CN219879956U - Single-cell high-throughput sorting microporous chip and proteome multi-group chemical analysis system - Google Patents
Single-cell high-throughput sorting microporous chip and proteome multi-group chemical analysis system Download PDFInfo
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Abstract
The utility model discloses a single-cell high-flux sorting microporous chip and a proteome multi-group analysis system consisting of a single-cell flow sorting unit, the single-cell high-flux sorting microporous chip and a liquid chromatograph-mass spectrometer analysis unit.
Description
Technical Field
The utility model discloses a micropore array chip, and belongs to the technical field of biological information and materials.
Background
The flow separation technology is a modern cell separation technology for qualitatively or quantitatively analyzing cell characteristics and functional states according to scattered light and emitted light intensity generated by cells under the excitation of high-energy laser, so that specific cell subpopulations are obtained through limiting various parameters, and the flow separation technology is commonly used for identifying, classifying, quantifying and separating complex sample cells. The flow sorter is equipped with a monoclonal sorting device (automated cell deposition unit, ACDU) which can take 96 or 384 standard pore plates as single cell receiving containers, and complete full-automatic single cell sorting by setting parameters such as pore plate type, sorting starting point, end point position and the like. The method has the advantages of high single cell acquisition precision, high flux and low cost, and can assist in verifying the cell map drawn by high-flux sequencing, so that the method is widely applied to single cell sequencing research. In addition to standard well plates, the ACDU stage is equipped with slide sample holders for making temporary smears of sorted cells, which effectively secures slide positions, providing the opportunity for flow sorting single cells to microwell chips.
In chinese patent No. CN113138249B, the inventors constructed a microporous array silicon-based chip for producing micro proteome data represented by single cells. The micropore array chip is used as a single-cell proteome sample processing platform and has various advantages: firstly, the device can be used as a container for tissue cell lysis, metabolite extraction and proteolysis, so that the loss caused by the transfer of samples between different containers is avoided; secondly, compared with a conventional centrifuge tube, the nano-upgrading micropore has the advantages that the surface area is reduced by hundreds of times, and the surface area of a sample contacted with a container is effectively reduced, so that the sample loss caused by nonspecific adsorption on the inner wall of the container is obviously reduced; furthermore, the micro-pore chip can obviously reduce the volume of a sample solution, effectively shorten the intermolecular diffusion distance while avoiding the dilution of a micro-sample, and improve the reaction concentration of the micro-sample, thereby enhancing the interaction between substrate protein and protease so as to improve the enzymolysis speed and efficiency. The chip partially solves the problem of analysis of a micro-cell proteome sample, but has the following problems in the actual use process: the lack of an automatic cell sorting scheme matched with the chip results in that cell sample sorting mainly depends on manual capillary micromanipulation, and the cell sample sorting has the advantages of low speed of obtaining single cells and micro cells, low flux and high technical requirements for operators, and limits the further development and application of the chip.
Based on the problems in the use process of the chip in the prior art, the cell capturing efficiency of automatic cell sorting is low, the flux is low, and the cell subpopulation with high heterogeneity is difficult to obtain.
The utility model aims to provide a microporous array chip which can be matched with a flow sorter with a slide sample clamp equipped on an objective table to establish a high-flux single-cell capturing system based on the microporous array chip, and solve the problems of low single-cell capturing efficiency, low flux, difficulty in obtaining high-heterogeneity cell subsets and the like in the sample pretreatment process of single-cell proteome analysis.
Disclosure of Invention
In view of the above, the present utility model provides a micro-hole array chip, which is rectangular with a specification of 25×75mm, and is divided into a blank area and a micro-hole array distribution area along a long axis of 75mm, wherein the area of 0-21.081mm along the long axis is the blank area, the area of 21.081-75mm is the micro-hole array distribution area, 5×12 micro-hole arrays are distributed on the micro-hole array distribution area, and the distance between the micro-holes is 2.503mm.
In a preferred embodiment, each microwell in the microwell array core has a diameter of 1.955mm and a microwell depth of 0.2mm.
In another preferred embodiment, the inner walls of the microwells are hydrophilically modified.
In a more preferred embodiment, the hydrophilic modification is a covalent modification using a silane derivative.
In a particularly preferred embodiment, the silane derivative is 2- [ methoxy (polyethyleneoxy) propyl ] trimethoxysilane.
Next, the present utility model provides a proteome multi-group chemical analysis system comprising the above-described microwell array chip, single cell flow sorting unit, and liquid chromatography-mass spectrometry unit.
In a preferred embodiment, the nozzle model of the single cell flow sorting unit is 85 μm; the position of the separation liquid flow is far left, and the separation mode is single cell. In one embodiment of the utility model, the single cell flow sorter unit is a commercially available BD company flow sorter (model: aria III). In practice, other flow sorters known in the art capable of performing single cell sorting may be used in the application of the present utility model.
In another preferred embodiment, the single cell flow sorting unit comprises a forward fluorescence microscope applied to quality control evaluation of single cell flow sorting effects.
In yet another preferred embodiment, the proteomic analysis comprises a metabolome, a proteome, and a phosphorylated proteome analysis.
In another preferred embodiment, the mass spectrometry is electrospray ionization mass spectrometry.
Aiming at the problems that the manual operation is low in efficiency and difficult to standardize in the sample pretreatment process of the cell proteome analysis, single-cell capturing efficiency is low, flux is low, high heterogeneous cell subsets are difficult to obtain and the like, the utility model provides a micropore array chip with unique design, single cells of single-cell flow sorting are obtained through the micropore array chip in a high flux manner, dead cells can be removed through the combination of various luciferins in the flow sorting, specific cell subsets with different surface markers and different states are obtained, a micropore chip processing platform can directly obtain the cell subsets in complex samples such as tissues and body fluids, the sample processing and extraction are completed in micropores, and the multi-group chemical characterization including metabolome, proteome and phosphorylated proteome is directly carried out on the single cells. In the proteome multi-group chemical analysis system provided by the utility model, the per-hour sorting quantity of the single flow sorting unit reaches more than 500 single cells, so that the sample preparation flux is greatly improved, the manual operation is reduced, and the parallelism among samples is ensured as much as possible. Compared with the sorting flux of 20 single cells per hour of manual micromanipulation, the sorting speed is improved by 25 times.
Drawings
FIG. 1A top plan view of a micro-hole array chip
FIG. 2 shows the primary cell microscopic examination result of EGFP reporter gene mice in microwell chips after flow sorting.
Detailed Description
The utility model will be further described with reference to specific embodiments, and advantages and features of the utility model will become apparent from the description. These examples are only exemplary and do not limit the scope of the utility model as defined by the claims.
Example 1 preparation of microwell array chips
In a specific embodiment of the present utility model, the micro-pore array chip is a silicon-based micro-pore array chip, and is prepared by the following steps:
a monocrystalline silicon wafer is used as a substrate, and a micro-pore chip is prepared by photoetching, silicon deep reactive ion etching and a micro-electromechanical technology (MEMS) of laser micro-cutting. The photoetching process adopts Hexamethyldisilazane (Hexamethyldisilazane) to modify the silicon wafer, and then the silicon wafer is pretreated by gluing, exposing and developing. In order to couple a chip with a sample injection platform of a flow sorter (BD, model: aria III), the chip takes the shape of a standard glass slide as a reference, and the silicon chip is cut to obtain the chip with the outer edge dimension of 25 multiplied by 75.149mm, and a non-array area is reserved on one side, so that the chip is convenient to hold, move and place. And etching deep silicon to form micropores with the diameter of 1.955mm and the depth of 0.20mm, wherein the micropore display number of each chip is 5 multiplied by 12, the interval of the outer edges of the micropores in the same vertical row is 2.555mm, and the interval of the outer edges of the micropores in the same horizontal row is 2.503mm. The micro-hole array chip is structurally shown in a top view in FIG. 1. According to the sorter setting, the microwells (lower left microwells as viewed in fig. 1) at the corners of the array region adjacent to the non-array region side were determined as sorting start positions, and the corresponding microwells (upper right microwells as viewed in fig. 1) along the diagonal ends of the array region from the start positions were determined as sorting end positions.
The microporous silicon-based surface is hydrophilically modified by covalent binding of highly hydrophilic silane derivatives to reduce the loss of protein by non-specific adsorption.
Immersing a microwell array chip into H 2 O:H 2 O 2 HCl (v/v/v) =5:1:1 solution was activated for 10-15min, the chips were washed with deionized water and dried under nitrogen, and the activated chips were immersed in the silane derivative 2- [ methoxy (polyethyleneoxy) propyl at room temperature]The surface hydrophilic modification is carried out by reacting in trimethoxysilane (Oligo-EG, gelest, cat. No. 65994-07-2) for 30min, the obtained chip is washed by deionized water and dried in nitrogen, and the modified chip can be stored for 8 weeks at normal temperature.
Example 2 application of microwell array chip
Primary liver parenchymal cells were obtained from EGFP mice as follows: after the mice are anesthetized, decoupling and exposing the inferior vena cava, liver and portal vein; inserting a 24G vein indwelling needle from the portal vein initial position until the portal vein root part, and extracting the needle head; after the venous indwelling needle is connected with a perfusion tube, 6ml/min of I perfusion liquid preheated at 37 ℃ is perfused (136mM NaCl,5.3mM KCl,0.5mM NaH) 2 PO 4 ·2H 2 O，0.4mM Na 2 HPO 4 ·7H 2 O，9.1mM HEPES，4.1mM NaHCO 3 5mM glucose, 0.5mM EGTA) for 8min. When the liver bulges, the inferior vena cava is interrupted rapidly when perfusion begins, and a large amount of blood flows out. The inferior vena cava is pressed during the period, so that the liver is continuously bulged, and the full perfusion is ensured. Thereafter, 5ml/min of the II perfusate preheated at 37℃was perfused for 5min. Stopping perfusion, removing liver, removing gallbladder, and placing into a container filled with II perfusate (136mM NaCl,5.3mM KCl,0.5mM NaH) 2 PO 4 ·2H 2 O，0.4mM Na 2 HPO 4 ·7H 2 O，9.1mM HEPES，4.1mM NaHCO 3 ，5mM CaCl 2 In a 0.05% collagenase IV) petri dish, gently shredding the liver with sterile ophthalmic forcepsA large number of cell dispersions can be seen. Liver parenchymal cells were enriched by centrifugation at 50g at 4 ℃. After labeling CD45 and CD31 antibodies, cell pellets were obtained by centrifugation. The cell pellet was resuspended in DMEM and the cell suspension density was adjusted to 10 by cell counting 7 And each mL. Sorting the sample into microporous chips by a flow sorter. Adjusting the parameters of the commercial BD company flow sorter (model: aria iii) used includes: the nozzle model is 85 mu m; the droplet oscillation frequency (Freq) was 46.8; the amplitude of vibration (amplitude) was 12.4; drop position (Drop 1) 221; drop spacing (Gap) of 12; a droplet delay value of 28.15; the position of the separation liquid flow is far left; the sorting mode is Single cell. And placing the chip for testing into a slide sample clamp, setting coordinates of a starting point and an end point, and observing whether the liquid drops accurately enter micropores of the chip by irradiating the chip with naked eyes through a flashlight. And repeatedly adjusting the initial point and the end point coordinates, and replacing the test chip with the formal chip after confirming the final coordinates. Applying the hepatic parenchymal cell suspension, and circling and selecting CD45 - CD31 - The hepatic parenchymal cells were used as a target cell population, the number of cells required per microwell was set, and sorting was performed in a 12-column×5-row mode. For details see the BD_FACSAria_III_user_guide manual. The sorted microwell chips were subjected to microscopic examination using a forward fluorescence microscope (Nikon ECLIPSE Ni-E) to confirm whether the sorting accuracy reached the standard (fig. 2).
Samples in the microporous chip were prepared as described in chinese patent No. CN113138249B for proteome samples. As can be seen from mass spectrometry, sample preparation efficiency can be greatly improved by using a flow sorting system based on a microporous chip (as shown in table 1).
TABLE 1 control of the number of single cell samples taken per hour from the microwell chip for both methods
| Manual operationMicromanipulation of microscopic operations | Flow type sorting system based on microporous chip | |
| Obtaining single cell number per hour | 20±5 | 500±10 |
As can be seen from table 1, three independent repeated experiments showed that manual micromanipulation and the constructed microporous chip-based flow sorting system obtained the number of microporous chip single cell samples per hour. Therefore, the constructed flow sorting system based on the microporous chip can improve the original manual micromanipulation efficiency by 25 times. Using microwell chips, sorting out each microwell chip takes about 6 minutes, and sorting 500 single cells takes about only 10 chips for 1 hour.
Three repeated experiments show that under the system, the identification amount of the Hela protein of the single human cell line can reach 1200; up to 1600 of 15; up to 2800 up to 75. The primary hepatic parenchymal cell protein identification amount of 15 mice can reach 500; up to 1400 out of 75 (as shown in table 2). The specific experimental process comprises the following steps: sorting corresponding number of cells into chip micropores by flow, adding trypsin, digesting protein at 37 ℃ for 2-6h (the mass ratio of protease to protein can be 1 (1-3), the concentration of protease can be 0.5-1 mug/mu L), collecting digestion liquid, and carrying out liquid chromatography-mass spectrometry analysis on the digestion liquid. In mass spectrometry, a data independent scan mode can be used to search a database containing only proteomic profiles. In the identification of peptide fragments and proteins, the false positive rate (FDR) of the protein was set to 1% at peptide fragment level and protein score (-10 lgP). Gtoreq.20, and the library was searched using PEAKS Studio X+ software.
TABLE 2 mass spectrometry identification results for samples prepared by flow sorting systems based on microporous chips
Table 2 shows the data of the micro-cell proteome such as single cell obtained by mass spectrum identification by sample preparation based on the flow sorting system of the microporous chip.
Claims (10)
1. The single-cell high-flux sorting microporous chip is characterized in that the single-cell high-flux sorting microporous chip is in a rectangle with the specification of 25 multiplied by 75mm, the single-cell high-flux sorting microporous chip is divided into a blank area and a microporous array distribution area along the long axis of 75mm, the area of 0-21.081mm along the long axis is the blank area, the area of 21.081-75mm is the microporous array distribution area, 5 multiplied by 12 microporous arrays are distributed on the microporous array distribution area, and the distance between micropores is 2.503mm.
2. The single cell high throughput sorting microwell chip of claim 1, wherein each microwell in the single cell high throughput sorting microwell chip has a diameter of 1.955mm and a microwell depth of 0.2mm.
3. The single cell high throughput sorting microwell chip of claim 1, wherein the inner walls of the microwells are hydrophilically modified.
4. The single cell high throughput sorting microwell chip of claim 3, wherein the hydrophilic modification is a covalent modification using a silane derivative.
5. The single cell high throughput sorting microporous chip of claim 4, wherein said silane derivative is 2- [ methoxy (polyethyleneoxy) propyl ] trimethoxysilane.
6. A proteome multi-component analysis system, comprising the single-cell high-throughput sorting microporous chip, single-cell flow sorting unit, and liquid chromatography-mass spectrometry unit of any one of claims 1-5.
7. The proteomic multi-component analysis system of claim 6, wherein the nozzle model of the single cell flow sorting unit is 85 μm; the position of the separation liquid flow is far left, and the separation mode is single cell.
8. The proteome multi-component analysis system of claim 6, wherein the single cell flow sort unit comprises a forward fluorescence microscope for quality control evaluation of single cell flow sort effects.
9. The proteome multi-component analysis system of claim 6, wherein the proteome multi-component analysis comprises metabolome, proteome, and phosphorylated proteome multi-component analysis.
10. The proteomic multi-component analysis system of claim 6, wherein the mass spectrometry is electrospray ionization mass spectrometry.
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