CN116296702B - Dead-living dye capable of customizing detection channel based on mass spectrometry detection technology and preparation method and application thereof - Google Patents
Dead-living dye capable of customizing detection channel based on mass spectrometry detection technology and preparation method and application thereof Download PDFInfo
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- CN116296702B CN116296702B CN202310375121.3A CN202310375121A CN116296702B CN 116296702 B CN116296702 B CN 116296702B CN 202310375121 A CN202310375121 A CN 202310375121A CN 116296702 B CN116296702 B CN 116296702B
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- 238000004458 analytical method Methods 0.000 claims abstract description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 7
- VOQPQBGCWBEYEV-AWEZNQCLSA-N (S)-1-(4-bromoacetamidobenzyl)EDTA Chemical compound OC(=O)CN(CC(O)=O)C[C@@H](N(CC(O)=O)CC(O)=O)CC1=CC=C(NC(=O)CBr)C=C1 VOQPQBGCWBEYEV-AWEZNQCLSA-N 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 9
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- -1 4-Isothiocyanatobenzyl Chemical group 0.000 claims description 5
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- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 2
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- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- DCPLOIFDMMEBQZ-UHFFFAOYSA-N 2-bromo-n-phenylacetamide Chemical compound BrCC(=O)NC1=CC=CC=C1 DCPLOIFDMMEBQZ-UHFFFAOYSA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
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- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 description 1
- WDLRUFUQRNWCPK-UHFFFAOYSA-N Tetraxetan Chemical compound OC(=O)CN1CCN(CC(O)=O)CCN(CC(O)=O)CCN(CC(O)=O)CC1 WDLRUFUQRNWCPK-UHFFFAOYSA-N 0.000 description 1
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
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Abstract
The invention provides a customizable detection channel dead-living dye based on mass spectrometry detection technology, which comprises a detection analysis reagent capable of customizing the dead-living state of a sample cell of the detection channel, wherein the detection analysis reagent capable of customizing the dead-living state of the sample cell of the detection channel comprises a cell binding group, a metal ion binding group and a skeleton structure of a bridging cell binding group and a metal ion binding group which may exist; wherein the cell binding groups bind to single or multiple binding sites on one or more sample cells and the metal ion binding groups bind to one or more, metal ions as detection tags, which are customizable. The invention also discloses a preparation method and application thereof. The invention not only can realize the use effect which can be compared with commercial dead-alive dye, but also can realize the customization of any metal channel which can be applied to mass spectrometry detection, thereby meeting the diversified detection application requirements.
Description
Technical Field
The invention relates to the technical field of mass spectrometry detection, in particular to a dead-living dye of a customizable detection channel based on mass spectrometry detection technology, and a preparation method and application thereof.
Background
The mass spectrometry detection technique is a technique of detecting rare metal-labeled antibodies at the single cell level by a mass spectrometer and performing multi-parameter, high-throughput qualitative analysis on each cell. Compared with the traditional flow cytometry, the technology has the advantages of more detection parameters and higher signal to noise ratio, is the most advanced single-cell flow detection technology at present, and is more and more widely applied in the fields of clinical medicine and biological research.
The mass spectrometry detection technology can be applied to the field of biological research of immune monitoring, and has been applied to clinical application for screening diseases closely related to the state of the immune system. However, clinical detection or pharmaceutical development applications such as immunomonitoring or vaccine immunogenicity often require detection of the expression levels of small-duty cell populations and their biological indicators in the sample cells. This places high demands on the sensitivity and reproducibility of the detection assay system. However, since dead cells lose the selective permeability of cell membranes, in the antibody-based detection technique, the dead cells adsorb a large amount of antibodies for staining a sample non-specifically, and not only an abnormal antibody staining result occurs on the dead cells, but also an excessively low antibody staining concentration on living cells is caused, causing significant detection errors, and it is difficult to stably reflect the actual immune state of the sample. The dead living dye can objectively remove the dying result of dead cells in the analysis process and evaluate the living rate state of the processed sample, thereby ensuring the objectivity and accuracy of the detection data.
Currently, a commercial dead-living dye applied to mass spectrometry is cis-diamminedichloroplatin (cislatin), which is a dye covalently bound to proteins. The principle of distinguishing dead and alive is that the signal value of the dead cells with the loss of cisplatin-binding cell membrane selective permeability is higher than that of the living cell population, so that the distinction of the dead and living cell population in the sample cells is realized. However, cisplatin can also cross-link with single-or double-stranded Deoxyribonucleotides (DNA), resulting in a higher staining signal for living cells during staining, and the possibility of death and difficulty in objective discrimination of living cell populations. In addition, the commercial dead-alive dye cannot meet the custom-made requirements of a plurality of metal channels, namely detection channels, of users and cannot meet more diversified detection design schemes.
Disclosure of Invention
The invention provides a dead living dye capable of customizing a detection channel based on a mass spectrometry flow detection technology, and a preparation method and application thereof, which can more objectively distinguish dead living cells, realize an application effect comparable to commercial dead living dye, realize customization and selection of a metal channel and meet diversified detection application requirements.
The invention adopts the following technical scheme:
the invention provides a customizable detection channel dead-alive dye based on mass spectrometry detection technology, which comprises a detection analysis reagent capable of customizing the dead-alive state of a sample cell of the detection channel, wherein the detection analysis reagent capable of customizing the dead-alive state of the sample cell of the detection channel comprises a cell binding group, a metal ion binding group and a skeleton structure of a bridging cell binding group and a metal ion binding group which may exist; wherein the cell binding groups bind to single or multiple binding sites on one or more sample cells and the metal ion binding groups bind to one or more, metal ions as detection tags, which are customizable.
Further, the cell binding groups and the binding sites of the cells are functional group structures of the cells, ribonucleotides and derivatives thereof, deoxyribonucleotides and derivatives thereof, including amino groups, mercapto groups, methyl groups, acetyl groups, phosphate groups, carbonyl groups, aldehyde groups, carboxyl groups, glycosyl groups, and bases;
the binding mode of the metal ion binding group and the metal ion comprises a binding mode that the metal ion is chelated to the metal ion binding group in a coordination mode, a binding mode that the metal ion is embedded in a high polymer because the ion ruler diameter is larger than the polymer polymerization pore diameter, a binding mode that the metal ion is in a metal organic framework mode and a binding mode that the metal is taken as a part of a molecule in an atomic mode;
and a framework structure which can bridge the cell binding group and the metal ion binding group and avoid failed cell binding caused by steric hindrance effect, wherein the framework structure comprises a ring-shaped, chain-shaped, branched-chain-shaped, star-shaped, sphere-shaped, spindle-shaped, ship-shaped and net-shaped framework structure.
Further, the metal ion binding groups include ethylenediamine tetraacetic acid, diethylenetriamine pentaacetic acid, 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid.
Further, the metal ion is an element having the following atomic number: 3. 4, 11-13, 19-32, 37-42, 44-51, 55-60, 62-83.
Further, the metal ions are palladium element and platinum element.
Further, the detection and analysis reagent capable of customizing the dead and alive states of the sample cells of the detection channel comprises (S) -1- (p-bromoacetamidobenzyl) ethylenediamine tetraacetic acid BABE, can be targeted to bind to free sulfhydryl groups on cells, and can be customized to bound metal ions; or 1- (4-isothiocyanatobenzyl) ethylenediamine-N, N-tetraacetic acid ITCBE, can be targeted to bind to free primary amine groups on cells, and the bound metal ions can be tailored.
Further, the metal ion is an element having the following atomic number: 3. 4, 11-13, 19-32, 37-42, 44-51, 55-60, 62-83.
Further, the metal ions are palladium element and platinum element.
The second aspect of the present invention provides a method for preparing a dead-living dye of a customizable detection channel based on mass spectrometry detection technology, comprising the following steps: dissolving BABE or ITCBE with buffer solution, adding customized metal ions, and obtaining the dead and alive dye.
The third aspect of the invention provides the application of the dead and alive dye of the customizable detection channel based on the mass spectrometry detection technology in distinguishing dead and alive cell populations in the mass spectrometry detection technology.
The beneficial effects of the invention are as follows:
mass spectrometry detection technology is a high-new detection technology with high throughput, high sensitivity and high accuracy for antibody staining of cell samples, delineating cell composition and detecting protein expression levels. Because the dead cell population in the sample has very strong nonspecific adsorption capacity to antibodies, the staining state of the dead cells in the sample cells cannot truly reflect the actual protein expression level of the sample cells. Therefore, it is necessary to distinguish between dead and living cell populations in the sample by a suitable method, and reject the staining data of dead cells, thereby obtaining real target sample staining data. By analyzing the dead and alive staining data, the activity state of the sample cells can be objectively evaluated, and the data quality of the obtained samples can be further evaluated.
The invention discloses a novel dead living dye which is applied to a mass spectrometry flow detection technology and can customize a detection channel, wherein the dead living dye can realize signal distinction through different dyeing rates of dead living cells (the dye is mainly dyed in a short time because of the selective permeability of living cell membranes or protein outside the cell membranes, but dead cells can be marked on proteins inside and outside the membrane because of the loss of the selective permeability of the dye, so that the signal of the dead cells is higher than that of the living cells), the use effect of the dye can be realized, and the effect of distinguishing the dead living dye is more remarkable in part of samples (blood or tissue samples with low cell viability). After comparative analysis of the same various antibody staining combinations, the self-made dead-living dye disclosed by the invention does not influence the antibody staining effect of the antibody on the sample cells, and has high consistency with the antibody staining effect of the commercial dead-living dye for treating the sample cells. In addition, the self-made dead-living dye disclosed by the invention can realize customization of any metal channel applicable to mass spectrometry detection, meets diversified detection application requirements, and has more flexible and wide application prospects.
Drawings
FIG. 1 shows the effect of commercial dead and living dyes on differentiating dead and living cell populations from homemade dead and living dyes.
FIG. 2 is an expression heat map of 40 antibody detection parameters and staining signal intensity.
FIG. 3 is a graph of sample cell grouping effect versus subpopulation ratio for home-made and commercial dead-living dyes for the same sample.
Fig. 4 is a graph of a sample statistical consistency analysis.
FIG. 5 is a graph showing the effect of differentiating dead and living cell populations from home-made dead and living dyes using different metal channels.
FIG. 6 is a graph showing the effect of itCBE on differentiating dead and living cell populations from dead and living dyes.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings, but the present invention is not limited to the following specific examples.
The examples provided below are not intended to limit the scope of the invention nor the order in which the steps are performed, i.e., aqueous solutions, where concentration is distinguished in units of different concentration types, e.g., "mg/mL" means the ratio of solute mass to solution volume, and so forth. The present invention is obviously modified by those skilled in the art in combination with the prior common general knowledge, and also falls within the scope of protection claimed by the present invention.
Example 1
Preparation of dead and alive dye capable of customizing detection channel based on mass spectrometry flow detection technology
Preparation of 110Pd-BABE dead-living dye
(1) Preparing (S) -1- (p-Bromoacetamidobenzyl) ethylenediamine tetraacetic acid (1- (p-bromoacetamidobenzol) ethyl-N, N' -tetraacetic acid, BABE) solution
The BABE powder is dissolved to 1pM to 100M by using all buffer solutions with buffer capacity such as Tris-HCl buffer, phosphate buffer, carbonate buffer, HEPES buffer and the like, the pH value is 4-10, preferably 7.2-7.4, and the BABE powder is fully and uniformly mixed. This example uses phosphate buffer (GNM 20012) to prepare a 2mM BABE solution.
(2) Preparation of 110Pd-BABE dead and alive dye
Diluting 50mM 110Pd metal ion stock solution (obtained by dissolving palladium metal halide salt in concentrated hydrochloric acid) with the above prepared 2mM BABE solution to 1mM, mixing thoroughly, storing at low temperature for use, wherein the storage temperature can be 2-8deg.C, -20deg.C, -80deg.C, etc., preferably storing at-20deg.C for a long period, and storing at 2-8deg.C for a short period. Other metal ions that can bind EDTA and do not occupy the detected cell signal can be selected in addition to the 110Pd metal ions.
Example 2
Use of 110Pd-BABE dead-living dye and performance comparison with commercial dead-living dye
Step one, preparing a PBMC single cell suspension with a cell viability of about 80%
(1) PBMC single cell suspension preparation
The single cells to be detected, which can be digested tissue single cells, split red blood samples and peripheral blood single nuclear cells, were human peripheral blood single nuclear cells (Peripheral Blood Mononuclear Cells, PBMC) and the PBMC single cell suspension was aliquoted into 2 parts for testing respectively as a control group, i.e. commercial dead-alive dye group (cisplatin, standard BioTools, 201194) and a test group, i.e. self-made dead-alive dye group (110 Pd-BABE dead-alive dye prepared in example 1).
(2) Preparation of dead cells
Cells with 20% cell number are separated from each part, and the cells are placed in a water bath kettle at 75 ℃ and heated for 15min. The cell viability of the cells of 20% of the cell number after heat treatment was 0% as measured by trypan blue staining.
(3) Preparation of PBMC Single cell suspension with fixed cell viability
The cells with the cell viability of 0% and the cell number of 20% and the cells with the cell viability of the remaining 80% are uniformly mixed, and trypan blue staining detection is carried out after uniformly mixing, so that the PBMC single cell suspension with the cell viability of about 80% is obtained.
(4) Each group takes 10 7 Single cells were centrifuged at 400g/5min at room temperature and the supernatant was discarded.
Step two, determining the death and the activity of the cells
(5) Commercial dead-living dye and self-made dead-living dye are respectively mixed with phosphate buffer solution (GNM 20012) according to the volume ratio of 1:99 to prepare 100uL dead-living dye. The corresponding 100uL of dead and alive dye was added to each group of EP tube cell pellet, mixed well and stained on ice for 5min.
(6) 1mL of pre-chilled phosphate buffer containing bovine serum albumin was added, the mixture was centrifuged at 4℃for 400g/5min, the supernatant was discarded, and the procedure was repeated 2 times. Wherein, the phosphate buffer solution containing bovine serum albumin is prepared from 0.5-2mg of bovine serum albumin and 100mL of phosphate buffer solution (GNM 20012).
Step three, cell fixation
(7) The fixation fluid was prepared using Fix and perm (Standard BioTools) and DNA intercalators 191/193Ir (Standard BioTools) in a volume ratio of 2000:1 or other equivalent ratios, and cells were resuspended in 200uL of fixation fluid in each set of EP tubes, thoroughly mixed and then placed in a refrigerator at 4℃overnight.
Step four, rinsing and on-machine detection
(8) After 1mL of water was added to each set of EP tubes to resuspend the cells, the cell suspension was aspirated with a pipette and transferred through a flow tube filter head 38um filter membrane into the corresponding flow tube. After washing the EP tube wall several times by adding 1mL of water again, the washing liquid is sucked by a pipette, and the same flow tube filter membrane is filtered and then put into the same flow tube.
(9) Centrifuging at 4deg.C for 800g/5min, and discarding supernatant.
(10) After 1mL of water was added to each flow tube to resuspend the cells, 10uL were individually counted.
(11) According to the cell count result, the cells of a control group and a test group with the same cell number are taken, the cells are respectively washed with water for a plurality of times, then a proper amount of balance magnetic beads are added, the cells are respectively detected on a Helios mass spectrum cell analyzer (Fluidigm), and the two groups of data are processed and analyzed. The control group and the test group can be independently put on the machine or mixed on the machine (the mixed machine needs to pass through the sample bar code mark).
Example 3
Influence of dead and alive dyes of customizable detection channels on antibody staining based on mass spectrometry detection technology
The phosphate buffer containing bovine serum albumin according to this example was prepared from 0.5-2mg of bovine serum albumin and 100mL of phosphate buffer (GNM 20012).
Step one, preparing PBMC single cell suspension
(1) The single cells tested in this example were human peripheral blood mononuclear cells (Peripheral Blood Mononuclear Cells, PBMC), and PBMC single cell suspensions were aliquoted into 2 parts for testing in a control group, i.e., commercial dead-alive dye group (cisplatin, standard BioTools, 201194), and in a test group, i.e., self-made dead-alive dye group (110 Pd-BABE dead-alive dye prepared in example 1).
(2) Each group takes 10 7 Single cells were centrifuged at 400g/5min at room temperature and the supernatant was discarded.
Step two, determining the death and the activity of the cells
(3) Commercial dead-living dye and self-made dead-living dye are respectively mixed with phosphate buffer solution (GNM 20012) according to the volume ratio of 1:99 to prepare 100uL dead-living dye. The corresponding 100uL of dead and alive dye was added to each set of EP tube cell pellet, mixed well and stained on ice for 5min.
(4) 1mL of pre-chilled phosphate buffer containing bovine serum albumin was added, the mixture was centrifuged at 4℃for 400g/5min, the supernatant was discarded, and the procedure was repeated 2 times.
Step three, sealing
(5) 50uL of blocking solution was added to each group to resuspend the cells and transferred to clean EP tubes, labeled, and incubated on ice for 20min. The sealing liquid is specifically prepared from 0.5-2mL human immunoglobulin solution (2 mg/mL), 0.5-2mL mouse immunoglobulin solution (2 mg/mL), 0.5-2mL rat immunoglobulin solution (2 mg/mL), 0.5-2mL hamster immunoglobulin solution (2 mg/mL), 0.5-2mg bovine serum albumin and 100mL phosphate buffer solution (GNM 20012).
Step four, extracellular antibody staining
(6) 50uL of extracellular antibody mixture (40 extracellular antigen concentrations of 0.5ug/uL in Table 1, each 0.5uL of pre-chilled bovine serum albumin-containing phosphate buffer solution 30 uL) was added to each group, and the mixture was thoroughly mixed and stained on ice for 30min.
TABLE 1
(7) 1mL of pre-chilled phosphate buffer containing bovine serum albumin was added, the mixture was centrifuged at 4℃for 400g/5min, the supernatant was discarded, and the procedure was repeated 2 times.
Step five, cell fixation
(8) The fixation fluid was prepared using Fix and perm (Standard BioTools) and DNA intercalators 191/193Ir (Standard BioTools) in a volume ratio of 2000:1 or other equivalent ratios, and cells were resuspended in 200uL of fixation fluid in each set of EP tubes, thoroughly mixed and then placed in a refrigerator at 4℃overnight.
Step six, sample bar code marking (only the step is needed to be selected when a plurality of samples to be detected are mixed for machine-on detection):
(9) The cell samples were removed from the refrigerator at 4℃and centrifuged at 800g/5min at 4℃and the supernatant discarded.
(10) To each set of EP tubes 1mL of phosphate buffer (GNM 20012) was added and centrifuged at 4℃for 800g/5min and the supernatant was discarded.
(11) 100uL of BC1 solution is added into a commercial dead-living dye set, 100uL of BC2 solution is added into a self-made dead-living dye set, and cells are resuspended and then placed on ice for incubation for 30min. The BC1 solution is prepared from 1mL of Pd104 (1.5-2 uM, fluidigm), 1mL of Pd105 (1.5-2 uM, fluidigm), 100mL of phosphate buffer (GNM 20012), and the BC2 solution is prepared from 1mL of Pd104 (1.5-2 uM, fluidigm), 1mL of Pd106 (1.5-2 uM, fluidigm), 100mL of phosphate buffer (GNM 20012).
(12) To each set of EP tubes 1mL of phosphate buffer containing bovine serum albumin was added and centrifuged at 4℃for 800g/5min, and the supernatant was discarded.
(13) To each set of EP tubes 1mL of water was added to resuspend the cells, centrifuged at 4℃for 800g/5min and the supernatant discarded.
Step seven, rinsing and on-machine detection
(14) After 1mL of water was added to each set of EP tubes to resuspend the cells, the cell suspension was aspirated with a pipette and transferred through a flow tube filter head 38um filter membrane into the corresponding flow tube. After washing the EP tube wall several times by adding 1mL of water again, the washing liquid is sucked by a pipette, and the same flow tube filter membrane is filtered and then put into the same flow tube.
(15) Centrifuging at 4deg.C for 800g/5min, and discarding supernatant.
(16) After 1mL of water was added to each flow tube to resuspend the cells, 10uL were individually counted.
(17) According to the cell count result, the cells of the control group and the test group with the same cell number are taken, after mixing, the cell samples are washed again with water for a plurality of times, a proper amount of balance magnetic beads are added, detection is carried out on a Helios mass spectrum cell analyzer (Fluidigm) machine, and the two groups of data are processed and analyzed.
In the present invention, we contrast stained PBMC prepared in step one of example 2 with a cell viability of about 80% using commercial dead-live dye and homemade dead-live dye (110 Pd-BABE dead-live dye with thiol group as cell binding site). As a result, it was found that the metal detection signal intensity of commercial dead-alive dyes was equivalent to the effect of homemade dead-alive dyes in the dead cell staining population; however, the signal intensity of commercial dead-living dyes was significantly higher than the staining effect of self-made dead-living dyes in terms of staining signal intensity of living cells (fig. 1). Therefore, the self-made dead living dye has better effect of distinguishing dead and living cell populations in the sample. Since the mass flow dead living staining step occurs before antibody staining, in order to evaluate whether self-made dead living dye can have a significant effect on the antibody staining effect of a sample, we used commercial dead living dye and self-made dead living dye to differentiate dead and living cell populations in the sample on the PBMC sample, and then used 40 extracellular antibody mixed solutions for staining. The results of the heat map and protein differential expression showed that the self-made dead-living dye did not significantly affect antibody expression compared to the commercial dead-living dye (fig. 2), and analysis of the results of the subpopulations typing and subpopulation duty cycle revealed that the self-made dead-living dye was able to detect each subpopulation of cells consistent with the commercial dead-living dye and that the duty cycle of the corresponding subpopulations remained highly consistent (fig. 3). The results of the correlation analysis showed that the results of the homemade dead-living dye analysis were highly consistent with the commercial dead-living dye effect, with no significant statistical differences (fig. 4).
In order to verify the self-made dead-living dye applied to the mass spectrometry detection technology disclosed by the invention, the purpose that a detection channel can be customized and a labeled target can target functional groups on cells such as sulfhydryl, amino and the like is achieved, and the following application and expansion are performed. We developed custom made dead-living dyes for different metal channels of 104Pd, 105Pd, 106Pd, 108Pd, 110Pd based on BABE: 104Pd-BABE dead-alive dye, 105Pd-BABE dead-alive dye, 106Pd-BABE dead-alive dye and 108Pd-BABE dead-alive dye, 110Pd-BABE dead-alive dye (prepared in the same manner as in example 1 except that metal ions were replaced), and a comparative test (cell viability was adjusted) with a commercial dead-alive dye (cisplatin, standard BioTools, 201194) according to the method in example 2. The results showed that, in addition to the self-made dead-alive dyes on the 110Pd metal channels, the 104Pd, 105Pd, 106Pd, 108Pd metal channels, also had a distinguishing effect on dead and living cell populations that was comparable to the commercial dead-alive dyes (fig. 5, the subjects were PBMCs with 20% cell viability). Furthermore, we have devised a novel metal-binding molecule 1- (4-isothiocyanatobenzyl) ethylenediamine-N, N' -tetraacetic acid (isothioxynobenzyl-EDTA, ITCBE) that targets primary amine groups in cell samples. By the same preparation as in example 1, the dead dye of ITCBE-104Pd was prepared by changing BABE to ITCBE and 110Pd to 104 Pd. And tested in comparison to commercial dead-alive dyes (cell viability was adjusted) as described in example 2. The results show that ITCBE-104Pd dead-living dye with amino groups (primary amine groups) as cell binding sites also has an effect comparable to that of dead, living cell populations of commercial dead-living dye (fig. 6,8:2, 2:8, 7:3, 3:7 represent the cell number ratio of living cells to dead cells). Therefore, the dead and alive dye of the customizable detection channel based on the mass spectrometry detection technology disclosed by the invention can be expanded to all cell binding groups with targets such as functional group structures, nucleic acids (ribonucleotides or deoxyribonucleotides) and derivatives thereof on cells, and metal ion binding groups, and has the potential of being developed into the mass spectrometry detection and being applied to distinguishing dead and alive cell populations in sample cells, and all the dead and alive dye falls into the protection scope of the invention.
Claims (5)
1. A customizable detection channel dead-alive dye based on mass spectrometry detection technology, which is characterized by comprising a detection analysis reagent capable of customizing the dead-alive state of a sample cell of the detection channel, wherein the detection analysis reagent capable of customizing the dead-alive state of the sample cell of the detection channel comprises (S) -1- (p-bromoacetamidobenzyl) ethylenediamine tetraacetic acid BABE, free sulfhydryl groups on a target binding cell can be customized, and bound metal ions can be customized; or alternatively1- (4-Isothiocyanatobenzyl) ethylenediamine-N, N, N, N-tetraacetic acidITCBE, which can target primary amine groups free on bound cells, and bound metal ions can be tailored;
the dead and alive dye is obtained by dissolving BABE or ITCBE with buffer solution and adding customized metal ions.
2. The mass flow detection technology based customizable detection channel dead-alive dye of claim 1, wherein the metal ions are elements with the following atomic numbers: 3. 4, 11-13, 19-32, 37-42, 44-51, 55-60, 62-83.
3. The customizable detection channel dead and alive dye based on mass spectrometry detection technology according to claim 2, wherein the metal ions are palladium element and platinum element.
4. A method for preparing a dead-living dye for a customizable detection channel based on mass spectrometry detection techniques according to any one of claims 1 to 3, comprising the steps of: dissolving BABE or ITCBE with buffer solution, adding customized metal ions, and obtaining the dead and alive dye.
5. Use of a dead living dye of a customizable detection channel based on mass spectrometry detection techniques according to any one of claims 1 to 3 in mass spectrometry detection techniques to distinguish between dead and living cell populations.
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| CN111189761A (en) * | 2020-01-10 | 2020-05-22 | 浙江普罗亭健康科技有限公司 | Cell signal channel detection kit based on mass spectrometry flow technology and detection method |
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