Microfluidic device and applications thereof

Abstract

The application provides a microfluidic device and application thereof, and the microfluidic device comprises a microfluidic chip and a reactor arranged in a microchannel of the microfluidic chip, wherein the reactor comprises a protein sample pretreatment filler and a solid-phase extraction membrane which are connected together; the microfluidic device integrates the operations of protein preconcentration, reduction, alkylation, enzymolysis, desalination of polypeptides, elution and high-pH value reverse phase fractionation by using air pressure as driving force, and can be used for proteomic qualitative and quantitative analysis of small amount of cell, tissue or body fluid (such as blood, urine) samples; the automatic proteomic sample pretreatment platform comprising the microfluidic device can realize automatic operation, and improves sample processing throughput and quantitative analysis accuracy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of proteomics, and relates to a microfluidic device and application thereof, in particular to a microfluidic device, a microfluidic device, an automatic proteomics sample pretreatment platform and application thereof. BACKGROUND

[0002] In proteomics research, proteins in a sample are enzymatically digested into polypeptides, which are then used for liquid chromatography-mass spectrometry analysis to obtain protein information. This separation and identification method is the most widely used proteomics research method at present.

[0003] Proteomics analysis requires complex sample pre-treatment, mainly including: cell lysis, protein component enrichment, protein disulfide bond opening and protection, protein enzymolysis and polypeptide component desalination. In the current proteomics research strategy, the above sample pre-treatment steps are all completed manually, which greatly reduces the sample processing throughput, increases the sample loss and reduces the reaction efficiency of the related reactions. Therefore, a highly integrated and automated protein sample pre-treatment technology is needed to minimize the sample loss caused by manual operation, improve the system sensitivity, sample processing throughput and quantitative analysis accuracy, and also greatly reduce the workload of the experimental personnel and avoid experimental errors caused by human factors. In order to develop an automated proteomics analysis platform, a new proteome reactor needs to be developed. The proteome reactor is a capillary filled with strong cation exchange resin, which realizes the processes of protein enrichment, reduction, alkylation and enzymolysis, and can identify 17 proteins from 300 cells (J. Proteome Res. 2006, 5, 2754-2759). The rare cell proteome reactor is based on a strong cation exchange resin capillary monolithic column, which realizes the processes of protein pre-enrichment, reduction, alkylation, enzymolysis and polypeptide fractionation, and identifies 409 and 2281 proteins from 5000 and 50000 cells, respectively (Mol. Cell. Proteomics 2011, 10, M110.000679). Both of the two proteome reactors are for protein processing in capillary columns, which integrate part of the protein sample pre-treatment process in the same device, effectively reducing sample loss. However, due to the limited filling material of the capillary column, it is only suitable for the processing of a small amount of protein sample, and the processing process does not include the desalination process of polypeptides. Anal. Chem. 2016, 88, 4864-4871 discloses an integrated proteomics technology, which realizes the whole process of protein pre-enrichment, reduction, alkylation, enzymolysis, polypeptide desalination, elution and high-pH reversed-phase fractionation by filling C18 membrane and SCX packing in a pipette tip, and can identify 1270 proteins from 2000 cells in 1.4h of mass spectrometry time and 7826 proteins from 100000 cells in 22h of mass spectrometry time. Although this proteome reactor has good effect, it needs to rely on a centrifuge for operation, which is not suitable for an automated proteomics sample pre-treatment platform driven by air pressure.

[0004] Therefore, it has become an urgent problem to develop a brand-new microfluidic device driven by air pressure for proteomics sample pre-treatment. SUMMARY

[0005] In view of the deficiencies of the prior art, the microfluidic device and application thereof provided by the present application can realize protein sample pretreatment, including the whole process of protein pre-enrichment, reduction, alkylation, enzymolysis, polypeptide desalting, elution and high-pH value reverse phase fractionation, and can be used for proteomic qualitative and quantitative analysis of small amount of cells, tissues or body fluid (such as blood and urine) samples; in addition, the microfluidic device of the present application can realize automatic operation, improve sample processing throughput and quantitative analysis accuracy.

[0006] To achieve the object of the present application, the following technical solutions are adopted in the present application:

[0007] One of the objects of the present application is to provide a microfluidic device, which comprises a microfluidic chip and a reactor arranged in a microchannel of the microfluidic chip, and the reactor comprises a protein sample pretreatment filler and a solid phase extraction membrane connected together.

[0008] In the present application, the material of the microfluidic chip is an organic solvent-resistant material, preferably polystyrene.

[0009] In the present application, the shape of the microfluidic chip is any one or a combination of at least two of trapezoidal tetrahedron, cuboid or cylinder, preferably trapezoidal tetrahedron.

[0010] In the present application, the number of the microchannels is 1-100, when the number of the microchannels is 2-100, the plurality of microchannels are each independent of each other, can include a reactor or not, and the specific selection of the reactor can be the same or different, and the person skilled in the art can adjust according to actual needs.

[0011] In the present application, the shape of the microchannel includes any one or a combination of at least two of a right circular truncated cone, a cylinder or a cuboid.

[0012] In the present application, the microchannel comprises a right circular truncated cone segment and a cylinder segment arranged in connection, the right circular truncated cone segment comprises a cross-section end and a bottom end, and the cross-section end of the truncated cone segment is connected with the cylinder segment.

[0013] In the present application, the reactor is arranged in the truncated cone segment of the microchannel.

[0014] In the present application, along the direction from the bottom end to the cross-section end, the right circular truncated cone segment is sequentially provided with a protein sample pretreatment filler and a solid phase extraction membrane.

[0015] In the present application, the protein sample pretreatment filler comprises a strong cation exchange resin filler and / or a strong anion exchange resin filler.

[0016] In the present application, the strong cation exchange resin filler is a sulfonic acid group strong cation exchange resin filler.

[0017] In the present application, the strong anion exchange resin filler is a quaternary amine group strong anion exchange resin filler.

[0018] In the present application, the solid phase extraction membrane is a C18 membrane.

[0019] The second object of the present application is to provide a microfluidic device comprising the microfluidic device of the first object.

[0020] In the present application, the microfluidic device comprises:

[0021] The holder comprises a housing and a receiving chamber inside the housing;

[0022] The microfluidic device is arranged in the receiving chamber of the holder;

[0023] The connecting pieces are embedded at both ends of the housing of the holder;

[0024] The capillary tube penetrates through the connecting pieces and is in communication with the microchannel of the microfluidic device.

[0025] The microfluidic device of the present application can realize automatic operation: for example, the microfluidic device can be used on an Agilent capillary electrophoresis instrument to automatically and high-throughput process samples.

[0026] In the present application, the housing is formed by butting a first housing and a second housing, i.e., the first housing and the second housing are detachably connected.

[0027] In the present application, the outer surface of the connecting piece is provided with a limiting piece for limiting the relative movement of the holder in the axial direction.

[0028] In the present application, the limiting piece is a boss, and a groove engaged with the boss is arranged on the contact surface of the holder at both ends of the connecting piece, and the relative movement of the holder in the axial direction is limited by the engagement of the boss and the groove.

[0029] The third object of the present application is to provide an application of the microfluidic device in the proteomic qualitative and quantitative analysis of the biological sample to be tested.

[0030] In the present application, the biological sample to be tested comprises any one or a combination of at least two of cells, tissues or body fluids, and the body fluids include blood or urine.

[0031] In the present application, the microfluidic device is used for the protein sample pretreatment of the biological sample to be tested and the high-pH value reverse phase fractionation of polypeptides.

[0032] In the present application, the protein sample is subjected to enzymolysis on a protein sample pretreatment filler, and after the enzymolysis is completed, the polypeptides generated are transferred to a solid-phase extraction membrane, and then high-pH value reverse fractionation is performed.

[0033] In the present application, the pH value of the high-pH value reverse fractionation is higher than 8.

[0034] In the present application, the application comprises the following steps:

[0035] (1) adding the biological sample to be tested into a microfluidic device along a capillary tube, and enriching the protein on the protein sample pretreatment filler to obtain a protein sample pretreatment filler enriched with the protein;

[0036] (2) washing away the surfactant and other impurities remaining in the microfluidic device using a solution containing an organic solvent or a pure organic solvent, and sequentially completing the reduction reaction, alkylation reaction and enzymolysis reaction of the protein under the action of a reducing agent, an alkylation reagent and an enzyme to obtain polypeptides;

[0037] (3) transferring the polypeptides obtained in step (2) from the protein sample pretreatment filler to a solid-phase extraction membrane through a salt solution;

[0038] (4) after desalination, eluting the polypeptides using a solution containing a high proportion of organic solvent or sequentially eluting the polypeptides using a high-pH value solution containing different proportions of organic solvent from low to high, and performing high-pH value reverse fractionation.

[0039] In the present application, the pH value of the solution used in the reverse fractionation is higher than 8.

[0040] In the present application, the biological sample to be tested is a cell or a tissue, and the step (1) further comprises pre-sequentially performing lysis and acidification on the cell or tissue sample.

[0041] In the present application, the lysis is performed under the action of a lysis solution.

[0042] In the present application, the lysis solution comprises a surfactant.

[0043] In the present application, the surfactant comprises n-dodecyl-β-D-maltoside and / or cholesteryl succinate monolaurate.

[0044] In the present application, the organic solvent in step (2) is acetonitrile and / or methanol.

[0045] In the present application, the aqueous solution containing an organic solvent in step (2) is a potassium citrate aqueous solution containing acetonitrile and / or a potassium citrate aqueous solution containing methanol.

[0046] In the present application, the volume of acetonitrile is 10-30% of the volume of the potassium citrate aqueous solution containing acetonitrile, for example, 10%, 12%, 15%, 18%, 20%, 22%, 25%, 27%, 30%, etc.

[0047] In the present application, the volume of methanol is 10-30% of the volume of the potassium citrate aqueous solution containing methanol, for example, 10%, 12%, 15%, 18%, 20%, 22%, 25%, 27%, 30%, etc.

[0048] In the present application, the concentration of potassium citrate in the potassium citrate aqueous solution containing acetonitrile or the potassium citrate aqueous solution containing methanol is 5-10 mmol / L, for example, 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, etc.

[0049] In the present application, the biological sample to be tested is a body fluid, and the step (1) further comprises diluting the body fluid in advance by adding a diluent.

[0050] In the present application, the diluent is N-(2-hydroxyethyl) piperazine-N-2 sulfonic acid aqueous solution.

[0051] In the present application, the pH of the diluent is 6-7.

[0052] In the present application, the concentration of the diluent is 10-30 mmol / L, for example, 10 mmol / L, 12 mmol / L, 15 mmol / L, 18 mmol / L, 20 mmol / L, 22 mmol / L, 25 mmol / L, 28 mmol / L, 30 mmol / L, etc.

[0053] In the present application, the reactor in the microfluidic device in step (1) is activated in advance before the protein sample is added.

[0054] In the present application, the enrichment of the protein to the protein sample pretreatment filler in step (1) is achieved by gas pressurization.

[0055] In the present application, the reducing agent in step (2) includes any one or a combination of at least two of dithiothreitol, phosphoric acid trichloroethyl ester, or β-mercaptoethanol.

[0056] In the present application, the alkylating agent in step (2) includes iodoacetic acid and / or iodoacetamide.

[0057] In the present application, the enzyme in step (2) is alkaline protease.

[0058] In the present application, the enzyme in step (2) includes trypsin, chymotrypsin or elastase.

[0059] In the present application, the salt solution in step (3) is ammonium formate and / or ammonium bicarbonate.

[0060] The fourth object of the present application is to provide an automated proteomic sample pretreatment platform, which comprises a closed system, the microfluidic device of the second object and a collection system, and the closed system and the collection system are connected with the capillary at both ends of the microfluidic device.

[0061] In the present application, the number of the microfluidic devices is 1-100, and the number of the microfluidic devices is 2-100, and the different microfluidic devices are arranged in parallel.

[0062] In the present application, the closed system comprises a closed container provided with a gas channel and a sample placing device in the closed container, and the sample placing device is used for placing the sample.

[0063] In the present application, the collection system comprises a collection device, which is used for collecting the sample after the reaction system.

[0064] The fifth object of the present application is to provide an application of the automated proteomic sample pretreatment platform of the fourth object in proteomic qualitative and quantitative analysis of the biological sample to be tested.

[0065] In the present application, the application comprises: introducing gas into the closed container provided with a gas channel to pressurize, so that the sample in the sample placing device passes through the capillary under the action of pressure, reaches the microfluidic device for reaction, and reaches the collection device through the capillary after the reaction, and the collection device collects the sample after the reaction.

[0066] Compared with the prior art, the present application has the following beneficial effects:

[0067] (1) The microfluidic device for proteomic sample pretreatment of the present application can complete the operations of pre-enrichment, reduction, alkylation, enzymolysis, desalination of polypeptides, elution and high-pH value reverse phase fractionation of proteins by gas pressure driving, so as to realize proteomic qualitative and quantitative analysis of a small amount of cell, tissue or body fluid (such as blood and urine) sample; in addition, the microfluidic device of the present application can realize automatic operation, improve sample processing throughput and quantitative analysis accuracy.

[0068] (2) The microfluidic device of the present application can realize batch production, and has low cost.

[0069] (3) The microfluidic device of the present invention applied to the pretreatment of proteomics samples can be connected in parallel to form a multi-channel reactor such as a two-channel, four-channel or eight-channel reactor, or multiple microchannels and reactors can be connected in parallel in a microfluidic chip to process multiple protein samples at the same time and improve the sample processing throughput. Attached Figure Description

[0070] Figure 1 This is a schematic diagram of the microfluidic device in an embodiment of the present invention;

[0071] Figure 2 This is a schematic diagram of the microfluidic device in an embodiment of the present invention;

[0072] Figure 3 This is a schematic diagram of the structure of the automated proteomics sample pretreatment platform in an embodiment of the present invention;

[0073] Among them, 100 is the protein sample pretreatment packing material, 101 is the solid phase extraction membrane, 102 is the microfluidic chip, 10 is the immobilizer, 11 is the microfluidic device, 12 is the connector, 13 is the capillary, 14 is the limiting component, 1 is the microfluidic device, 2 is the closed system, 3 is the collection system, 21 is the gas channel, 22 is the closed container, and 23 is the sample placement device.

[0074] Figure 4 The graph shows the flow rate of the microfluidic device in Example 1 as water passes through the automated protein reaction system under different pressures.

[0075] Figure 5 This is a flow rate diagram of the microfluidic device in Example 1 flowing through the automated reaction system of proteins in different solvents at a pressure of 3 bar.

[0076] Figure 6 This is a chromatogram of HEK 293T cell samples processed by the microfluidic device in Example 1;

[0077] Figure 7 The graph shows the enzymatic hydrolysis efficiency and alkylation efficiency of the microfluidic device in Example 1 after three consecutive processing of protein samples.

[0078] Figure 8 This is a statistical chart showing the number of proteins and peptides identified by the microfluidic device in Example 1 when plasma samples were processed in a non-grading and high pH grading manner.

[0079] Figure 9 The image shows the methanol and acetonitrile tolerance test results of the microfluidic chip material in Example 2. Detailed Implementation

[0080] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0081] This embodiment provides a microfluidic device, such as Figure 1 As shown, from Figure 1 As can be seen, the microfluidic device includes a microfluidic chip 102 and a reactor disposed in the microchannel of the microfluidic chip 102. The reactor includes a protein sample pretreatment packing material 100 and a solid-phase extraction membrane 101 connected together. The microchannel includes a frustum section and a cylindrical section connected from left to right. The frustum section includes a cross-sectional end and a bottom end. The cross-sectional end of the frustum section is connected to the cylindrical section. The protein sample pretreatment packing material and the solid-phase extraction membrane are located in the frustum section of the microchannel.

[0082] This embodiment provides a microfluidic device, such as Figure 2 As shown,

[0083] The device includes a retainer 10 (including a housing and a accommodating chamber), a microfluidic device 11 disposed in the accommodating chamber inside the retainer (wherein the microfluidic device 11 is the microfluidic device provided above), connectors 12 embedded at both ends of the retainer, and a capillary 13 penetrating the connector 12. The capillary 13 is connected to the microchannel of the microfluidic device 11. The housing is formed by the mating of a first housing and a second housing. A limiting member 14 is provided on the outer surface of the connector 12 to limit the relative movement of the retainer 10 in the axial direction. The limiting member is a boss. Grooves that engage with the boss are opened on the contact surfaces of the retainer 10 and the connector at both ends. The relative movement of the retainer 10 in the axial direction is limited by the engagement of the boss and the groove.

[0084] This embodiment also provides an automated proteomics sample pretreatment platform, such as... Figure 3 As shown, it includes a closed system 2, a microfluidic device 1 (wherein the microfluidic device is the microfluidic device provided above) and a collection system 3. Both the closed system 2 and the collection system 3 are connected to capillaries at both ends of the microfluidic device 1. The closed system 2 includes a closed container 22 with a gas channel 21 and a sample placement device 23 located in the closed container 22.

[0085] Example 1

[0086] The parameters of the microfluidic device in this embodiment are set as follows: the microfluidic chip is a trapezoidal quadrilateral with a height of 8.57 mm, a width of 7.00 mm, an upper base length of 5.35 mm, and a lower base length of 6.32 mm; the bottom diameter of the right circular truncated cone segment is 1.46 mm, the cross-sectional diameter is 0.8 mm, and the height is 7.27 mm; the bottom diameter of the cylindrical segment is 1.46 mm, and the height is 1.3 mm; the protein sample pretreatment filler in this embodiment is a combination of sulfonic acid-based strong cation exchange resin filler and quaternary amine-based strong anion exchange resin filler, and the amount used is 1 mg; the solid-phase extraction membrane is a C18 membrane with 5 layers; the inner diameter of the capillary is 200 μm, the outer diameter is 360 μm, and the length is 15 cm.

[0087] The microfluidic device is used to test the volume of water flowing through the protein reactor under different pressures (2 bar, 4 bar, 6 bar, 8 bar, and 10 bar) at the same time (2 min), Figure 4 The pressure and flow rate curve of water passing through the protein reactor in this embodiment are shown in FIG. 6. Figure 4 It can be seen that the pressure and flow rate of the microfluidic device of the present application have a good linear relationship.

[0088] The microfluidic device is used to test the flow rate of different solvents in the protein group reactor under a pressure of 3 bar. Figure 5 It can be seen that the flow rate in methanol, acetonitrile, and solutions with high acetonitrile content is relatively fast, and the flow rate of water is slow.

[0089] It can be seen from FIG. 8 that the flow rate of the solvent in the microfluidic device can be controlled by pressure in this embodiment. Figure 4 Figure 5 It can be seen that the microfluidic device can process protein samples (HEK 293T cell samples) without affecting the enzymatic efficiency in this embodiment.

[0090] The enzymatic efficiency of the microfluidic device in processing HEK 293T cell samples is tested, Figure 6 The chromatographic peak graph of the microfluidic device processing HEK 293T cell samples is shown in FIG. 10. Figure 6 It can be seen that the chromatographic peak width is narrow and the distribution is uniform, indicating that the enzymatic efficiency of the sample is high.

[0091] The enzymatic efficiency and alkylation efficiency of the microfluidic device in processing HEK 293T cell samples are tested three times in succession, Figure 7 It can be seen that the alkylation efficiency of the three repeated experiments is close to 1, and the enzymatic efficiency is more than 97%.

[0092] It can be seen from FIG. 12 that the microfluidic device in this embodiment does not affect the enzymatic efficiency in processing protein samples, and has good sample processing effect. Figure 6 Figure 7 It can be seen that the microfluidic device in this embodiment does not affect the enzymatic efficiency in processing protein samples, and has good sample processing effect. ​​

[0093] The microfluidic device of this embodiment and the existing SISPOT proteomics reactor were used to process the same 10 μg plasma sample, and then the results were analyzed by liquid chromatography-mass spectrometry (LC-MS). The results are shown in Table 1.

[0094] Table 1

[0095]

[0096]

[0097] As shown in Table 1, when processing the same 10 μg plasma sample (excluding high pH reverse phase fractionation of peptides), the microfluidic device of this embodiment can identify 2550 peptides and 215 proteins, which is comparable to the SISPOT proteomics reactor, indicating that the microfluidic device of this embodiment has good sensitivity.

[0098] The microfluidic device of this embodiment was used to process trace amounts of bovine serum albumin, and the results were obtained by liquid chromatography-mass spectrometry (LC-MS) analysis:

[0099] Table 2

[0100] BSA amount Coverage (%) Amount of peptides PSMs 2 ng 7.58 5 5 20 ng 8.50 6 6 200 ng 29.41 17 20 2 μg 80.81 85 170

[0101] As shown in Table 2, the microfluidic device of this embodiment can still identify 5 peptides and achieve a coverage of 7.58% when the protein amount is as low as 2 ng, and can achieve a coverage of 80.81% when the protein amount reaches 2 μg. The results show that the microfluidic device of this embodiment has a good effect on processing trace protein samples.

[0102] The microfluidic device in this embodiment also integrates high-pH reverse-phase fractionation of peptides, thereby increasing the identification capacity of peptides and proteins, such as... Figure 3 As shown:

[0103] Table 3

[0104]

[0105]

[0106] As shown in Table 3, 20 μg of plasma sample was treated using the proteomics reactor of this embodiment. The peptides were eluted sequentially using 5 mmol / L ammonium formate solutions containing 3%, 6%, 9%, 15%, and 80% acetonitrile at pH 10. Five fractions were performed, and a total of 4154 peptides and 317 proteins were identified.

[0107] like Figure 8As shown, the number of peptides and the number of proteins identified by the microfluidic device in the present embodiment at high pH value reversed phase fractionation are 1.7 times and 1.5 times of those without fractionation, respectively.

[0108] Example 2

[0109] Considering that methanol and acetonitrile, two common organic solvents, are used in the pretreatment process of protein samples, the material of the microfluidic chip inside the microfluidic device applied to the pretreatment of proteomic samples needs to be able to withstand general organic solvents and have certain mechanical strength and the like. Four materials were selected as test objects according to the needs, namely polystyrene (abbreviated as PS), polypropylene (abbreviated as PP), polycarbonate (abbreviated as PC), and polymethyl methacrylate (PMMA).

[0110] Methanol and acetonitrile resistance tests were performed on the particles of the above four materials, and the test results are shown in Figure 9 As shown, the four materials were sequentially soaked in methanol and acetonitrile, respectively, and no obvious change was observed after short-time soaking. After more than 30 min, the four materials were still not obviously changed after methanol soaking, and the PC material became white and the transparency decreased after being soaked in acetonitrile. The PMMA material also became white, the transparency decreased, and it became soft. The PP and PS materials were not obviously changed. However, because the transparency of the PS material is better than that of the PP material, the PS material was finally selected as the material of the microfluidic chip.

[0111] Example 3

[0112] The present embodiment provides an automated proteomic sample pretreatment platform, and the microfluidic device in the automated proteomic sample pretreatment platform is the same as that in Example 1.

[0113] The present embodiment also provides an application of the automated proteomic sample pretreatment platform in qualitative and quantitative analysis of protein samples, which includes the following steps:

[0114] (1) The protein concentration of the plasma sample with a concentration of about 60 μg / μL was diluted to 1 μg / μL by using 20 mmol / L N-(2-hydroxyethyl) piperazine-N-2 sulfonic acid aqueous solution (HEPES) with a pH of 7.4;

[0115] (2) The various required reagents are moved into the reagent tubes and placed in a sealed container, the two-way valve is opened, nitrogen is introduced, and the sample or reagent in the reagent tube is made to flow through the microfluidic device by nitrogen pressure, the microfluidic device is first activated by 80 μL of methanol and 40 μL of 10 mmol / L HEPES (pH 7.4) aqueous solution, respectively; after activation, a protein sample with a concentration of 1 μg / μL is placed in the reagent tube, and the protein sample is made to flow through the microfluidic device by nitrogen pressure, so that the protein sample is enriched on the SCX / SAX mixed packing;

[0116] (3) The microfluidic device is washed with 10 mmol / L HEPES aqueous solution containing 20% acetonitrile and pure acetonitrile, 50 mmol / L dithiothreitol (DTT) in 20 mmol / L HEPES (pH 8.0) aqueous solution is added, and the reduction of the protein is completed at room temperature for 30 minutes. Then, 20 μL of 20 mmol / L HEPES (pH 8.0) is added to wash away the DTT, and 10 mmol / L iodoacetamide solution containing trypsin is added, and the alkylation and enzymolysis of the protein are completed at room temperature and in the dark for 60 minutes;

[0117] (4) The generated polypeptide is transferred from the SCX / SAX mixed packing to the C18 membrane using 60 μL of 500 mmol / L ammonium formate aqueous solution and 60 μL of 500 mmol / L sodium chloride aqueous solution;

[0118] (5) 60 μL of 1% formic acid aqueous solution is added for desalination, and then 0.5% acetic acid aqueous solution containing 80% acetonitrile is used to elute the polypeptide from the C18 membrane, and the eluted polypeptide is collected in a collection tube and then placed in a freeze dryer for freeze-drying and then re-dissolved in 0.1% formic acid aqueous solution, which can be detected by liquid chromatography-mass spectrometry.

[0119] The number of polypeptides and proteins in 10 μg of plasma sample detected by liquid chromatography-mass spectrometry is shown in Table 4:

[0120] Table 4

[0121] Number of experiments Amount of peptides identified Amount of proteins identified Experiment 1 2442 214 Experiment 2 2657 215

[0122] As can be seen from Table 4, more than 2400 polypeptides and 210 proteins can be identified in both repeated experiments, indicating that the protein sample pretreatment by the microfluidic device for proteomics sample pretreatment in this embodiment has good stability.

[0123] Example 4

[0124] This embodiment provides an automated proteomics sample pretreatment platform, and the microfluidic device in the automated proteomics sample pretreatment platform uses the same microfluidic device as in Example 1.

[0125] The present embodiment also provides the application of the automated proteomic sample pretreatment platform in the qualitative and quantitative analysis of protein samples, including the following steps:

[0126] (1) The HEK 293T cell sample is lysed using a compatible lysis solution (the lysis solution contains 10 mmol / L HEPES, pH 7.4, 600 mmol / L guanidine hydrochloride, 1% DDM, 1 mmol / L Na3VO4 and protease inhibitors), and after the lysis is completed, 0.1% formic acid is added to acidify the sample solution to a pH of 2;

[0127] (2) The various required reagents are moved into the reagent tubes and placed in a sealed container, a two-way valve is opened, nitrogen gas is introduced, and the sample or reagent in the reagent tube is made to flow through the microfluidic device by nitrogen gas pressure. The microfluidic device is first activated by 80 μL of methanol, 20 μL of 100 mmol / L potassium citrate aqueous solution and 20 μL of 10 mmol / L potassium citrate aqueous solution, respectively; after activation, the protein sample is placed in the reagent tube, and the sample is made to slowly flow through the microfluidic device by nitrogen gas pressure, so that the protein is enriched on the SCX / SAX mixed packing;

[0128] (3) 8 mmol / L potassium citrate aqueous solution containing 20% acetonitrile is used to wash away the surfactant DDM bound to the C18 membrane; then, 10 mmol / L tris(2-carboxyethyl) phosphine hydrochloride (TCEP) solution is added, and the reduction of the protein is completed at room temperature for 15 minutes; then, 20 μL of 20 mmol / L HEPES, pH 8.0 is added to wash away the TCEP, and then 10 mmol / L iodoacetamide solution containing trypsin is added, and the alkylation and enzymolysis of the protein are completed at room temperature and in a dark environment for 60 minutes;

[0129] (4) 60 μL of 500 mmol / L ammonium formate aqueous solution and 60 μL of 500 mmol / L sodium chloride aqueous solution are added to transfer the generated polypeptides from the SCX / SAX mixed packing to the C18 membrane;

[0130] (5) 60 μL of 1% formic acid aqueous solution is added for desalination. Finally, 0.5% acetic acid aqueous solution containing 80% acetonitrile is used to elute the polypeptides from the C18 membrane 3; the eluted polypeptides are collected in a collection tube, and then placed in a freeze dryer for freeze-drying and then re-dissolved in 0.1% formic acid aqueous solution, which can be detected by liquid chromatography-mass spectrometry.

[0131] The number of polypeptides and proteins detected by liquid chromatography-mass spectrometry is shown in Table 5:

[0132] Table 5

[0133] Number of experiments Amount of peptides identified Amount of proteins identified Experiment 1 31244 4553 Experiment 2 31837 4426 Experiment 3 31855 4489

[0134] From Table 5, more than 30000 polypeptides and 4500 proteins can be identified in 3 repeated experiments, indicating that the automatic proteomic sample pretreatment platform of the embodiment has good reproducibility in processing protein samples.

[0135] Embodiment 5

[0136] The embodiment provides an automatic proteomic sample pretreatment platform, and the microfluidic device in the automatic proteomic sample pretreatment platform is selected from the same microfluidic device as in Embodiment 1.

[0137] The embodiment also provides an application of the automatic proteomic sample pretreatment platform in qualitative and quantitative analysis of protein samples, which comprises the following steps:

[0138] (1) The HEK 293T cell sample is lysed by using a compatible lysis solution (the lysis solution comprises 10 mmol / L HEPES, pH 7.4, 600 mmol / L guanidine hydrochloride, 1% DDM, 1 mmol / L Na3VO4 and protease inhibitors), and after the lysis is completed, 0.1% formic acid is added to acidify the sample solution to pH 2;

[0139] (2) Various required reagents are moved into the reagent tube and placed in a sealed container, a two-way valve is opened, nitrogen is introduced, and the sample or reagent is made to flow through the microfluidic device by nitrogen pressure. The microfluidic device is first activated by 80 μL of methanol, 20 μL of 100 mmol / L potassium citrate aqueous solution and 20 μL of 10 mmol / L potassium citrate aqueous solution, respectively; after the activation, the protein sample is placed in the reagent tube, and the sample is made to slowly flow through the microfluidic device by nitrogen pressure, so that the protein is enriched on the mixed packing of strong cation exchange resin / strong anion exchange resin;

[0140] (3) 8 mmol / L potassium citrate aqueous solution containing 20% acetonitrile is used to wash away the surfactant DDM combined to the surface of C 18 (4) 60 μL of 500 mmol / L ammonium formate aqueous solution and 60 μL of 500 mmol / L sodium chloride aqueous solution are added to transfer the generated polypeptides from the SCX / SAX mixed packing to the C18 membrane;

[0141] (4) 60 μL of 500 mmol / L ammonium formate aqueous solution and 60 μL of 500 mmol / L sodium chloride aqueous solution are added to transfer the generated polypeptides from the SCX / SAX mixed packing to the C18 membrane;

[0142] (5) Add 60 μL of 5 mmol / L ammonium formate aqueous solution for desalination. Finally, use 5 mmol / L ammonium formate solution containing 3%, 6%, 9%, 15%, 80% acetonitrile respectively with pH value of 10 to elute the polypeptides in turn, that is, high pH value reverse phase fractionation; the eluted polypeptides are collected in a collection tube, and then placed in a freeze dryer to be freeze-dried and then re-dissolved in 0.1% formic acid aqueous solution, which can be detected by liquid chromatography-mass spectrometry.

[0143] A 20 μg HEK 293T cell protein sample was processed using the process described in this example and subjected to high pH value reverse phase fractionation, and the number of polypeptides and proteins identified is shown in Table 6:

[0144] Table 6

[0145] Fractionation Amount of peptides identified Amount of proteins identified 3% ACN 15501 4869 6% ACN 24283 5694 9% ACN 23068 5511 15% ACN 30923 5860 80% ACN 33707 5797 Combined results 92412 8010

[0146] As can be seen from Table 6, the number of polypeptides and proteins identified in each fractionation is relatively average, and a total of 92412 polypeptides and 8010 proteins are identified in the final combined results, indicating that the protein automatic reaction system of this example has good sensitivity in processing protein samples.

[0147] Comparative Example 1

[0148] The scheme of this comparative example is CN106770814A. By comparing the schemes of this comparative example and Example 1, in Comparative Example 1, the sample is made to enter the proteome reactor under the action of centrifugal force by centrifugation, but this method cannot timely control the start and end of the reaction, and the action of centrifugal force is to throw the sample to the inner wall surface of the pipette tip, which is not easy to enter the exchange resin packing, and is not conducive to the continuous reaction; in Example 1, the microfluidic device can ensure the continuous reaction under the action of air pressure, and in addition, by controlling the air pressure, the occurrence and interruption of the reaction can be adjusted at any time. In addition, Comparative Example 1 only uses strong cation exchange resin packing, which cannot effectively bind proteins with isoelectric point lower than 2, resulting in sample loss; and Example 1 uses strong cation / anion exchange resin mixed packing, which has complementary effect and can effectively bind proteins, almost without causing sample loss.

[0149] The applicant declares that the above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto. It should be understood by those skilled in the art that any changes or replacements within the technical scope disclosed by the present application can be easily thought of by those skilled in the art, and all fall within the protection scope and disclosure scope of the present application.

Claims

1. A microfluidic device, characterized in that, The microfluidic device comprises: a holder comprising a housing and a receiving chamber inside the housing; a microfluidic device arranged in the receiving chamber of the holder; the microfluidic device is driven by air pressure to perform protein sample pretreatment; a connector embedded at both ends of the housing of the holder; a capillary tube penetrating through the connector, the capillary tube being in communication with the microchannels of the microfluidic device; the microfluidic device comprises a microfluidic chip and a reactor arranged in the microchannels of the microfluidic chip, the reactor comprising a protein sample pretreatment filler and a solid phase extraction membrane connected together; the number of the microchannels is 1-100, when the number of the microchannels is 2-100, the plurality of microchannels are independent of each other; the material of the microfluidic chip is polystyrene; the microchannels comprise a circular truncated cone segment and a cylindrical segment arranged in connection, the circular truncated cone segment comprises a cross-section end and a bottom end, the cross-section end of the circular truncated cone segment being connected with the cylindrical segment; the reactor is arranged in the circular truncated cone segment of the microchannels; from the bottom end to the cross-section end, the circular truncated cone segment is sequentially provided with the protein sample pretreatment filler and the solid phase extraction membrane; the protein sample pretreatment filler is a combination of sulfonic acid-based strong cation exchange resin filler and quaternary amine-based strong anion exchange resin filler; the solid phase extraction membrane is a C18 membrane.

2. The microfluidic device of claim 1, wherein, the shape of the microfluidic chip is any one or a combination of at least two of trapezoidal quadrilateral, cuboid or cylinder.

3. The microfluidic device of claim 2, wherein, the shape of the microfluidic chip is trapezoidal quadrilateral.

4. The microfluidic device of claim 1, wherein, the housing is formed by butt joint of a first housing and a second housing.

5. The microfluidic device of claim 1, wherein, the outer surface of the connector is provided with a limiting piece, the limiting piece being used to limit the relative movement of the holder in the axial direction.

6. The microfluidic device of claim 5, wherein, the limiting piece is a boss, and grooves engaged with the boss are arranged on the contact surfaces of the two ends of the holder and the connector, and the relative movement of the holder in the axial direction is limited by the engagement of the boss and the groove.

7. The microfluidic device according to any one of claims 1-6 is applied to the proteomic qualitative and quantitative analysis of a biological sample to be tested.

8. Use according to claim 7, characterized in that, The biological sample to be tested comprises any one or a combination of at least two of cells, tissues or body fluids, and the body fluids include blood or urine.

9. Use according to claim 7, characterized in that, The microfluidic device is used for protein sample pretreatment and high-pH value reversed phase fractionation of polypeptides in the biological sample to be tested.

10. Use according to claim 9, characterized in that, The protein sample is subjected to enzymolysis on the protein sample pretreatment filler, and after the enzymolysis is completed, the generated polypeptides are transferred to the solid phase extraction membrane, and then high-pH value reversed phase fractionation is performed.

11. Use according to claim 10, characterized in that, The pH value of the high-pH value reversed phase fractionation is higher than 8.

12. Use according to claim 7, characterized in that, The application comprises the following steps: (1) adding the biological sample to be tested along the capillary tube into the microfluidic device, so that the proteins are enriched on the protein sample pretreatment filler, to obtain the protein sample pretreatment filler enriched with proteins; (2) under the action of a reducing agent, an alkylating agent and an enzyme, the protein sample pretreatment filler enriched with proteins obtained in step (1) is sequentially subjected to reduction reaction, alkylation reaction and enzymolysis reaction, to obtain polypeptides; (3) the polypeptides obtained in step (2) are transferred from the protein sample pretreatment filler to the solid phase extraction membrane by a salt solution; (4) After desalting, polypeptides on the solid-phase extraction membrane are eluted by organic solvent solutions with different concentrations in turn, and high-pH value reversed-phase fractionation is performed, wherein the pH value of the solution used in the reversed-phase fractionation is higher than 8.

13. An automated proteomic sample pre-treatment platform characterized by, The automatic proteomic sample pretreatment platform comprises a closed system, the microfluidic device according to any one of claims 1-6, and a collection system, and the closed system and the collection system are connected with the capillary tubes at two ends of the microfluidic device; The closed system comprises a closed container provided with a gas channel and a sample placing device in the closed container, and the sample placing device is used for placing a sample; The collection system comprises a collection device used for collecting the sample after the reaction system; The number of the microfluidic devices is 1-100, and the different microfluidic devices are connected in parallel.

14. Application of the automatic proteomic sample pretreatment platform according to claim 13 in proteomic qualitative and quantitative analysis of a biological sample to be tested.

15. Use according to claim 14, characterized in that, The application comprises: introducing a gas into the closed container provided with a gas channel to pressurize, so that the sample in the sample placing device passes through the capillary tubes under the action of the pressure, reaches the microfluidic device to react, and after the reaction, reaches the collection device through the capillary tubes, and the collection device collects the sample after the reaction.

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