Cell lysis buffer based on digital microfluidic system, quantitative pipetting device and pre-treatment method for protein

Abstract

The present invention relates to a cell lysis buffer, comprising the following active components: 0.08%-0.2% of DDM, 35-45 mM of CAA, 5-15 mM of TCEP, 85-100 mM of TEAB, 0-0.25% of ProteaseMAX, and 0-0.25% of RapiGest SF. The present invention also provides a pre-treatment method for a single-cell protein based on a digital microfluidic system. The cell lysis buffer is used for performing lysis, and a relatively high quantity of a detectable protein and a relatively high quantity of detectable peptide fragments can be obtained, and the method can be used for single-cell proteomics analysis. In addition, the present invention provides a quantitative pipetting device, which is used for stably and precisely preparing the obtained single-cell protein solution into a sample solution suitable for liquid chromatography detection.

Description

Cell lysis solution, quantitative pipetting device and protein pretreatment method based on digital microfluidic system

[0001] The present invention claims priority to Chinese patent application No. 202310941147X, filed with the Patent Office of China on July 28, 2023, entitled “Cell lysis solution and protein pretreatment method based on digital microfluidic system”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of biotechnology, and in particular relates to a cell lysate and protein pretreatment method based on a digital microfluidic system. Background Art

[0003] Cells are the fundamental units of structure and function in organisms. Due to factors such as the stochastic expression of genetic information and microenvironmental variations, heterogeneity between cells is ubiquitous. However, since analyses based on populations of cells reflect averages and fail to truly capture the differences between individual cells, heterogeneity analysis at the single-cell level is necessary. Proteins, as the direct executors of biological functions within cells, can sense and respond to external and internal stimuli through the dynamic changes in proteins and their post-translational modifications, thereby influencing the function and state of the entire organism. Consequently, single-cell proteomics has garnered increasing attention from researchers, particularly in areas such as cancer, immunity, development, and evolution. Uncovering the depth of cellular heterogeneity has become a hot topic and a significant opportunity. However, because the protein content of individual cells is very low and the number of protein types within a single cell is vast, uncovering cellular heterogeneity and functional characteristics within complex populations requires analyzing a large number of different protein species within a large number of cells. Consequently, single-cell proteomics places extremely high demands on sensitivity and throughput.

[0004] Among the various methods currently available for analyzing single-cell proteomics, those based on liquid chromatography coupled to mass spectrometry are able to obtain more comprehensive information about proteins within individual cells. Protein quantification is categorized into relative and absolute quantification. While absolute quantification appears to be more ideal, it is more complex and expensive, while relative quantification often meets scientific research needs. Therefore, non-targeted relative quantification, which compares chromatographic peaks of identical peptides with the same ionization efficiency in different samples, is more commonly used. Non-targeted relative quantification is further divided into label-free quantification and label-based quantification techniques. Label-free quantification involves enzymatically cleaving cells into peptides and then directly delivering them to mass spectrometry for detection. Compared to label-based methods, this method eliminates the need for expensive labeling reagents and simplifies the operation. The commonly used labeling reagent for label-based quantification is Thermo Fisher's TMT label. The TMT labeling method involves reacting peptides with a labeling reagent based on a non-labeled sample and then mixing them for mass spectrometry analysis. It offers low systematic error, good reproducibility, and high sensitivity.

[0005] The single-cell non-labeled quantitative sample pre-processing process on the microfluidic chip includes single-cell separation, cell lysis, protease cleavage, and sample output.

[0006] The single-cell TMT labeling quantitative sample pre-processing process includes single-cell isolation, cell lysis, protease cleavage, TMT labeling, termination labeling, and sample output.

[0007] Both label-free and label-based quantitative techniques require single-cell isolation to obtain suitable samples. However, there are still huge challenges in processing trace single-cell samples to obtain peptide solutions before mass spectrometry analysis, avoiding the introduction of mass spectrometry-incompatible reagents, reducing sample volume, and effectively transferring peptides to the mass spectrometry detector.

[0008] The pre-treatment of single-cell proteomics samples generally includes processes such as cell lysis, reduction, alkylation, enzymatic hydrolysis, TMT labeling, and termination. Currently, the most commonly used processing method is to use a cell sorter to sort single cells into a 96-well plate, and then manually add and transfer reagents to achieve pre-treatment for single-cell proteomics.

[0009] Commonly used methods for single-cell sorting technology include infinite dilution, precision-controlled manual picking, and laser acquisition microdissection (LCM). These are manual operations with low efficiency. Fluorescence activated cell sorting (FACS) and magnetic separation technology (MACS) improve sorting efficiency but are relatively expensive.

[0010] The current single-cell protein sample pretreatment system, CellenONE, utilizes droplet imaging technology for single-cell sorting. An ultra-micro-nanoliter reagent dispensing system dispenses mixed solutions (including lysis buffer and enzyme reagents) onto standard well plates or chips. The reaction proceeds with temperature-controlled incubation, and the system connects to a liquid-phase injection system for direct sample injection. The system achieves a final reaction volume of approximately 200 nL, with a throughput of 12 groups of 16 markers, or 192 samples per plate.

[0011] Traditional single-cell sorting technology cannot meet the high-throughput requirements of single-cell proteomics; because it relies on manual operations, it may cause errors and interference in the analysis results, and cannot meet the high-sensitivity requirements of single-cell proteomics; moreover, there are defects such as more reaction reagents required, which will increase the experimental cost, reduce the reaction efficiency, and cannot be parallelized when processing the same batch of samples. There is a time difference between the addition and mixing of the first sample reagent and the last sample reagent, and the reaction time cannot be parallelized and consistent.

[0012] Digital microfluidics systems can directly manipulate cell suspensions, separating large droplets into smaller ones to isolate single cells. They can also operate on multiple droplets simultaneously, enabling the full process of high-throughput label-free quantification of single-cell protein samples and pre-processing for TMT-labeled quantification. These systems offer advantages such as full automation, streamlined workflows, and controllable operations. Digital microfluidics technology is relatively new and has its own system technology requirements. In particular, achieving high numbers of detectable proteins and peptides is a key requirement for digital microfluidics systems.

[0013] Summary of the Invention

[0014] Based on this, the purpose of the present invention is to provide a cell lysis solution based on a digital microfluidics system and a single-cell protein pretreatment method using the cell lysis solution. Through such a treatment method, the time required for single-cell protein pretreatment is greatly shortened, and a higher number of detectable proteins and detectable peptides can be obtained.

[0015] The above objectives are achieved by the following technical solutions.

[0016] The first aspect of the present invention is to provide a cell lysis solution based on a digital microfluidic system, which includes the following active components: 0.08% to 0.2% DDM, 35 to 45 mM CAA, 5 to 15 mM TCEP, 85 to 100 mM TEAB, 0 to 0.25% ProteaseMAX, and 0 to 0.25% RapiGest SF.

[0017] In some embodiments thereof, it comprises the following active ingredients: 0.08%-0.15% DDM, 38-42mM CAA, 8-12mM TCEP, 90-100mM TEAB, and 0.05-0.2% ProteaseMAX; or it comprises the following active ingredients: 0.08%-0.15% DDM, 38-42mM CAA, 8-12mM TCEP, 90-100mM TEAB and 0.05-0.2% RapiGest SF.

[0018] In some preferred embodiments, it includes the following active ingredients: 0.09% to 0.12% DDM, 38 to 42 mM CAA, 8 to 12 mM TCEP, 90 to 100 mM TEAB, and 0.12 to 0.18% ProteaseMAX; or it includes the following active ingredients: 0.09% to 0.12% DDM, 38 to 42 mM CAA, 8 to 12 mM TCEP, 90 to 100 mM TEAB, and 0.12 to 0.18% RapiGest SF.

[0019] A second aspect of the present invention provides a quantitative pipetting device, comprising a body, a capillary tube, a liquid inlet tube, a suction and discharge device, and a selection valve;

[0020] The body is provided with a fixing part, a liquid outlet pipe and a supporting seat, the supporting seat is used to place the sample injection container, the capillary tube is detachably mounted on the fixing part, and when the capillary tube is mounted on the fixing part, the liquid outlet pipe, the capillary tube and the sample injection container on the supporting seat are sequentially connected;

[0021] The liquid inlet pipe, the liquid outlet pipe and the suction and discharge device are all connected to the selection valve, and the selection valve can be switched to connect the liquid inlet pipe to the suction and discharge device, or to connect the suction and discharge device to the liquid outlet pipe.

[0022] In some embodiments, the fixing member is movably arranged on the main body, and the quantitative pipetting device also includes a first driving device, the output end of which is connected to the fixing member and drives the fixing member to move between the liquid outlet tube and the supporting seat relative to the main body.

[0023] In some embodiments, the supporting seat is movably arranged on the main body, and the quantitative pipetting device also includes a second driving device, the output end of which is connected to the supporting seat and drives the supporting seat to move back and forth in a direction close to or away from the fixing member.

[0024] In some embodiments, the liquid outlet pipe and the supporting seat are arranged opposite to each other in the height direction of the main body, and the liquid outlet pipe is located above the supporting seat.

[0025] In some embodiments, the fixing member includes a connector and two clamping members arranged on the connector; the connector is connected to the output end of the first driving device, the clamping member is an elastic structure, and the two clamping members cooperate to form a non-closed elastic clamping structure.

[0026] In some embodiments, the supporting seat includes a supporting plate and a side plate vertically connected to the supporting plate; the side plate is connected to the output end of the second driving device, and a placement groove is provided on the supporting plate, and the groove wall of the placement groove is inclined toward one side of the side plate.

[0027] In some embodiments, the suction and discharge device includes a third driving device and a syringe, the syringe has an injection body and a push rod arranged in the injection body, the output end of the third driving device is connected to the push rod and drives the push rod to move back and forth in the injection body to perform suction or discharge operations.

[0028] In some embodiments, an observation window is further provided on the main body, a placement hole is provided through the main body corresponding to the observation window, the capillary is detachably plugged into the placement hole, and light-emitting bodies are provided on opposite sides of the observation window corresponding to the placement hole.

[0029] In some embodiments, the wall of the placement hole is further provided with a sensor for sensing the state of the capillary tube being inserted into the placement hole.

[0030] The third aspect of the present invention is to provide a quantitative pipetting assembly, which includes a body, a liquid inlet tube, a suction and discharge device, and a selection valve;

[0031] The main body is provided with a fixing part, a liquid outlet pipe and a supporting seat, the liquid outlet pipe and the fixing part are arranged correspondingly, and the fixing part and the supporting seat are arranged correspondingly;

[0032] The liquid inlet pipe, the liquid outlet pipe and the suction and discharge device are all connected to the selection valve, and the selection valve can be switched to connect the liquid inlet pipe to the suction and discharge device, or to connect the suction and discharge device to the liquid outlet pipe.

[0033] A fourth aspect of the present invention provides a single-cell protein pre-treatment method based on a digital microfluidics system, the method comprising the following steps:

[0034] S1. Prepare cell suspension;

[0035] S2. Inject the cell suspension onto the digital microfluidic chip and tear it into single-cell droplets;

[0036] S3. Inject any of the above lysates onto the digital microfluidic chip, tear the lysate into single droplets of lysate, merge with the single cell droplets, perform cell lysis, and obtain protein solution droplets;

[0037] S4. Injecting the enzymatic hydrolysate onto the digital microfluidic chip, tearing the enzymatic hydrolysate into single droplets, merging them with the protein solution droplets, and reacting to obtain peptide solution droplets;

[0038] S5. Mark or not mark as needed;

[0039] S6. If labeling is not required, the protein sample is directly obtained; if labeling is required, after the labeling reaction is completed, labeling is terminated to obtain the protein sample.

[0040] In some embodiments, the cell solubility in the cell suspension injected in step S2 is 5×105 cells / mL to 8×105 cells / mL. More preferably, the cell solubility in the injected cell suspension is 5.5×105 cells / mL to 6.5×105 cells / mL. Most preferably, the cell solubility in the injected cell suspension is 6×105 cells / mL, at which time the single cell acquisition efficiency is the highest.

[0041] In some embodiments, the enzymatic hydrolysis solution includes 0.05 ug / uL to 0.1 ug / uL Trypsin / Lys-C Mix and 90 mM to 110 mM TEAB. In this case, a larger number of detectable proteins and peptides are obtained.

[0042] In some embodiments, the protein sample can be obtained in step S6 by dipping with a gun tip or by capillary aspiration, and capillary aspiration can obtain a higher number of detectable proteins and peptides.

[0043] In some preferred embodiments, the capillary is the capillary in any one of the quantitative pipetting devices of the present invention. Furthermore, the protein sample is a sample whose volume is fixed to 2-10 uL after being processed by the quantitative pipetting device.

[0044] In some embodiments, the tearing method is to tear the droplet into half with equal volume. Using this method to separate single droplets can quickly and controllably form single cell droplets or reaction liquid droplets.

[0045] In some embodiments, the temperature for cell lysis in step S3 is 52° C.-60° C., preferably 54° C.-58° C.; and / or the lysis time is 13-17 min.

[0046] In some embodiments, the reaction temperature in step S4 is 35° C. to 39° C., and the reaction time is 1.5 h to 2.5 h.

[0047] The present invention has found that in a digital microfluidic system, when cells are lysed using a suitable lysis solution (0.08% to 0.2% DDM + 35 to 45mM CAA + 5 to 15mM TCEP + 85 to 100mM TEAB + 0 to 0.25% ProteaseMAX + 0 to 0.25% RapiGest SF), lysis is most complete and a high number of detectable proteins and peptides can be obtained. The single-cell protein pretreatment method of the present invention, through the above-mentioned treatment method, greatly shortens the time required for single-cell protein pretreatment, enables all parallel samples to react simultaneously, and obtains a high number of detectable proteins and peptides.

[0048] Furthermore, using 0.05ug / uL-0.1ug / uL Trypsin / Lys-C Mix and 90mM-110mM TEAB as the enzymatic digestion solution can effectively complete the enzymatic cleavage reaction, thereby obtaining a higher number of detectable proteins and peptides, which can meet the high sensitivity and throughput requirements of single-cell proteomics.

[0049] Furthermore, the protein droplet sample of the single-cell protein pre-treatment is at the nL level and cannot be directly introduced into the liquid chromatography. The present invention is achieved through the quantitative pipetting device, which is arranged by a main body, a capillary, a liquid inlet pipe, a suction and discharge device and a selection valve. The capillary can be used to absorb the sample liquid obtained by protein pre-treatment, and the capillary is detachably mounted on the fixing part of the main body. When the capillary is mounted on the fixing part, the liquid outlet pipe on the main body, the capillary and the injection container placed on the supporting seat are connected in sequence. At the same time, the selection valve is switched to connect the liquid inlet pipe with the suction and discharge device or to connect the suction and discharge device with the liquid outlet pipe. The quantitative solution can be sucked into the capillary through the liquid inlet pipe, the suction and discharge device and the liquid outlet pipe in sequence, driving the sample liquid in the capillary to be synchronously flushed out to the injection container, thereby quantitatively preparing the sample liquid to a predetermined volume (for example, the commonly used 2 to 10 uL) for liquid-mass detection. Therefore, when this quantitative pipetting device is used in a single-cell protein pretreatment system, the sample liquid aspirated by the capillary can be prepared into a quantitative volume in the device and then transferred to the injection container of the liquid chromatograph. Compared with the traditional manual pipetting and preparation of sample liquid, it has the characteristics of greater stability, higher accuracy, higher sample output efficiency, and is more suitable for liquid-mass spectrometry testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 is a schematic diagram of the microfluidic core machine in the digital microfluidic system.

[0051] FIG2 is a schematic diagram of the imaging system in the digital microfluidic system.

[0052] Figure 3 is a diagram showing the internal structure of the temperature control system in the digital microfluidic system.

[0053] FIG4 is a schematic diagram of the structure of a digital microfluidic chip.

[0054] FIG5 is a schematic diagram of the partitioning of the digital microfluidic chip during the experiment.

[0055] FIG6 is an experimental flow chart of a single-cell protein pre-treatment method according to an embodiment of the present invention.

[0056] 7A to 7I are schematic diagrams of the steps of the single-cell protein pretreatment method in Example 1.

[0057] FIG8 is a schematic diagram of a droplet being torn in half.

[0058] FIG9 is a schematic structural diagram of a quantitative liquid transfer device.

[0059] FIG10 is a schematic structural diagram of the quantitative pipetting device of FIG9 with the outer shell removed.

[0060] FIG11 is a schematic diagram of a partial structure of the quantitative pipetting device of FIG10 .

[0061] FIG12 is a schematic structural diagram of the capillary tube, the fixing member and the liquid outlet tube of FIG10 .

[0062] FIG13 is a schematic structural diagram of the capillary tube of FIG10 . DETAILED DESCRIPTION

[0063] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0064] Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those in Molecular Cloning: A Laboratory Manual (4th edition, edited by Green and Sambrook, published in 2013), or according to manufacturer recommendations. All commonly used chemical reagents used in the examples were commercially available.

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0066] It should be noted that, in this document, “fixed to” or “connected to” may mean directly fixing or connecting to an element, or indirectly fixing or connecting to an element.

[0067] Below are some definitions of terms.

[0068] PBS Phosphate buffer saline: phosphate buffer saline.

[0069] TMT Tandem mass tags: Tandem mass tags.

[0070] DDM n-Dodecyl-β-D-maltoside: n-Dodecyl β-D-maltoside, a non-ionic detergent, destroys membrane glycoproteins and the phospholipid bilayer, releasing proteins from the cell membrane. DDM also stabilizes membrane proteins and dissolves them in solution.

[0071] CAA Chloracetamide: Chloroacetamide, a sulfhydryl protecting agent, can prevent repeated oxidation of disulfide bonds and the formation of new disulfide bonds.

[0072] TCEP triethylcarboxyurea: a reducing agent that reduces disulfide bonds, making the reduced proteins easier to cleave and dissociate.

[0073] TEAB triethylammonium bicarbonate buffer: pH 8.5 ± 0.1, is a highly effective alkaline protein lysis agent that promotes protein lysis in an anhydrous environment and releases ions in the ion trap, thereby increasing ion signal intensity. Additionally, the buffering properties of TEAB can help stabilize protein structure, making it easier to maintain high-quality protein.

[0074] The single-cell protein pretreatment method described in the present invention is based on the design of a chip using digital microfluidics technology. It has a fully automated process and requires almost no manual intervention. The experimental cycle is greatly shortened, and samples can be quantitatively distributed. It has modular integration, flexible structure, and diverse functions, which can be adjusted according to needs. By using digital microfluidics technology to control samples and reagents, react and transfer to the upper cover detection point, the experimental environment is relatively independent and the risk of contamination is low. After process optimization, the method for single-cell proteomics pretreatment based on digital microfluidics technology, wherein the TMT-labeled single-cell proteomics sample pretreatment scheme includes the following experimental process: single cell separation, cell lysis, protease cleavage, TMT labeling, termination labeling, and sample output, including the following steps:

[0075] (1) Preparation of cell suspension: HeLa cells were cultured in a 37°C incubator with 5% CO2. The concentrations of fetal bovine serum and penicillin-streptomycin were 10% and 1%, respectively. When the cell density reached 80% of the entire culture dish, the cells were collected into a centrifuge tube and centrifuged at 100 × g, the supernatant discarded, and the cells were washed three times with 1 × PBS. The cells were then resuspended and counted.

[0076] (2) Single cell isolation: Inject the chip with the medium hexadecane, load the chip into the core machine, and set the corresponding parameters. Inject the cell suspension, enter the tearing path, and tear into single cell droplets. After tearing into single cell droplets, confirm the single cell in the single droplet and calculate the single cell yield.

[0077] (3) Cell Lysis: The lysis solution is introduced into the lysis pathway and injected with the cell lysis solution described herein. The lysis solution is then run to split the lysis solution in half into single droplets, which are then merged with the single cell droplets to obtain single cell lysis droplets. The temperature is controlled at 56°C and the lysis is performed for 10 minutes. This step completes the cell lysis, alkylation, and reduction reactions simultaneously.

[0078] (4) Protease cleavage: introduce the enzymatic solution path, inject the enzymatic solution of the present invention, run, tear the enzymatic solution into half into single droplets, merge with the single cell lysis droplets, control the temperature at 37°C, and maintain for 2 hours.

[0079] (5) TMT labeling: inject TMT reagent, mix with peptide solution, and incubate at room temperature for 1 h;

[0080] (6) Stop labeling: inject the stop reagent, mix with the reaction solution, and incubate at room temperature for 15 minutes;

[0081] (7) Sample output: Move the sample to the sample outlet, lower the temperature to 15°C, and solidify the hexadecane medium. Aspirate the sample solution into a liquid phase injection vial for mass spectrometry analysis.

[0082] After protein pre-treatment in the digital microfluidic chip, the treated single-cell fluid needs to enter the liquid chromatograph for chromatographic analysis. Since the injection volume of liquid chromatography usually needs to be 2-10uL, and the sample droplet volume after protein pre-treatment using a microfluidic chip is very small (eg. 200nL), the sample droplet after protein pre-treatment cannot be directly injected into the liquid chromatography. Even if the sample liquid is directly aspirated using the liquid chromatograph's injection needle, there may be a problem of not being able to aspirate due to the small sample volume. Even after the sample liquid is aspirated, the sample liquid still needs to be quantitatively prepared to 2-10uL. Currently, quantitative preparation is generally performed manually, which is prone to the situation where the preparation volume is too large or too small. When the preparation volume is less than the preset volume, the liquid chromatograph will have the risk of inhaling air, and when the quantitative preparation volume is larger than the preset volume, it may cause incomplete sampling, affecting the detection effect. Therefore, the processed single-cell droplet can be moved to the sample outlet, and the sample droplet can be manually aspirated with a capillary, and then made into 2uL (2-10uL) through a quantitative pipetting device and discharged into a liquid chromatography injection vial for liquid-mass chromatography detection.

[0083] In some embodiments, a quantitative pipetting device 100 is provided. As shown in FIG. 9 to FIG. 13 , the quantitative pipetting device 100 includes a body 1 , a capillary 2 , a liquid inlet tube 3 , a suction and discharge device 4 , and a selection valve 5 .

[0084] The main body 1 is provided with a fixing part 11, a liquid outlet pipe 12 and a supporting seat 13. The supporting seat 13 is used to place the sampling container 200. The capillary tube 2 can be detachably installed on the fixing part 11. When the capillary tube 2 is installed on the fixing part 11, the liquid outlet pipe 12, the capillary tube 2 and the sampling container 200 on the supporting seat 13 are connected in sequence.

[0085] The liquid inlet pipe 3, the liquid outlet pipe 12, and the aspiration and discharge device 4 are all connected to a selector valve 5, and the selector valve 5 can be switched to connect the liquid inlet pipe 3 to the aspiration and discharge device 4, or to connect the aspiration and discharge device 4 to the liquid outlet pipe 12. The liquid inlet pipe 3 can be connected to an external solution bottle for quantitative preparation with the sample liquid or a solution bottle for cleaning. Different solution bottles can be connected according to actual needs and are not particularly limited here. When the selection valve 5 is switched to connect the liquid inlet pipe 3 with the suction and discharge device 4, the solution in the solution bottle connected to the liquid inlet pipe 3 can be sucked in by the suction and discharge device 4. Then, the selection valve 5 is switched to connect the suction and discharge device 4 with the liquid outlet pipe 12. The suction and discharge device 4 can discharge the sucked quantitative solution into the capillary 2 through the liquid outlet pipe 12, driving the sample liquid in the capillary 2 to be synchronously flushed out to the sampling container 200, thereby quantitatively preparing the sample liquid in the sampling container 200 to a predetermined volume, wherein the sampling container 200 can be adapted to a liquid chromatograph so as to be able to perform liquid-mass detection on the sample liquid with a predetermined volume.

[0086] Specifically, when the quantitative pipetting device 100 is applied to a single-cell protein pretreatment system, a digital microfluidic chip obtains approximately 200 nL of sample liquid after protein pretreatment, and the sample liquid is moved to the sample outlet of the chip. The capillary 2 is used, and the capillary 2 can be manually grabbed or grabbed by an automatic grabbing device to absorb the sample liquid in the sample outlet. The capillary 2 with the sample liquid is then fixed to the fixing member 11. At this time, the liquid outlet pipe 12, the capillary 2 and the injection container 200 on the supporting seat 13 are connected in sequence. According to the configuration requirements of the sample liquid, if necessary, 200nL of sample liquid is added to formic acid solution to prepare 2uL, and the formic acid solution can be connected externally at the liquid inlet pipe 3. When the selection valve 5 is switched to the liquid inlet pipe 3 and connected to the suction and discharge device 4, the suction and discharge device 4 quantitatively absorbs 1.8uL of formic acid solution. Then, when the selection valve 5 is switched to the suction and discharge device 4 and connected to the liquid outlet pipe 12, the suction and discharge device 4 can discharge the quantitatively absorbed formic acid solution into the capillary 2 through the liquid outlet pipe 12, driving the sample liquid in the capillary 2 to be simultaneously flushed out into the sampling container 200, and obtaining a quantitative 2uL sample preparation liquid, so that the sample liquid can be quantitatively prepared to a predetermined volume for liquid-mass detection.

[0087] When the liquid aspiration is completed and cleaning is required, the capillary tube 2 is removed from the fixing member 11, the liquid outlet pipe 12 is connected to the cleaning liquid, and the liquid inlet pipe 3 is connected to the cleaning liquid. The selector valve 5 is switched to the suction and discharge device 4 to be connected to the liquid outlet pipe 12. The suction and discharge device 4 draws the cleaning liquid through the liquid outlet pipe 12 and the suction and discharge device 4 for cleaning, and then the cleaning liquid is discharged through the suction and discharge device 4 through the liquid outlet pipe 12. Similarly, the selector valve 5 is switched to the suction and discharge device 4 to be connected to the liquid inlet pipe 3. The suction and discharge device 4 draws the cleaning liquid through the liquid inlet pipe 3 and the suction and discharge device 4 for cleaning, and then the cleaning liquid is discharged through the suction and discharge device 4 through the liquid inlet pipe 3.

[0088] In summary, the quantitative pipetting device 100 is provided with a main body 1, a capillary 2, a liquid inlet tube 3, a suction and discharge device 4 and a selection valve 5, wherein the capillary 2 can be used to absorb the sample liquid obtained by protein pretreatment, and the capillary 2 can be detachably installed on the fixing part 11 of the main body 1. When the capillary 2 is installed on the fixing part 11, the liquid outlet tube 12, the capillary 2 and the sampling container 200 on the supporting seat 13 are connected in sequence. At the same time, the selection valve 5 is switched to the liquid inlet tube 3 to be connected with the suction and discharge device 4 or to the suction and discharge device 4 to be connected with the liquid outlet tube 12. The quantitative solution can be sucked into the capillary 2 after passing through the liquid inlet tube 3, the suction and discharge device 4 and the liquid outlet tube 12 in sequence, driving the sample liquid in the capillary 2 to be synchronously flushed out to the sampling container 200, thereby quantitatively preparing the sample liquid to a predetermined volume for liquid-quality detection. Therefore, when the quantitative pipetting device 100 is used in a single-cell protein pretreatment system, the sample liquid drawn by the capillary 2 can be dispensed into a quantitative volume in the device and then transferred to the injection container 200 of the liquid chromatograph. Compared with the traditional manual pipetting and sample liquid preparation operation, it has the characteristics of greater stability, higher accuracy, and higher sample output efficiency.

[0089] In some embodiments, as shown in Figures 10 and 11, a fixing member 11 is movably mounted on the body 1, and the quantitative pipetting device 100 further includes a first drive device 6. The output end of the first drive device 6 is connected to the fixing member 11 and drives the fixing member 11 to move relative to the body 1 between the liquid outlet 12 and the support base 13. It is understood that by providing the first drive device 6 to drive the fixing member 11 to move, when the capillary tube 2 is mounted on the fixing member 11, the first drive device 6 can drive the capillary tube 2 toward the liquid outlet 12 until it is tightly fitted with the liquid outlet 12, facilitating the entry of the solution from the liquid outlet 12 into the capillary tube 2. When it is necessary to remove the capillary tube 2 from the fixing member 11 of the body 1, the first drive device 6 drives the capillary tube 2 to move in the opposite direction, thereby removing the capillary tube 2 from the liquid outlet 12. At this point, the capillary tube 2 can be removed, allowing the capillary tube 2 to be flexibly positioned and operated simply and conveniently. The first driving device 6 may be a device such as a screw slide motor that can drive the fixing member 11 to move linearly, and no special limitation is imposed here.

[0090] Specifically in this embodiment, as shown in Figure 11, the first driving device 6 includes a first driving motor 61, a first screw rod 62 and a first slider 63; the first screw rod 62 is arranged on the main body 1 along the vertical direction of the main body 1, the first driving motor 61 is arranged on the main body 1, and the first driving motor 61 drives the first slider 63 to slide back and forth along the first screw rod 62, and the fixing member 11 is arranged on the first slider 63.

[0091] In some embodiments, as shown in Figures 10 and 11, the support base 13 is movably disposed on the body 1, and the quantitative pipetting device 100 further includes a second drive device 7, the output end of which is connected to the support base 13 and drives the support base 13 to move back and forth in a direction close to or away from the fixed member 11. It can be understood that since the capillary 2 can move back and forth under the drive of the first drive device 6 and its position can be flexibly changed, the second drive device 7 is provided to drive the support base 13 to move back and forth in a direction close to or away from the fixed member 11, thereby adapting to the position change of the capillary 2 and avoiding interference with the position of the capillary 2 during loading and unloading. Therefore, when the capillary 2 moves upward to a state of close fit with the liquid outlet pipe 12, the sample injection container 200 placed on the support base 13 can still be against the liquid outlet end of the capillary 2 to smoothly receive the solution discharged from the capillary 2. Among them, the second drive device 7 can be a device such as a screw slide motor that can drive the linear motion of the support base 13, and is not particularly limited here.

[0092] Specifically in this embodiment, as shown in Figure 11, the second driving device 7 includes a second driving motor 71, a second screw rod 72 and a second slider 73; the second screw rod 72 is arranged on the main body 1 along the vertical direction of the main body 1, the second driving motor 71 is arranged on the main body 1, and the second driving motor 71 drives the second slider 73 to slide back and forth along the second screw rod 72, and the supporting seat 13 is arranged on the second slider 73.

[0093] In some embodiments, as shown in Figure 11, the liquid outlet pipe 12 and the supporting seat 13 are arranged relative to each other in the height direction of the main body 1, and the liquid outlet pipe 12 is located above the supporting seat 13, so that when the capillary 2 is installed on the fixing member 11, the solution can be discharged from the liquid outlet pipe 12 located at a high position through the capillary 2 under the action of gravity to the sampling container 200 on the supporting seat 13, thereby improving the smoothness of the solution discharge.

[0094] In some embodiments, as shown in FIG10 , the fixing member 11 includes a connector 111 and two clamping members 112 provided on the connector 111; the connector 111 is connected to the output end of the first drive device 6, and the clamping member 112 is an elastic structure, and the two clamping members 112 cooperate to form a non-closed elastic clamping structure. The capillary 2 is elastically clamped by the two clamping members 112, so that the capillary 2 can be firmly fixed and has the characteristics of convenient loading and unloading. In addition, it should be noted that, as shown in FIG12 and FIG13 , the capillary 2 includes a tube body 21 and a connecting tube 22 connected to the tube body 21, the outer wall of the connecting tube 22 can be clamped by the clamping member 112, and the tube mouth of the connecting tube 22 can form a tight fit with the liquid outlet pipe 12.

[0095] In some embodiments, as shown in FIG10 , the support base 13 includes a support plate 131 and a side plate 132 vertically connected to the support plate 131; the side plate 132 is connected to the output end of the second driving device 7. The support plate 131 is provided with a placement groove 133, the groove wall of which is inclined toward one side of the side plate 132. After the sample injection container 200 is placed in the placement groove 133, the inclined groove wall can tilt the sample injection container 200 toward the side of the side plate 132, so that the inner side wall of the sample injection container 200 abuts against the liquid outlet end of the capillary tube 2, thereby facilitating the transfer of solution from the capillary tube 2 to the sample injection container 200 and effectively preventing the solution from splashing.

[0096] In some embodiments, as shown in Figures 10 and 11, the aspiration and dispensing device 4 includes a third drive device 41 and a syringe 42. The syringe 42 has an injection body and a push rod 421 disposed within the injection body. The output end of the third drive device 41 is connected to the push rod 421 and drives the push rod 421 to move back and forth within the injection body to perform aspiration or dispensing operations. The output end of the third drive device 41, such as a screw slide motor, drives the push rod 421 of the syringe 42 to move to automatically perform aspiration or dispensing operations, thereby enabling automatic quantitative solution preparation operations for the sample liquid drawn by the capillary 2.

[0097] Specifically, in this embodiment, as shown in FIG11 , the third drive device 41 includes a third drive motor 411, a third screw rod 412, and a third slider 413. The third screw rod 412 is disposed on the body 1 along the vertical direction of the body 1. The third drive motor 411 is disposed on the body 1, and the third drive motor 411 drives the third slider 413 to slide back and forth along the third screw rod 412. The push rod 421 is disposed on the third slider 413. Furthermore, the end of the third slider 413 is L-shaped, and the end of the push rod 421 is connected to the L-shaped structure.

[0098] In some embodiments, as shown in Figures 9 and 10, the body 1 is further provided with an observation window 14. A placement hole 141 is formed through the body 1 corresponding to the observation window 14. The capillary tube 2 is detachably inserted into the placement hole 141. Light emitters 142 are provided on opposite sides of the observation window 14 corresponding to the placement hole 141. It will be appreciated that by providing the observation window 14 and the light emitters 142 on the observation window 14, a bright area can be formed on the observation window 14 at a position corresponding to the placement hole 141. Inserting the capillary tube 2 into the placement hole 141 places the capillary tube 2 in the bright area, facilitating observation of the liquid level within the capillary tube 2.

[0099] In some embodiments, as shown in FIG10 , the wall of the placement hole 141 is further provided with a sensor 143 for sensing the insertion of the capillary tube 2 into the placement hole 141. When the sensor 143 senses that the capillary tube 2 is inserted into the placement hole 141, the luminous element 142 emits light to form a bright area to illuminate the capillary tube 2, making it easier to observe the liquid level in the capillary tube 2.

[0100] In some embodiments, as shown in Figure 10, the liquid inlet pipe 3, the suction and discharge device 4 and the selection valve 5 are all arranged at the appropriate position of the main body 1. Specifically in this embodiment, the liquid inlet pipe 3 and the selection valve 5 are all arranged above the suction and discharge device 4, and the liquid inlet pipe 3, the suction and discharge device 4 and the liquid outlet pipe 12 are located on the same plane of the main body 1.

[0101] In some embodiments, as shown in FIG. 9 , the quantitative pipetting device 100 further includes a protective shell 8 . The protective shell 8 covers the body 1 , and the fixing member 11 and the supporting seat 13 are exposed outside the protective shell 8 .

[0102] In some embodiments, as shown in FIG9 , a button 81 is further provided on the protective shell 8 , and the button 81 is used to control the opening and closing state of the entire quantitative pipetting device 100 .

[0103] The present invention further provides a quantitative liquid transfer assembly, as shown in FIG9 to FIG11 , which includes a body 1 , a liquid inlet tube 3 , a suction and discharge device 4 and a selection valve 5 .

[0104] The main body 1 is provided with a fixing part 11 , a liquid outlet pipe 12 and a supporting seat 13 . The liquid outlet pipe 12 and the fixing part 11 are arranged correspondingly, and the fixing part 11 and the supporting seat 13 are arranged correspondingly.

[0105] The liquid inlet pipe 3 , the liquid outlet pipe 12 and the suction and discharge device 4 are all connected to the selection valve 5 , and the selection valve 5 can be switched to connect the liquid inlet pipe 3 with the suction and discharge device 4 , or to connect the suction and discharge device 4 with the liquid outlet pipe 12 .

[0106] The present invention also provides a single-cell protein pretreatment system, which includes a microfluidic chip and the above-mentioned quantitative pipetting device 100. The microfluidic chip has a sample outlet hole, and the capillary 2 can be inserted into the sample outlet hole to absorb the sample liquid. The capillary 2 after sampling is installed on the fixing part 11 to quantitatively prepare the sample liquid and can conveniently transfer the quantitatively prepared sample liquid to a liquid chromatograph for detection.

[0107] In summary, the single-cell protein pretreatment system is equipped with the quantitative pipetting device 100, which can well absorb the sample liquid obtained by protein pretreatment of the microfluidic chip, and prepare the absorbed sample liquid into a quantitative volume in the device, and can be conveniently transferred to the injection container 200 of the liquid chromatograph. Compared with the traditional operation of completely manual pipetting and preparation of sample liquid, it has the characteristics of greater stability, higher accuracy and higher sample output efficiency.

[0108] In the following embodiments, the digital microfluidic chip used is: a digital microfluidic A3B4 chip, on which a certain voltage is applied to perform droplet control and tearing.

[0109] In the following embodiments, the tearing method is tearing the droplet volume in half: by controlling the power on and power off of the electrodes on the chip, the droplets are manipulated; the droplets will move toward the adjacent powered electrode and away from the powered-off electrode; in this way, the equal-volume droplets are controlled to move in the opposite direction, and the equal-volume tearing is performed in half, and each group obtains two droplets after being torn in half; according to the above method, the droplets after being torn in half are torn in half again for a second round to obtain four droplets, and then these four droplets are torn in half for a third round to obtain eight droplets. See Figure 8, in which the ordinary framed square represents the unpowered electrode, the bold black rounded frame represents the droplet, and the oblique shaded square represents the powered electrode. By controlling the power on and power off of the electrode, the droplet control and tearing in half are achieved to meet the experimental needs.

[0110] In the following examples, the experimental reagents used are as follows:

[0111] Take the configuration of one of the lysis buffers as an example:

[0112] First prepare 10× stock solution:

[0113] 1% DDM: Weigh 100 mg and dissolve in 1 mL 100 mM TEAB and mix thoroughly.

[0114] 100mM TCEP: Weigh 28.6mg and dissolve in 1mL 100mM TEAB and mix thoroughly.

[0115] 400mM CAA: Weigh 37.4mg and dissolve in 1mL 100mM TEAB and mix thoroughly.

[0116] 1% RapiGest SF: Dissolve 1 mg of lyophilized powder in 100 μL of 100 mM TEAB and mix thoroughly.

[0117] 1mL lysis buffer 0.1% DDM + 40mM CAA + 10mM TCEP + 100mM TEAB + 0.15% RapiGest SF preparation:

[0118] 100uL 1% DDM+100uL 100mM TCEP+100uL 400mM CAA+150uL1% RapiGest SF+550uL

[0119] 100mM TEAB, prepared after thorough mixing.

[0120] The present invention is further described in detail below with reference to specific embodiments.

[0121] Example 1

[0122] Since the non-labeled sample processing steps are the same as the first half of the TMT labeled sample preparation, we will use half of the chip to display the TMT labeled sample as an example, as shown in Figure 6. The specific steps are as follows:

[0123] 1. Collect the normally cultured cells in a 15 mL centrifuge tube and centrifuge at 100 × g for 5 minutes to remove the culture medium; wash the cell pellet three times with 1 × PBS buffer and then count the cells to obtain a cell suspension of a certain concentration.

[0124] 2. Use a 200 μL pipette tilted 45° to inject hexadecane into the oil filling hole on the upper cover of the digital microfluidic chip to create an environment suitable for the movement of single-cell droplets, and load the chip into the core machine.

[0125] 3. Turn on the core machine and open the microfluidic system interface of the host computer. The internal structure of the system is shown in Figure 1. Set the corresponding parameters, such as the corresponding row and column voltages, row hold time, frame delay time, etc.; open the camera interface. The internal structure of the imaging system is shown in Figure 2, which includes high- and low-magnification microscopes and a light source. At the same time, calibrate the chip position under a high-magnification microscope.

[0126] 4. Select stage 1 in the microfluidic system, inject 512 nL of cell suspension and cell lysate (0.08%-0.2% DDM + 35-45 mM CAA + 5-15 mM TCEP + 85-100 mM TEAB + 0-0.25% ProteaseMAX + 0-0.25% RapiGest SF) into different injection holes, tear them into single droplets, and run. The system moves to the specified position according to the edited path and completes the tearing. The movement of the droplets and cell lysate can be observed under a low-power microscope.

[0127] 5. Execute Phase 2, screening for single-cell droplets and performing single-cell identification. The system automatically switches to a high-power microscope, identifies single-cell droplets, and automatically moves them along a pre-set path to the bottom of the successfully lysed solution. Non-single-cell droplets and excess lysate are automatically moved to the waste area.

[0128] 6. Open the temperature control interface. The internal structure of the temperature control system is shown in Figure 3. Set the temperature to 56°C for 15 minutes and execute the merge function. Simultaneously, the single droplet of lysate is merged with the single cell droplet to obtain a single-cell lysate mixed droplet. Cell lysis is performed, and cell membrane rupture can be observed under the microscope. Cell lysis, alkylation, and reduction reactions are completed simultaneously. See Figures 5 and 7A. As shown in Figure 5, the lysate is located in the center area of ​​the chip. After the cells and other reagents are broken into single droplets, they are moved to the lysate area for mixing to complete each reaction step.

[0129] 7. After the lysis reaction is complete, inject 512 nL of the digestion solution (0.05-0.15 μg / μL Trypsin / Lys-C Mix + 100 mM TEAB) through the injection port using a 10 μL pipette. The digestion solution is automatically fragmented into single droplets. Automatic identification is performed, selecting the successfully fragmented single droplet of digestion solution and moving it below the protein solution droplet formed after cell lysis of the single cell lysis solution droplet until all digestion solutions are run. See Figure 7B.

[0130] 8. Open the temperature control interface and reset the temperature to 37°C for 2 hours. Then, combine the corresponding protein droplets and the enzyme digestion solution simultaneously, mix them, and react to obtain the peptide solution. See Figure 7C.

[0131] 9. After the digestion reaction is complete, use the automated sample injection device to inject 16 TMT reagents into the sample injection wells, perform droplet injection and tearing, and the system will identify and move the successfully torn TMT droplets below the peptide solution until all TMT reagents are run. See Figure 7D.

[0132] 10. Combine the TMT reagent droplet and the single-cell peptide solution droplet and mix thoroughly. Incubate at room temperature for 1 hour. See Figure 7E.

[0133] 11. After the TMT reaction is completed, inject 0.45-0.55% hydroxylamine solution, perform droplet injection and tearing, and move the successfully torn hydroxylamine solution to the bottom of the above liquid after the system recognizes it, until all hydroxylamine reagents are run.

[0134] The TMT labeling termination reaction was performed at room temperature for 15 minutes, and the hydroxylamine solution was mixed with the above reaction solution. See Figures 7F and 7G.

[0135] After the reaction is terminated, the automated mixing output path is executed, and each sample set contains peptide solutions obtained from 16 TMT-labeled single cells (see Figure 7H).

[0136] Transfer the sample to the sample outlet, open the temperature control interface, set the temperature to 15°C, and allow the hexadecane medium to solidify. Using the automated sample dispenser, remove the sample to a liquid phase vial, dilute with 0.1% formic acid to the minimum injection volume, and analyze on the analyzer. See Figure 7I.

[0137] The above experimental process only takes half of the area as an example, and 256 single-cell reactions can be performed simultaneously.

[0138] Each example was tested in triplicate.

[0139] Experimental Group 1: The lysis buffer consisted of 0.1% DDM + 40mM CAA + 10mM TCEP + 100mM TEAB + 0.15% ProteaseMAX, and the enzymatic agent was 0.1ug / uL Trypsin / Lys-C Mix + 100mM TEAB. Capillary aspiration was performed using a quantitative pipette to prepare a fixed volume (e.g., 2uL) and dispense it into a liquid chromatography vial. Following the above method, cell suspensions of varying concentrations were loaded onto the slides, and the single-cell yield was calculated. Results are shown in Table 1.

[0140] Table 1.

[0141] The results showed that when the cell suspension concentration on the slide was 6×10^5 cells / mL, the single cell yield was higher. When the concentration was less than 4×10^5 or greater than 8×10^5 cells / mL, the single cell yield was lower. After multiple experimental analyses, the optimal range was 4-8×10^5 cells / mL.

[0142] Experimental Group 2: Different concentrations of lysing agents were tested (prepared by weighing each component according to concentration and dissolving it in TEAB). A cell suspension with a loading concentration of 6 × 10^5 cells / mL was used, and 0.05 μg / μL Trypsin / Lys-C Mix + 100 mM TEAB was used as the enzymatic agent. Samples were drawn through the capillary tube of the quantitative pipetting device described herein, and a fixed volume (e.g., 2 μL) was prepared in the device and discharged into a liquid chromatography injection vial. The sample was then transferred to a liquid chromatography-mass spectrometry instrument for protein and peptide analysis. The results are shown in Table 2.

[0143] Table 2.

[0144] Results showed no significant differences in sample pretreatment between the lysis reagents 0.1% DDM + 40mM CAA + 10mM TCEP + 0 / 0.15% RapiGest SF / 0.15% RapiGest SF. However, lowering the DDM concentration to 0.01%, increasing the CAA concentration to 200mM, or increasing the TCEP concentration to 80mM may result in incomplete cell lysis, thus affecting the number of protein and peptide identifications. While increasing the RapiGest SF concentration had no effect on the number of protein identifications, high concentrations are not recommended due to reagent cost and the impact on the mass spectrometer's ion source.

[0145] Experimental Group 3: Different concentrations of enzymatic hydrolysis agents were tested. A cell suspension with a loading concentration of 6 × 10^5 cells / mL was used, along with a lysis agent consisting of 0.1% DDM, 40mM CAA, 10mM TCEP, 100mM TEAB, and 0.15% RapiGest SF. Samples were drawn through the capillary tube of the quantitative pipetting device described herein, and a fixed volume (e.g., 2 μL) was prepared within the device and discharged into a liquid chromatography injection vial. The sample was then transferred to a liquid chromatography instrument for protein and peptide analysis. The results are shown in Table 3.

[0146] The experimental results showed that there was no significant difference in sample pretreatment between 0.05 / 0.1ug / uL Trypsin / Lys-C Mix; however, when the concentration of the enzymatic solution was reduced to 0.005ug / uL, the number of protein and peptide identifications may be affected due to insufficient enzymatic hydrolysis.

[0147] Experimental Group 4: During the pretreatment step, the cell suspension was plated at a concentration of 6 × 10^5 cells / mL. A lysis buffer of 0.1% DDM, 40mM CAA, 10mM TCEP, 100mM TEAB, and 0.15% RapiGest SF was used, and a 0.05ug / uL Trypsin / Lys-C Mix with 100mM TEAB was used as the enzymatic digestion agent. The effects of different sampling methods on the number of detected proteins and peptides were investigated. The results are shown in Table 4.

[0148] In the capillary aspiration experiment, the capillary in the quantitative pipetting device is used to take samples, a quantitative volume is prepared in the quantitative pipetting device, and the sample is discharged into a liquid chromatography injection vial for detection on the machine.

[0149] After the tip of the gun is dipped, the sample volume is manually diluted to the same multiple as in the capillary aspiration experiment, and then transferred to a liquid chromatography injection vial for detection on the machine.

[0150] The experimental results show that capillary sampling is better than pipette sampling.

[0151] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A cell lysis solution for a digital microfluidic system, characterized in that: It includes the following active ingredients: 0.08%-0.2% DDM, 35-45mM CAA, 5-15mM TCEP, 85-100mM TEAB, 0-0.25% ProteaseMAX, and 0-0.25% RapiGest SF.

2. The cell lysis solution according to claim 1, characterized in that It includes the following active ingredients: 0.08% to 0.15% DDM, 38 to 42 mM CAA, 8 to 12 mM TCEP, 90 to 100 mM TEAB, and 0.05 to 0.2% ProteaseMAX; or it includes the following active ingredients: 0.08%-0.15% DDM, 38-42 mM CAA, 8-12 mM TCEP, 90-100 mM TEAB and 0.05-0.2% RapiGest SF.

3. The cell lysis solution according to claim 1, characterized in that It includes the following active ingredients: 0.09%-0.12% DDM, 38-42mM CAA, 8-12mM TCEP, 90-100mM TEAB, 0.12-0.18% ProteaseMAX; or it includes the following active components: 0.09%-0.12% DDM, 38-42mM CAA, 8-12mM TCEP, 90-100mM TEAB, and 0.12-0.18% RapiGest SF.

4. A quantitative liquid transfer device, characterized in that: The quantitative liquid transfer device comprises a body, a capillary, a liquid inlet tube, a suction and discharge device and a selection valve; The main body is provided with a fixing part, a liquid outlet pipe and a supporting seat, the supporting seat is used to place a sample injection container, the capillary tube is detachably mounted on the fixing part, and when the capillary tube is mounted on the fixing part, the liquid outlet pipe, the capillary tube and the sample injection container on the supporting seat are connected in sequence; The liquid inlet pipe, the liquid outlet pipe and the suction and discharge device are all connected to the selection valve, and the selection valve can be switched to connect the liquid inlet pipe with the suction and discharge device, or to connect the suction and discharge device with the liquid outlet pipe.

5. The quantitative liquid transfer device according to claim 4, characterized in that: The fixing part is movably arranged on the body. The quantitative liquid transfer device further comprises a first driving device. The output end of the first driving device is connected to the fixing part and drives the fixing part to move between the liquid outlet pipe and the supporting seat relative to the body.

6. The quantitative liquid transfer device according to claim 5, characterized in that: The supporting seat is movably arranged on the main body. The quantitative liquid transfer device also includes a second driving device. The output end of the second driving device is connected to the supporting seat and drives the supporting seat to reciprocate in a direction close to or away from the fixing member.

7. The quantitative liquid transfer device according to claim 5, characterized in that: The liquid outlet pipe and the supporting seat are arranged opposite to each other in the height direction of the main body, and the liquid outlet pipe is located above the supporting seat.

8. The quantitative liquid transfer device according to claim 5, characterized in that: The fixing member includes a connecting body and two clamping members arranged on the connecting body; the connecting body is connected to the output end of the first driving device, the clamping member is an elastic structure, and the two clamping members cooperate to form a non-closed elastic clamping structure.

9. The quantitative liquid transfer device according to claim 6, characterized in that: The bearing seat includes a bearing plate and a side plate vertically connected to the bearing plate; the side plate is connected to the output end of the second driving device, and a placement groove is opened on the bearing plate, and the groove wall of the placement groove is inclined toward one side of the side plate.

10. The quantitative liquid transfer device according to claim 4, characterized in that: The suction and discharge device includes a third driving device and a syringe, wherein the syringe has an injection body and a push rod arranged in the injection body, and the output end of the third driving device is connected to the push rod and drives the push rod to reciprocate in the injection body to perform suction or discharge operations.

11. The quantitative liquid transfer device according to claim 4, characterized in that: The main body is also provided with an observation window, a through placement hole is opened in the main body corresponding to the observation window, the capillary is detachably plugged into the placement hole, and luminous bodies are provided on opposite sides of the observation window corresponding to the placement hole.

12. The quantitative liquid transfer device according to claim 11, characterized in that: The hole wall of the placement hole is also provided with a sensor for sensing the state of the capillary being inserted into the placement hole.

13. A quantitative liquid transfer assembly, characterized in that: The quantitative liquid transfer device comprises a body, a liquid inlet pipe, a suction and discharge device and a selection valve; The main body is provided with a fixing part, a liquid outlet pipe and a bearing seat, the liquid outlet pipe and the fixing part are arranged correspondingly, and the fixing part and the bearing seat are arranged correspondingly; The liquid inlet pipe, the liquid outlet pipe and the suction and discharge device are all connected to the selection valve, and the selection valve can be switched to connect the liquid inlet pipe with the suction and discharge device, or to connect the suction and discharge device with the liquid outlet pipe.

14. A single cell protein pretreatment method based on a digital microfluidics system, characterized in that: The following steps are involved: S1. Prepare cell suspension; S2. Inject the cell suspension into the digital microfluidic chip and tear it into single cell droplets; S3. injecting the lysate according to any one of claims 1 to 3 onto the digital microfluidic chip, tearing the lysate into single droplets of the lysate, merging the single droplets with the single cell droplets, performing cell lysis, and obtaining protein solution droplets; S4. Injecting the enzymatic hydrolysate onto the digital microfluidic chip, tearing the enzymatic hydrolysate into single droplets, merging the droplets with the protein solution, and reacting to obtain a peptide solution; S5. Mark or not mark as needed; S6. Obtain protein samples.

15. The single cell protein pretreatment method based on the digital microfluidic system according to claim 14, characterized in that: The cell concentration of the cell suspension injected in step S2 is 5×10 5 / mL~8×10 5 Pieces / mL.

16. The single cell protein pretreatment method based on the digital microfluidic system according to claim 15, characterized in that: The cell concentration of the cell suspension injected in step S2 is 5.5×10 5 / mL~6.5×10 5 Pieces / mL.

17. The single cell protein pretreatment method based on the digital microfluidic system according to claim 14, characterized in that: The enzymatic hydrolysis solution includes 0.05ug / uL-0.1ug / uL Trypsin / Lys-C Mix and 90mM-110mM TEAB.

18. The single cell protein pretreatment method based on a digital microfluidic system according to any one of claims 14 to 17, characterized in that: In step S6, a protein sample is obtained by capillary aspiration.

19. The single cell protein pretreatment method based on the digital microfluidic system according to claim 18, characterized in that: The capillary is the capillary in the quantitative pipetting device according to any one of claims 4 to 12.

20. The single cell protein pretreatment method based on the digital microfluidic system according to claim 19, characterized in that: The protein sample is a sample that is processed by the quantitative liquid transfer device according to any one of claims 4 to 12 and then fixed to a volume of 2 to 10 uL.

21. The single cell protein pretreatment method based on a digital microfluidic system according to any one of claims 14 to 17, characterized in that: The tearing method is to tear the droplet into half with equal volume.

22. The single cell protein pretreatment method based on a digital microfluidic system according to any one of claims 14 to 17, characterized in that: The temperature for cell lysis in step S3 is 52° C. to 60° C., preferably 54° C. to 58° C.; and / or the lysis time is 13 to 17 min.

23. The single cell protein pretreatment method based on a digital microfluidic system according to any one of claims 14 to 17, characterized in that: The reaction temperature in step S4 is 35° C. to 39° C., and the reaction time is 1.5 h to 2.5 h.

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