation status of signal proteins of acute myelogenous leukemia cells under stimulation of various cytokines(18). Sachs et al have examined phosphorylation levels of 11 proteins and lipids in human CD4+ T cells using flow cytofluorescent sorting(19). However, the fluorescence flow cytometry cannot remove the false positive cells, so that the true protein expression change is annihilated in the subsequent experiment to draw a wrong conclusion.
Bendall and Nolan et al, combined with flow cytometry and Mass spectrometry, propose a novel Mass cytometry (Mass cytometry)(20). 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 is 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 very wide atomic weight detection range (88-210 Da), extremely high resolution and interference between adjacent channels<0.3%, the number of channels is increased while avoiding back-off calculations. Thus not only simplifying the experimental process, but also saving the samples andand (3) a reagent. Evan W.Newell et al carefully analyzed 25 surface markers and functional protein expression of human CD8T cells and the binding of three different viral epitopes by mass spectrometry flow cytometry, showed a continuous process of T cell maturation, and revealed the existence of complex functional polymorphisms in T cell populations(21). However, detection methods that rely solely on the binding of antigen-antibody to recognize target protein molecules are susceptible to binding specificity, may have high false positives, and are limited in application by the type of specific probe.
To obtain spatial information on protein expression, Giesen C et al(22)Immunohistochemistry and mass spectrum flow type are organically combined by using a laser ablation technology, and subcellular localization images (the image resolution can reach 1 mu m) of 32 proteins in the same visual field are obtained. The authors utilize the imaging mass spectrometry flow technology to study human breast cancer slice samples, and according to the analysis of the obtained image information, the authors realize the subgroup typing of cells and the polymorphism research of tumor tissue cells, and will strongly promote the research of tissue functions and polymorphisms, and provide a technical basis for personalized molecular targeted therapy. Isolight single cell functional information multiple detection system of IsoPlexis company(23)The method is the most advanced and indispensable solution in the single cell analysis field at present, can provide complete immune cell functional response analysis under the resolution and sensitivity of single cells to help predict and understand the complex patient response of cancer immunotherapy, and establish direct correlation between the immune functional response and 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 later-stage monitoring. However, in flow cytometry, direct detection of intracellular and nuclear proteins is difficult, and detection application mainly aims at secreted proteins, and cannot detect cell surface, transmembrane proteins, intracellular and nuclear proteins.
Western blotting (Western Blot) is a commonly used method for protein determination in cell and molecular biology and immunogenetics. The specific process is to use gel electricityProteins in a sample are separated by electrophoresis, then transferred to a membrane (typically nitrocellulose or PVDF), and then detected 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(24-26)。
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 in situ immobilization of proteins using light-induced blotting technique and detection of 11 protein targets in a single cell by detection of fluorescent antibodies(27). Based on this technique, Herr et al monitored neural stem cell differentiation and response to mitogen stimulation in a single rat(28). 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, due to the limitation of a photosensitive reagent in the gel, the photosensitive immobilization efficiency is low, the lower limit of detection is 27000 single-cell protein molecules, the application to the detection of low-abundance proteins is difficult, a large promotion space still exists, and scWB has high background fluorescence when detecting target proteins, and the sensitivity is low, which is not satisfactory for detecting low-abundance proteins. In addition, the short excitation wavelength (UVB, 280-320 nm) can cause the inactivation of protein antigen sites, thereby causing false negative effect. And are also difficult to apply to single cell protein analysis of rare cell populations.
Therefore, those skilled in the art have been devoted to develop a high-throughput, high-sensitivity protein detection chip to solve the problem of protein analysis of single cells, especially rare cell populations, after transfection.
Disclosure of Invention
In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to develop a single-cell transfection analysis chip based on the in-situ microchip immunoblotting method, so as to rapidly, accurately and efficiently analyze the expression of exogenous genes in transfected cells, especially the detection of protein expression level under the condition of low co-expression and the analysis of the relation between the reporter gene and the target gene expression level, as well as the gene expression detection analysis of other rare cells.
In order to achieve the aim, the invention provides a single-cell transfection protein analysis chip with high flux and high sensitivity, which consists of a carrier and a light-sensitive protein fixed gel coating, wherein the coating is divided into more than one detection area by a hydrophobic separation band; a cell trap is disposed in the detection region.
Furthermore, the light-sensitive protein fixing gel is polyacrylamide gel with tetrazole as a light-sensitive group.
Furthermore, the polyacrylamide gel selects molecules of which the electron withdrawing group in the structural formula is 1, 2-dimethyl-1H-pyrrole, 2-methyl-1H-pyrrole, 2-methylthiophene, 2-methylfuran or toluene.
Further, the hydrophobic separation band is constructed by adopting hydrophobic separation pen division or adopting hydrophobic coating separation.
Further, the diameter size of the cell well is set according to the size of the cells to be detected.
The invention also provides a method for detecting cell expression protein by using the single cell transfection protein analysis chip with high flux and high sensitivity, which comprises the following steps:
and 5, adding an antibody probe, incubating, detecting a signal, and analyzing the expression of the target protein.
Further, the antibody probe in the step 5 is marked by fluorescence, enzyme, colloidal gold or superparamagnetic microspheres.
Further, the signal detection object in step 5 is an antibody probe or a reagent including an aptamer, a nanobody, and a lectin.
Further, the wavelength range of the irradiation in the step 4 is 300-340 nm, and the time range is 30 s-10 min.
Further, the application objects of the method comprise single-cell or multi-cell protein expression, extracted protein solution or purified protein solution, DNA-protein and RNA-protein; wherein the protein solution may include one or more protein analyte substances.
Compared with the prior art, the invention at least has the following beneficial technical effects:
(1) the research on the expression correlation between the reporter gene and the target gene can be carried out, and particularly, a powerful platform is provided for evaluating the reliability of the transfection strategy under the condition of low co-expression;
(2) the regionalization design of the detection range can carry out simultaneous analysis of multiple targets, and the simultaneous research on the expression of the fusion protein and the downstream protein is realized;
(3) the true single cell up-sampling can be carried out, not only the low-abundance protein of the transfected cell can be detected, but also the related gene expression of the rare cell group can be researched;
(4) the portability is high, the cost is low, the result is visual, and the storage stability is good;
(5) the functional stability is good, the storage period reaches 3 months, and the signal has no obvious change.
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 illustrating the basic appearance and principle of a chip according to a preferred embodiment of the present invention;
FIG. 2 is a diagram showing the results of the loading localization and reporter gene examination of transfected cells according to a preferred embodiment of the present invention;
FIG. 3 is a schematic diagram illustrating the operation of UV-activated cross-linked protein in the light-sensitive protein-immobilized gel according to a preferred embodiment of the present invention;
FIG. 4 is a synthesis scheme of a light-sensitive protein-immobilized gel according to a preferred embodiment of the present invention;
FIG. 5 is a NMR spectrum of ethyl 2- (1-methyl-1H-pyrrol-2-yl) -2H-tetrazole-5-carboxylate, a product of a procedure for synthesizing a light-sensitive protein immobilized gel according to a preferred embodiment of the present invention;
FIG. 6 is a mass spectrometric identification of a synthetic light-sensitive protein immobilized gel N- (3-methacrylamidoylpropyl) -2- (1-methyl-1H-pyrrol-2-yl) -2H-tetrazole-5-carboxamide of a preferred embodiment of the present invention;
FIG. 7 is a NMR spectrum of synthetic light-sensitive protein-immobilized gel N- (3-methacrylamidoylpropyl) -2- (1-methyl-1H-pyrrol-2-yl) -2H-tetrazole-5-carboxamide in accordance with a preferred embodiment of the present invention;
FIG. 8 is a photograph of different concentrations of light-sensitive protein immobilized gel immobilized protein according to a preferred embodiment of the invention;
FIG. 9 is a graph showing the efficiency of capturing proteins by UV excitation of light-sensitive protein-immobilized gels of different concentrations in accordance with a preferred embodiment of the present invention;
FIG. 10 is a graph showing the effect of different UV irradiation times on the efficiency of protein capture by a light-sensitive protein immobilization gel according to a preferred embodiment of the present invention;
FIG. 11 is a diagram showing the UV absorption spectrum of the light-sensitive protein immobilized gel in accordance with the 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.
The invention establishes a regional small-sized locatable chip based on acrylamide gel with tetrazole as a photosensitive group, and comprises the following specific steps:
after the gel had formed, the chip was gently lifted from the mold. The chip is covered with a layer of template, and due to the design of the template, the isolated areas are free of gel. Hydrophobic spacer pens create hydrophobic areas along the barrier. To accurately locate transfected cells, we assigned trap coordinates for each cell. Different transfected cells were loaded onto the chip simultaneously, and in addition to a cylinder with a diameter of 30 μm, a 2mm wide isolation region was designed on the mold. In this study, the chip was divided into three isolated domains according to experimental requirements.
As shown in FIG. 1, after loading the cells, it takes 5-10 minutes for the cells to settle into the cell well. After washing excess cells with phosphate buffer, transfected cells expressing the re-reporter gene were placed under a confocal fluorescence microscope and coordinates were recorded. The whole process takes 5 minutes to ensure that the protein status in the cells is normal. After positioning, electrophoresis was performed according to scWB electrophoresis program. The size of the target protein determines the electrophoresis time. Immediately after electrophoresis, proteins were immobilized by UV cross-linking. Under the irradiation of ultraviolet rays, the protein and MMP gel will undergo a cross-linking reaction, and the process is shown in FIG. 3. And then the targeted protein can be specifically and quantitatively or semi-quantitatively detected by detecting the fluorescence intensity of the antibody probe (as shown in fig. 2).
Due to the efficient and rapid nature of MMP gels, exposure for 60 seconds can meet the fixation requirements for low abundance proteins. In the antibody detection phase, multiple antibodies can be incubated simultaneously in each region due to the presence of hydrophobic separation zones. We removed the extra cells on the surface and observed the cells falling into the cell trap under a confocal fluorescence microscope. Our data indicates that in some traps there may be 2-3 cells and their coordinates recorded so that they can be discarded in subsequent antibody detection assays. Then, electrophoresis was performed. After 60 seconds of UV irradiation, the proteins were immobilized with MMP gel. The fluorescent signal is detected after incubation with the fluorescent antibody.
Example 1 Synthesis of light-sensitive protein-immobilized gel N- (3-Methylamidoaminopropyl) -2- (1-methyl-1H-pyrrol-2-yl) -2H-tetrazole-5-carboxamide
Step one, synthesizing 2- (1-methyl-1H-pyrrole-2-yl) -2H-tetrazole-5-carboxylic acid ethyl ester, comprising the following steps:
1.1. dissolving methyl-1 hydro-pyrrole (5g, 12.5mmol) in 30mL of trifluoroethanol;
1.2. iodobenzene diacetate (20g, 62.5mmol) was added to the 1.1 solution at-40 deg.C;
1.3. stirring for 2 hours at-40 ℃ under the protection of nitrogen;
1.4. the product was concentrated to a black oil and dissolved in dichloromethane;
1.5. add 5-Ethyl formate tetrazole (3g, 21.6mmol), copper (II) trifluoromethanesulfonate (3.1g, 8.6mmol), and triethylamine (16ml, 108mmol) to the solution in 1.4;
1.6. stirring for 24 hours at room temperature under the protection of nitrogen;
1.7. after the reaction is finished, washing the product by using saturated ammonium chloride and brine, drying by using anhydrous magnesium sulfate, filtering and drying;
1.8. the product was further purified by flash chromatography on silica gel (eluent PE: EA ═ 5: 1) to afford product I as a brown oil. The yield of the product I is 450mg and 9.6%, and the nuclear magnetic resonance hydrogen spectrum characterization of the product I is shown in FIG. 5.
Step two, synthesizing 2- (1-methyl-1H-pyrrole-2-yl) -2H-tetrazole-5-carboxylic acid, comprising the following steps:
2.1. dissolve product I from step one (450mg, 2mmol) in 1: 1MeOH/H2O (20 ml);
2.2. lithium hydroxide (850mg, 10mmol) was added to the 2.1 solution at 0 deg.C;
2.3. placing the mixture obtained in the step 2.2 at room temperature, and stirring and reacting for 2 hours under the protection of nitrogen;
2.4. after the reaction is finished, adding 2N HCL at 0 ℃, and adjusting the pH to 4-5;
2.5. extraction with ethyl acetate solution and washing of the combined organic phases with brine, drying over anhydrous sodium sulfate, filtration and concentration to give product II as a brown solid. The yield of the product II was 370mg, 94%.
Step three, synthesizing the light-sensitive protein fixed gel N- (3-methacrylamide propyl) -2- (1-methyl-1H-pyrrole-2-yl) -2H-tetrazole-5-formamide, comprising the following steps:
3.1. dissolving N- (3-aminopropyl) methacrylate (370mg, 1.67mmol) in 20mL tetrahydrofuran;
3.2. 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCl. HCl, 650mg, 3.34mmol), 1-hydroxybenzotriazole (HOBt, 220mg, 1.67mmol) were added at 0 ℃ and reacted for 30 min;
3.3. adding the product II (300mg, 1.67mmol) and triethylamine (850mg, 8.35mmol) in the step two into the mixed solution in the step 3.2, and stirring and refluxing for reaction overnight;
3.4. after the reaction was complete, the product was purified by preparative HPLC to give white powder III with a yield of 41mg and 6.7%. The mass spectrum identification result of the product III is shown in FIG. 6, and the nuclear magnetic resonance hydrogen spectrum characterization thereof is shown in FIG. 7.
Example 2 efficiency of immobilization of different concentrations of light-sensitive protein-immobilized gels on bovine serum albumin BSA
1. Dissolving the product III in example 1 in dimethyl sulfoxide DMSO to prepare a 100mM storage solution S1;
2. in a 1.5ml Ep tube, 25. mu.L of 1.5M Tris-HCl pH8.8, 166.7. mu.L of 30% acrylamide/methylene bisacrylamide (29: 1), 265.3. mu.L ddHO, and 15. mu.L (3%), 7.5. mu.L (1.5%), 3.75. mu.L (0.75%), 0. mu.L (0%) S1, respectively, were added to prepare a gel precursor;
3. and 10. mu.l of 5% SDS, 10. mu.l of 5% TritonX-100, 4. mu.l of APS and 4. mu.l of TEMED were added thereto, and the mixture was gently shaken to prepare S1 solutions having different concentrations. Dripping the solution on a porous microarray mold, slightly covering a glass slide to avoid bubbles, standing for 20min, and stripping the mold after the gel is solidified to prepare porous microarray gel;
4. RIPA-like lysate (also a Radio-immune lysis assay buffer) was prepared according to 0.5% SDS, 0.1% v/v Triton X-100, 0.25% sodium deoxycholate, 12.5mM Tris, 96mM glycine, pH 8.3, and stored at 4 ℃;
5. heating lysis solution/electrophoresis solution in water bath to 50-55 deg.C, and turning on ultraviolet in advance to stabilize light source;
6. placing the chip in a new dish with the glue surface facing upwards, dropwise adding 200 μ l BSA solution (5.12mg/mL), gently shaking the slide to make the BSA solution distributed uniformly, and standing for 3 min;
7. placing the gel in an electrophoresis tank, and pouring the gel into 10ml of RIPA-like lysate preheated in water bath at 55 ℃ from one corner of the electrophoresis tank gently as soon as possible;
8. immediately turning on a voltage power supply, starting a voltage of 200V (E ═ 40V/cm2), and carrying out protein electrophoretic separation for 30 s;
9. stopping voltage immediately, and performing ultraviolet exposure for 10 min;
10. after the exposure, the gel was taken out and placed in Coomassie brilliant blue dye solution (0.1g of Coomassie brilliant blue powder dissolved in 20mL of methanol +16mL of water +4mL of acetic acid) for dyeing for 5 min;
11. taking a picture and recording the protein fixation condition;
shaking and washing TBST, changing the liquid once every 15min for 2h, and then shaking and washing overnight;
13. pictures were taken and protein fixation was recorded.
The photograph of the protein fixed is shown in FIG. 8. Statistics is carried out to draw a change curve (shown in figure 9) of the capture efficiency of the light-sensitive protein fixed gel with different concentrations to the protein after ultraviolet irradiation, which indicates that the S1 light-sensitive protein fixed gel with a low concentration level has stronger and higher fixation efficiency to the protein in the gel after ultraviolet excitation.
Example 3 efficiency of immobilizing light-sensitive protein-immobilized gel to bovine serum albumin BSA at different UV irradiation times
1. Dissolving the product III in example 1 in dimethyl sulfoxide DMSO to prepare a 100mM storage solution S1;
2. in a 1.5ml Ep tube, 25. mu.L of 1.5M Tris-HCl pH8.8, 166.7. mu.L of 30% acrylamide/methylene bisacrylamide (29: 1), 265.3. mu.L ddHO, 3.75. mu.L (0.75%) S1, formulated as colloidal precursor;
3. adding 10 μ l of 5% SDS, 10 μ l of 5% TritonX-100, 4 μ l of APS and 4 μ l of TEMED, shaking up gently, dripping the solution on a porous microarray mold, slightly covering a slide glass to avoid bubbles, standing for 20min, and stripping the mold after the gel is solidified to prepare porous microarray gel;
4. RIPA-like lysate (also a Radio-immune lysis assay buffer) was prepared according to 0.5% SDS, 0.1% v/v Triton X-100, 0.25% sodium deoxycholate, 12.5mM Tris, 96mM glycine, pH 8.3, and stored at 4 ℃;
5. heating lysis solution/electrophoresis solution in water bath to 50-55 deg.C, and turning on ultraviolet in advance to stabilize light source;
6. placing the chip in a new dish with the glue surface facing upwards, dropwise adding 200 μ l BSA solution (5.12mg/mL), gently shaking the slide to make the BSA solution distributed uniformly, and standing for 3 min;
7. placing the gel in an electrophoresis tank, and pouring the gel into 10ml of RIPA-like lysate preheated in water bath at 55 ℃ from one corner of the electrophoresis tank gently as soon as possible;
8. immediately turning on a voltage power supply, starting a voltage of 200V (E ═ 40V/cm2), and carrying out protein electrophoretic separation for 30 s;
9. immediately stopping voltage and ultraviolet exposure, wherein the exposure time is respectively set to 10min, 6min, 4min, 2min, 1min, 30s and 15 s;
10. after the exposure, the gel was taken out and placed in Coomassie brilliant blue dye solution (0.1g of Coomassie brilliant blue powder dissolved in 20mL of methanol +16mL of water +4mL of acetic acid) for dyeing for 5 min;
11. taking a picture and recording the protein fixation condition;
shaking and washing TBST, changing the solution once every 15min for 2h, and then shaking and washing overnight;
13. pictures were taken and protein fixation was recorded.
Experiments on chicken ovalbumin, trypsin inhibitor and lysozyme were carried out in the same manner. The curve (as shown in fig. 10) is statistically drawn by the change of the capture efficiency of the light-sensitive protein-immobilized gel on several proteins with the uv exposure time, which indicates that the light-sensitive protein-immobilized gel has a stronger and higher immobilization efficiency on the in-gel protein after uv excitation in a shorter time (within 30 s).
Example 4 detection of light absorption Properties
The ultraviolet-visible absorption spectrum of the synthesized compound in the embodied example 1 was measured with an ultraviolet-visible spectrophotometer manufactured by agilent technologies (china) limited. The instrument model is Evolution 220UV-Vis, and the spectrum scanning range is from 300nm to 800 nm. The ultraviolet-visible absorption spectrum shown in FIG. 11 was obtained, and the light absorption characteristics representative of the light-sensitive protein-immobilized gel according to the present invention were obtained.
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.
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Claims (10)
1. a single cell transfection protein analysis chip with high flux and high sensitivity is characterized in that the chip consists of a carrier and a light sensitive protein fixed gel coating, wherein the coating is divided into more than one detection area by a hydrophobic separation band; a cell trap is disposed in the detection region.
2. The high-throughput high-sensitivity single-cell transfected protein analysis chip of claim 1, wherein the light-sensitive protein immobilization gel is polyacrylamide gel using tetrazole as a photosensitive group.
3. The high-throughput high-sensitivity single-cell transfected protein assay chip of claim 2, wherein said polyacrylamide gel is selected from molecules having the structural formula wherein the electron withdrawing group is 1, 2-dimethyl-1H-pyrrole, 2-methyl-1H-pyrrole, 2-methylthiophene, 2-methylfuran, or toluene.
4. The high throughput high sensitivity single cell transfected protein analysis chip according to claim 1, wherein said hydrophobic spacer is constructed by dividing with a hydrophobic spacer pen or by partitioning with a hydrophobic coating.
5. The high throughput high sensitivity single cell transfected protein analysis chip of claim 1, wherein said cell well is sized in diameter according to the size of the cells to be detected.
6. A method for detecting protein expressed by cells by using the single-cell transfected protein assay chip with high throughput and high sensitivity of claims 1-5, wherein the method comprises the following steps:
step 1, loading cells, and settling the cells into the cell trap;
step 2, after washing excessive cells, positioning and recording coordinates of transfected cells expressing the target genes and the reporter genes by using a confocal fluorescence microscope;
step 3, performing electrophoresis according to the size of the target protein;
step 4, immediately performing Ultraviolet (UV) irradiation to crosslink and fix the protein;
and 5, adding an antibody probe, incubating, detecting a signal, and analyzing the expression of the target protein.
7. The method of claim 6, wherein the antibody probe in step 5 is labeled with a fluorescent, enzyme, colloidal gold, or superparamagnetic microsphere.
8. The method of claim 6, wherein the signal detection object in step 5 is an antibody probe or a reagent including an aptamer, a nanobody, or a lectin.
9. The method of claim 6, wherein the irradiation in step 4 is performed at a wavelength ranging from 300 to 340nm for a time ranging from 30s to 10 min.
10. The method of claim 6, wherein the objects of application of the method include single-cell or multi-cell protein expression, extracted protein solution or purified protein solution, and DNA-protein, RNA-protein; wherein the protein solution may include one or more protein analyte substances.
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