Extracellular vesicle in-situ microreactor and preparation and application thereof

Abstract

The invention relates to an extracellular vesicle in-situ microreactor and preparation and application thereof, the inner wall of a capillary is subjected to silanization treatment, and the surface of the capillary is modified with epoxy groups and amphoteric molecules through free radical polymerization reaction, so that a capillary open tubular column is prepared; and then modifying polyethyleneimine and iodoacetic acid N-hydroxysuccinimide ester in sequence through covalent bonding to prepare the amphoteric solid-phase alkylation capillary microreactor. The method has the advantages that the extracellular vesicles are captured in situ, the vesicle proteome sample is treated, and the method is suitable for proteome sample treatment of trace extracellular vesicle samples. In addition, the method is resistant to various surfactants and strong cracking reagents and has good sample compatibility.

Description

TECHNICAL FIELD

[0001] The present application relates to a micro extracellular vesicle in-situ microreactor and its preparation and application, belonging to the field of extracellular vesicle proteomics, which can be applied to the proteomic sample processing of micro extracellular vesicle samples. BACKGROUND

[0002] Extracellular vesicles, as one of the important means of intercellular communication (Science, 2020, 367, eaau6977), can reflect the physiological state of the parent cells, and have considerable potential in disease detection (Cell, 2020, 182, 4, 1044-1061), therapeutic intervention (Nat Rev Drug Discov, 2013, 12, 347-357) and drug delivery (Adv Drug Deliv Rev, 2018, 130, 12-16) and many other aspects. Therefore, it is of great significance to analyze the information carried by extracellular vesicles from the proteomic level. However, in actual research, due to the limitations of analysis strategies and detection means, it is difficult to carry out proteomic analysis on some extracellular vesicle samples that are difficult to collect in large quantities. At present, the analysis method of extracellular vesicles for small or micro extracellular vesicle samples is mainly based on antibody method, such as immunomagnetic bead method (Meth in Mol Biol, 2015, 1218, pp. 465-81) and Vn96 peptide affinity capture (J Extracell Vesicles, 2017, 6(1): 1313091). This kind of method has the common shortcomings that the number of detectable targets is limited, non-targeted global analysis of proteins in extracellular vesicles cannot be achieved, and the detection effect is limited by the specificity of the antibody, which cannot meet the needs of proteomic analysis of micro extracellular vesicle samples.

[0003] In view of the above problems, the present application provides a micro extracellular vesicle in-situ microreactor, which realizes in-situ pretreatment of micro extracellular vesicle proteomic samples in a zwitterionic solid-phase alkylated capillary microreactor, and at the same time completes the rapid removal of other interfering reagents (such as lysis reagent, surfactant, reducing agent, etc. Small molecules), the limited effect of the open column is also conducive to improving the reaction efficiency of the proteins in the extracellular vesicles, providing an important means for proteomic analysis of micro extracellular vesicles. SUMMARY

[0004] The epoxy group and the amphoteric molecule are modified in the inner surface of the capillary by free radical polymerization reaction, and then the polyethyleneimine and iodoacetic acid N-hydroxysuccinimidyl ester are modified in sequence by covalent bond to prepare the micro extracellular vesicle in-situ microreactor. In addition, the microreactor is used for pretreatment of microvesicle protein samples such as cell culture medium, body fluid, lavage fluid, etc.

[0005] To achieve the above object, the technical scheme adopted by the present application is:

[0006] (1) A quartz capillary tube with a polyacetylimide coating with an inner diameter of 100-200 μm and an outer diameter of 360 μm is used, the capillary tube is first activated to expose the hydroxyl groups on the inner wall, and the capillary tube is subjected to alkali washing, acid washing and alcohol washing in sequence using sodium hydroxide, hydrochloric acid and methanol.

[0007] (2) The above capillary tube is subjected to silanization treatment, a silanization reagent with a double bond is used to perform a silanization combination reaction with the silicon hydroxyl groups to introduce a double bond on the inner surface of the capillary tube, and then copolymerization is performed with a methyl methacrylic acid monomer and a [3-(methyl methacrylamido) propyl] dimethyl (3-thiopropyl) ammonium hydroxide monomer. The silanization reagent used is 3-(methyl methacryloyloxy) propyl trimethoxysilane (γ-MAPS) or vinyl trimethoxysilane (VMTS).

[0008] (3) The epoxy group and the amphoteric molecule are modified on the inner wall of the capillary tube through a free radical polymerization reaction: the initiator, the two kinds of monomers and the pore-forming agent are uniformly dispersed in a solution, the solution is degassed, and the silanization-treated capillary tube is introduced, the polymerization is initiated by the initiator, and the reaction is performed at 60°C for 5-7 hours to form a coating structure on the inner surface of the capillary tube. The mass ratio of the initiator, the epoxy group monomer and the amphoteric molecule monomer in the polymerization solvent is in the range of 1:25:75-1:75:25, and the mass ratio of the total mass of the monomers and the pore-forming agent is 1:5-1:20. The monomers used are two or one of the epoxy group-containing acryl-based monomers such as glycidyl methacrylate (GMA) and glycidyl acrylate, and the amphoteric molecule containing positive and negative charges such as [3-(methyl methacrylamido) propyl] dimethyl (3-thiopropyl) ammonium hydroxide (SPP); the initiator used to initiate the polymerization reaction includes one of azobisisobutyronitrile (AIBN) and azobisisoheptyl nitrile (ABVN); and the pore-forming agent is a solvent for promoting the formation of pores in the polymer, including one or two or more of ethylene glycol, n-propanol, butanediol and n-dodecanol.

[0009] (4) Introducing hydrophilic compound polyethyleneimine (PEI) on the surface of the capillary open column, further modifying N-hydroxysuccinimidyl iodoacetate through covalent bonding, the specific reaction process is as follows: passing PEI solution into the capillary open column, reacting at 60°C for 4 to 6 hours, after the reaction is completed, removing the residual PEI in the open column, and washing with water until neutral. Then dissolving N-hydroxysuccinimidyl iodoacetate in a mixed system of methanol and phosphate buffer, adding the capillary open column modified with PEI, reacting at 40°C for 24 hours (adding once every 4 to 6 hours), finally washing with methanol and blowing dry with nitrogen, thereby obtaining the amphoteric solid-phase alkylated capillary open column. The final concentration of the polyethyleneimine solution is 10 mg / mL to 100 mg / mL; the final concentration of the N-hydroxysuccinimidyl iodoacetate solution is 10 mg / mL to 100 mg / mL, and the volume ratio of the phosphate buffer to methanol is 1:5 to 1:20, wherein the concentration of the phosphate buffer is 0.01 mol / L to 0.1 mol / L, and the pH value is 7 to 9.

[0010] (5) Applying it to the pretreatment of proteomic samples of trace extracellular vesicles, the specific process is as follows: passing about 1 μL of extracellular vesicle sample liquid into the microreactor for overnight incubation, and pushing out the sample liquid after the incubation is completed. Passing the lysis solution into the microreactor to completely cover the incubation position of the sample liquid. The lysis solution includes a surfactant such as sodium dodecyl sulfate (SDS) and a reducing agent tris(2-carboxyethyl)phosphine (TCEP), the concentration of SDS is 0.01% (w / w) to 0.5% (w / w), and the concentration of TCEP is 25 mmol / L to 100 mmol / L. Ultrasonic treatment at 4°C for 5 to 15 minutes makes the extracellular vesicles adsorbed on the inner wall of the microreactor completely lysed, and then water bath heating at 90°C to 95°C for 5 to 10 minutes quickly realizes the denaturation, reduction and alkylation of proteins. Then washing with 50% methanol (v / v) or acetonitrile to remove the residual lysis solution and reducing agent in the open column, and replacing with a weakly alkaline buffer system such as ammonium bicarbonate (ABC) or hydroxyethylpiperazine ethanesulfonic acid (HEPES), the concentration is 25 mmol / L to 100 mmol / L, and the pH value is 7 to 9. Finally, passing a protease such as trypsin, staphylococcus aureus V8 protease (Glu-C) or lysyl endopeptidase (Lys-C) into the amphoteric solid-phase alkylated open column, the amount of the protease added is 10 ng to 100 ng, and the enzymolysis is carried out at 37°C for 12 to 15 hours, and the enzymolysis product is stored at a low temperature of -60°C to -80°C or directly subjected to liquid chromatography-mass spectrometry analysis.

[0011] (6) Liquid chromatography coupled with tandem mass spectrometry analysis was performed using an Orbitrap Exploris 480 (Thermo Fisher Scientific) mass spectrometer (San Jose, CA) with a FAIMS Pro interface coupled with an Easy-nano LC 1000 system (Thermo Fisher Scientific). A self-made C18 analytical column was used for polypeptide separation, and the flow rate was set to range from 0.1 μL / min to 0.3 μL / min. A microcapillary aspirator (P-2000, Sutter Instrument, CA) was used to prepare the needle tip. Mobile phase A and B were formic acid aqueous solution and acetonitrile aqueous solution, respectively.

[0012] Mass spectrometry measurements were performed in data-independent acquisition mode (DIA). The electrospray voltage was set to range from 2.10 kV to 2.35 kV, and the ion transfer tube temperature was set to range from 320 °C to 340 °C. For full MS scans, the Orbitrap resolution was set to 60,000 or 90,000, the normalized AGC target was set to range from 200% to 400%, and the maximum injection time was set to range from 100 ms to 150 ms. The exclusion time was set to range from 30 s to 60 s, and the intensity threshold was set to range from 3,000 to 6,000. The HCD collision energy was set to range from 20% to 40%, and the isolation window (m / z) was set to range from 1.2 to 2.0. The MS / MS scan resolution was 15,000 or 22,500, the AGC target was set to range from 3e5 to 6e5, and the maximum injection time was 100 ms to 150 ms. FAIMS was set to default settings, with a total carrier gas flow rate set to range from 3 L / min to 5 L / min, and a compensation voltage (CV) of -45 V and -65 V. Single-cell analysis was performed in data-independent acquisition (DIA) mode. Precursor ions were collected at m / z 400 to 1200, the isolation window (m / z) was set to range from 10 to 30, the normalized collision energy was set to range from 20% to 50%, and the resolution for MS / MS scans was set to range from 20,000 to 40,000.

[0013] The present application prepares a capillary open column by silanizing the inner wall of a capillary and modifying epoxy groups and amphiphilic molecules on the surface thereof through a free radical polymerization reaction, and then sequentially modifies polyethyleneimine and iodoacetic acid N-hydroxysuccinimidyl ester through covalent bonding to prepare an amphiphilic solid-phase alkylated capillary microreactor.

[0014] The specific sample processing process is as follows: first, the extracellular vesicle solution is introduced into the microreactor for incubation, and the sample liquid is pushed out after the incubation is completed. Then, the lysis solution is introduced into the microreactor to cover the incubation position of the vesicle solution; then, the vesicles captured on the inner wall of the microreactor are lysed by ultrasonic crushing; meanwhile, under high temperature conditions, the denaturation, reduction and alkylation reactions of the proteins are rapidly realized, so that the proteins are covalently fixed on the inner surface of the microreactor; finally, the microreactor is washed with solvents such as methanol and ammonium bicarbonate to remove interfering substances such as surfactants, lipids and sugars; an appropriate amount of protease is added, and enzymolysis is carried out at an appropriate temperature. The enzymolysis product can be directly subjected to liquid chromatography-mass spectrometry analysis.

[0015] The advantage of the present application is that the capture of extracellular vesicles and the processing of vesicle proteome samples are carried out in situ, and the proteome sample processing is suitable for micro extracellular vesicle samples. In addition, the present method is resistant to various surfactants and strong lysis reagents, and has good sample compatibility.

[0016] The present application has the following advantages:

[0017] 1. The micro extracellular vesicle in-situ microreactor can realize the protein sample processing of ultramicro vesicles, providing an important idea for the proteome analysis and research of micro vesicle samples.

[0018] 2. The epoxy group and the amphoteric molecule are modified on the inner wall surface of the capillary through a free radical polymerization reaction to prepare a capillary open column, and then polyethyleneimine and iodoacetic acid N-hydroxysuccinimidyl ester are sequentially modified through covalent bonding. The preparation process is easy to control, and it is easy to obtain amphoteric solid-phase alkylated capillary microreactors with uniform quality.

[0019] 3. The prepared micro extracellular vesicle in-situ microreactor can be compatible with various surfactants for the processing of extracellular vesicle proteome samples, improve the extraction efficiency of micro extracellular vesicle proteins, avoid the loss of micro samples caused by multiple transfers, and improve the recovery rate of micro sample processing.

[0020] 4. The micro extracellular vesicle in-situ microreactor can realize the simultaneous performance of multiple reactions (including protein denaturation, reduction and alkylation). Due to the confinement effect of the capillary open column, the reaction volume (nanoliter) is reduced, which is conducive to improving the efficiency of sample processing.

[0021] The beneficial effects that can be produced by the present application include:

[0022] The micro extracellular vesicle in-situ microreactor provided by the present application can realize the capture and crushing of micro extracellular vesicles in the capillary open column, and realize the selective enrichment and in-situ enzymolysis of the proteins in the vesicles. Combined with high-sensitivity liquid chromatography-mass spectrometry technology, the analysis of the proteome of micro extracellular vesicles can be realized. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Schematic diagram of preparation of micro extracellular vesicle in-situ microreactor;

[0024] Figure 2 Scanning electron microscope characterization diagram of micro extracellular vesicle in-situ microreactor;

[0025] Figure 3 Flowchart of in-situ pretreatment of intravesicular protein based on micro extracellular vesicle in-situ microreactor;

[0026] Figure 4 Extracellular vesicle proteomic analysis of 0.83 μL human embryonic stem cell culture solution enriched with vesicles by ultracentrifugation (5.5 x 10 11 particles / mL) using micro extracellular vesicle in-situ microreactor;

[0027] Figure 5 Extracellular vesicle proteomic analysis of 0.83 μL human adipose stem cell culture solution using micro extracellular vesicle in-situ microreactor;

[0028] Figure 6 Extracellular vesicle proteomic analysis of 0.83 μL human embryonic stem cell culture solution enriched with vesicles by ultracentrifugation (5.5 x 10 11 particles / mL) using micro extracellular vesicle in-situ microreactor, LC-MS / MS analysis and identification of total number of proteins, number of proteins matched with ExoCarta exosome protein library and number of proteins matched with TOP100 exosome protein library in three parallel measurements;

[0029] Figure 7 Extracellular vesicle proteomic analysis of 0.83 μL human adipose stem cell culture solution using micro extracellular vesicle in-situ microreactor, LC-MS / MS analysis and identification of total number of proteins, number of proteins matched with ExoCarta exosome protein library and number of proteins matched with TOP100 exosome protein library in three parallel measurements; DETAILED DESCRIPTION

[0030] The application will be described in greater detail below with reference to the embodiments. However, the application is not limited to these embodiments.

[0031] Unless otherwise specified, the raw materials in the embodiments of the application are obtained through commercial channels.

[0032] Example 1

[0033] Silanization treatment of the inner wall of the capillary microreactor

[0034] 1. First, a 5 m long quartz capillary tube (inner diameter 200 μm, outer diameter 360 μm, Polymicro TECHNOLOGIES) coated with polyacetylimine was cut, and 1 mol / L NaOH aqueous solution was made to flow through the capillary tube by pushing it from one open end and letting it flow out from the other end using a high-precision syringe pump (Harvard) combined with a syringe at a speed of 16.66 μL / min for 3 hours. Then, the remaining NaOH solution in the capillary tube was pushed out and removed by manually using a syringe to suck air. Next, H2O was made to flow through the capillary tube by pushing it from one open end and letting it flow out from the other end using a high-precision syringe pump (Harvard) combined with a syringe at a speed of 16.66 μL / min for 2 hours. Then, the remaining H2O in the capillary tube was pushed out and removed by manually using a syringe to suck air. Next, 1 mol / L hydrochloric acid was made to flow through the capillary tube by pushing it from one open end and letting it flow out from the other end using a high-precision syringe pump (Harvard) combined with a syringe at a speed of 16.66 μL / min for 2 hours. Then, the remaining hydrochloric acid in the capillary tube was pushed out and removed by manually using a syringe to suck air. Next, H2O was made to flow through the capillary tube by pushing it from one open end and letting it flow out from the other end using a high-precision syringe pump (Harvard) combined with a syringe at a speed of 16.66 μL / min for 2 hours. Then, the remaining H2O in the capillary tube was pushed out and removed by manually using a syringe to suck air. Finally, methanol was made to flow through the capillary tube by pushing it from one open end and letting it flow out from the other end using a high-precision syringe pump (Harvard) combined with a syringe at a speed of 16.66 μL / min for 1 hour. After the injection was completed, the capillary tube was completely dried by blowing nitrogen.

[0035] 2. The methanol solution (50%, v / v) of 3-(methacryloyloxy)propyltrimethoxysilane was introduced into the above activated capillary tube by manually using a syringe until the solution filled the inside of the capillary tube. Then, the two open ends of the capillary tube were plugged with rubber plugs, and the capillary tube was placed in an oven at 50°C for 24 hours. After the reaction was completed, the remaining solution in the capillary tube was pushed out and removed by manually using a syringe to suck air. Then, methanol was made to flow through the capillary tube by pushing it from one open end and letting it flow out from the other end using a high-precision syringe pump (Harvard) combined with a syringe at a speed of 16.66 μL / min for 1 hour, and the capillary tube was completely dried by blowing nitrogen. Thus, the silanization treatment of the inner wall of the capillary tube was completed.

[0036] Example 2

[0037] Amino functionalization treatment of the inner wall of the capillary tube

[0038] 1. A solution was prepared by mixing azobisisobutyronitrile (AIBN), glycidyl methacrylate (GMA), [3-(methacryloylamido)propyl]dimethyl(3- thio propyl)ammonium hydroxide (SPP), ethylene glycol and n-propanol (1:50:50:180:670, mass ratio) uniformly, and then deoxygenated by nitrogen. The solution was injected into the silanized capillary obtained in Example 1 using a syringe until the solution filled the capillary, and then the two ends of the capillary were plugged with rubber plugs and placed in a 60°C oven for 6 hours to polymerize and form a coating structure on the inner surface of the capillary. After the reaction was completed, the remaining solution in the capillary was pushed out by manually using a syringe to suck air, and then methanol was pushed through the capillary from one open end and flowed out from the other open end by a high-precision syringe pump (Harvard) combined with a syringe at a speed of 16.66 μL / min for 1 hour, and then completely dried by nitrogen. The average wall thickness of the inner wall coating of the capillary open column prepared was 2 μm.

[0039] 2. A 10 mg / mL aqueous solution of polyethyleneimine was injected into the capillary open column obtained in step 1 using a syringe until the solution filled the open column, and then the two ends of the capillary open column were plugged with rubber plugs and placed in a 50°C oven for 6 hours. After the reaction was completed, the remaining aqueous solution of polyethyleneimine in the open column was pushed out by manually using a syringe to suck air, and then H2O was pushed through the capillary from one open end and flowed out from the other open end by a high-precision syringe pump (Harvard) combined with a syringe at a speed of 16.66 μL / min for 2 hours; and then the remaining H2O in the capillary was pushed out by manually using a syringe to suck air. Finally, methanol was pushed through the capillary from one open end and flowed out from the other open end by a high-precision syringe pump (Harvard) combined with a syringe at a speed of 16.66 μL / min for 1 hour; after the injection was completed, it was completely dried by nitrogen, and the amino functionalization treatment of the inner wall of the capillary was completed.

[0040] Example 3

[0041] Iodoacetic acid group functionalization treatment of the inner wall of a capillary microreactor

[0042] A 10 mg / mL solution of iodoacetic acid N-hydroxysuccinimidyl ester was prepared in a 1:4 mixture of phosphate buffer (0.05 mol / L, pH = 8) and methanol. The 10 mg / mL solution of iodoacetic acid N-hydroxysuccinimidyl ester was manually injected into the amino-functionalized capillary open tubular column obtained in Example 2 using a syringe until the solution filled the interior of the open tubular column. Rubber stoppers were then used to seal the ends of the capillary open tubular column, and the column was placed in a 40°C oven for 24 hours. The solution was replaced every 6 hours by injecting a fresh solution of iodoacetic acid N-hydroxysuccinimidyl ester. The methanol was then pushed through the capillary by a high-precision syringe pump (Harvard) at a rate of 16.66 μL / min for 1 hour. After the injection, the column was flushed with nitrogen gas to dry the column. The iodoacetyl-functionalized capillary open tubular column was then obtained.

[0043] Example 4

[0044] Example 3 obtained the amphoteric solid-phase alkylated capillary open tubular column was used for the pretreatment of 0.83 μL of extracellular vesicle proteome sample of human embryonic stem cell culture solution enriched by ultracentrifugation

[0045] 1. After the growth density of human embryonic stem cells reached 80%-90%, the cell culture solution of human embryonic stem cells was collected. The cell culture solution was then centrifuged at 500g for 10 minutes and at 4000g for 20 minutes. The solution was then centrifuged at 100,000g for 4 hours to obtain a vesicle-enriched solution. The concentration of vesicles in the vesicle-enriched solution was then measured by nanoparticle tracking analysis (NTA) to be 5.5 x 10 11 particles / mL.

[0046] 2. Sodium dodecyl sulfate (0.0005 g / mL) and reducing agent tris(2-carboxyethyl) phosphine (50 mmol / L) were prepared as a lysis solution (solvent: water). A 6 cm long amphoteric solid-phase alkylated open tubular column obtained in Example 3 was used. The 830 nL of vesicle-enriched solution (5.5 x 10 11The capillary column was capped with rubber plugs at both ends, and then incubated for 12 hours. After the incubation, the sample position was marked (marking the position of the solution at both ends of the capillary column, the same below), the solution in the open column was pushed out and drained manually using a syringe to suck air, and then 850 nL of lysis solution was introduced into the open column using a high-precision injection pump (Harvard) combined with a syringe, so that it completely covered the original sample mark position. The two ends of the open column were capped with rubber plugs during the incubation.

[0047] 3. The open column was placed in a micro-sample ultrasonic disruptor, and the extracellular vesicles adsorbed on the inner wall of the open column micro-reactor were lysed at 4°C and 250W for 5 minutes. Then the sample was heated in a water bath at 90°C for 5 minutes to quickly realize the denaturation, reduction and alkylation of the vesicle proteins. After the reaction was completed, the sample position was marked again (marking the position of the solution at both ends of the micro-reactor, the same below).

[0048] 4. A high-precision injection pump (Harvard) combined with a syringe was used to introduce 300 μL of 50% methanol aqueous solution into the open column to remove residual surfactants and other interference substances in the column, and then 300 μL of 50 mmol / L ammonium bicarbonate solution (pH=8) was introduced into the open column for cleaning. A high-precision injection pump (Harvard) combined with a syringe was used to introduce 900 nL of protease solution (0.005 ng / nL Trypsin protease and 0.005 ng / nL Lys-C protease mixed enzyme solution, solvent: 0.05 mol / L ammonium bicarbonate solution) into the open column, so that it completely covered the sample mark position. The two ends of the open column were capped with rubber plugs, and the enzyme was placed in a water bath at 37°C for 12 hours. The enzyme digestion product was taken out and stored at -80°C or directly subjected to liquid chromatography-mass spectrometry analysis.

[0049] 5. The mass spectrometry detection result is shown in Figure 4 The ion intensity of the mass spectrometry signal peak is 4.8E8, and the average ion intensity is about 2E8. The processed sample is 0.83 μL of enriched vesicle solution (vesicle concentration 5.5×10 11 particles / mL). The average number of identified proteins in three mass spectrometry detections is 525, of which the average number of proteins matched with the ExoCarta exosome protein library is 475, and the enrichment selectivity is 90.48%.

[0050] Therefore, the vesicle micro-reactor has the ability to capture extracellular vesicles and proteins in the vesicle solution.

[0051] Example 5

[0052] The zwitterionic solid-phase alkylated capillary open-tubular column obtained in Example 3 was used for pretreatment of an extracellular vesicle proteome sample of 0.83 μL human adipose stem cell cell culture solution

[0053] 1. After the human adipose stem cells reached 80%-90% of the growth density, the cell culture solution of the human embryonic stem cells was collected, and then the cell culture solution was centrifuged at 500 g for 10 minutes and at 4000 g for 20 minutes.

[0054] 2. Sodium dodecyl sulfate (0.0005 g / mL) and reducing agent tris(2-carboxyethyl) phosphine (50 mmol / L) were configured as a lysis solution (solvent: water). A 6 cm long zwitterionic solid-phase alkylated open-tubular column obtained in Example 3 was used, and 830 nL of human adipose stem cell cell culture solution was introduced into the open-tubular column using a high-precision syringe pump (Harvard) combined with a syringe. The two ends of the capillary open-tubular column were plugged with rubber plugs, and then incubated for 12 hours. After the incubation was completed, the sample position was marked (the two end positions of the cell culture solution were marked, and the same below), the solution in the open-tubular column was manually pushed out and drained using a syringe to suck air, and then 850 nL of lysis solution was introduced into the open-tubular column using a high-precision syringe pump (Harvard) combined with a syringe to completely cover the original sample marking position. The two ends of the open-tubular column were plugged with rubber plugs during the incubation.

[0055] 3. The above open-tubular column was placed in a micro-sample ultrasonic disruptor, and ultrasonic treatment was performed at 4°C and 250 W for 5 minutes to lyse the extracellular vesicles adsorbed on the inner wall of the micro-reactor of the open-tubular column. Then the sample was heated in a water bath at 90°C for 5 minutes to rapidly realize denaturation, reduction and alkylation of the vesicle proteins. After the reaction was completed, the sample position was marked again (the two end positions of the solution in the micro-reactor were marked, and the same below).

[0056] 4. 300 μL of 50% methanol aqueous solution was introduced into the open-tubular column using a high-precision syringe pump (Harvard) combined with a syringe to wash away the residual surfactants and other interference substances in the column, and then 300 μL of 50 mmol / L ammonium bicarbonate solution (pH = 8) was introduced into the open-tubular column for washing. 900 nL of protease solution (0.005 ng / nL Trypsin protease and 0.005 ng / nL Lys-C protease mixed enzyme solution, solvent: 0.05 mol / L ammonium bicarbonate solution) was introduced into the open-tubular column using a high-precision syringe pump (Harvard) combined with a syringe to completely cover the sample marking position, and then the two ends of the open-tubular column were plugged with rubber plugs and placed in a water bath at 37°C for enzymatic hydrolysis for 12 hours. The enzymatic hydrolysate was stored at -80°C or directly subjected to liquid chromatography-mass spectrometry analysis.

[0057] 5. The mass spectrometry detection results are as follows Figure 5As shown, the ion intensity of the mass spectrometry signal peak is 1.6E9, and the average ion intensity is about 3E8. The sample processed is 0.83 μL of human fat stem cell culture solution. The average number of identified proteins in two mass spectrometry detections is 235, of which the average number of proteins matched with the ExoCarta exosome protein library is 223, and the enrichment selectivity is 94.89%.

[0058] Therefore, the vesicle microreactor has the capability of capturing extracellular vesicles and intravesicular proteins in an ultramicro cell culture solution sample.

[0059] Example 6

[0060] The amphoteric solid-phase alkylated capillary open column obtained in Example 3 is used for pretreatment of an extracellular vesicle proteome sample of 1 μL of human alveolar lavage fluid

[0061] 1. The human alveolar lavage fluid is centrifuged at 500g for 10 minutes to remove cells and impurities, and then centrifuged at 4,000g for 20 minutes to remove cell debris and impurities.

[0062] 2. Sodium dodecyl sulfate (0.0005 g / mL) and reducing agent tris(2-carboxyethyl) phosphine (50 mmol / L) are configured as a lysis solution (solvent is water). A 6 cm long amphoteric solid-phase alkylated open column obtained in Example 3 is used, and a 1 μL human alveolar lavage fluid treated as described above is introduced into the open column using a high-precision syringe pump (Harvard) combined with a syringe. The ends of the capillary open column are plugged with rubber plugs, and then incubated for 12 hours. After incubation, the sample position is marked (the same as the position of the cell culture solution at both ends, the same below), the solution in the open column is pushed out and drained manually using a syringe, and then 1.1 μL of lysis solution is introduced into the open column using a high-precision syringe pump (Harvard) combined with a syringe, so that it completely covers the original sample mark position. The ends of the open column are plugged with rubber plugs during incubation.

[0063] 3. The above open column is placed in a micro-sample ultrasonic disruptor, and ultrasonicated at 4°C and 250W for 5 minutes to lyse the extracellular vesicles adsorbed on the inner wall of the open column microreactor; then the sample is heated in a water bath at 90°C for 5 minutes to rapidly realize denaturation, reduction and alkylation of the vesicle proteins. After the reaction is completed, the sample position is marked again (the same as the position of the solution at both ends in the microreactor, the same below).

[0064] 4. Use high-precision syringe pump (Harvard) combined with syringe to inject 300 μL of 50% methanol aqueous solution into the open tubular column to remove residual surfactants and other interference substances, and then inject 300 μL of 50 mmol / L ammonium bicarbonate solution (pH = 8) into the open tubular column for cleaning; inject 900 nL of protease solution (0.005 ng / nL Trypsin protease and 0.005 ng / nL Lys-C protease mixed enzyme solution, solvent is 0.05 mol / L ammonium bicarbonate solution) into the open tubular column through the high-precision syringe pump (Harvard) combined with the syringe, so that it completely covers the sample labeling position, and then plug the two ends of the open tubular column with rubber plugs and place it in a water bath at 37°C for enzymatic hydrolysis for 12 hours. Take out the enzymatic hydrolysis product and store it at -80°C or directly perform liquid chromatography-mass spectrometry analysis.

[0065] Example 7

[0066] Use of the amphoteric solid-phase alkylated capillary open tubular column obtained in Example 3 for pretreatment of an extracellular vesicle proteome sample of 1 μL human cerebrospinal fluid

[0067] 1. Centrifuge the human cerebrospinal fluid at 500g for 10 minutes to remove cells and impurities, and then centrifuge at 4,000g for 20 minutes to remove cell debris and impurities.

[0068] 2. Configure sodium dodecyl sulfate (0.0005 g / mL) and reducing agent tris(2-carboxyethyl) phosphine (50 mmol / L) as a lysis solution (solvent is water). Cut a 6 cm long amphoteric solid-phase alkylated open tubular column obtained in Example 3, use a high-precision syringe pump (Harvard) combined with a syringe to inject 1 μL of human cerebrospinal fluid treated as described above into the open tubular column, plug the two ends of the capillary open tubular column with rubber plugs, and then incubate for 12 hours. After incubation, mark the sample position (mark the position of the two ends of the cell culture solution, the same below), use a syringe to manually push out and drain the solution in the open tubular column, and then inject 1.1 μL of lysis solution into the open tubular column through the high-precision syringe pump (Harvard) combined with the syringe, so that it completely covers the original sample labeling position. Plug the two ends of the open tubular column with rubber plugs during incubation.

[0069] 3. Place the above open tubular column in a micro-sample ultrasonic disruptor, and ultrasonically treat it at 4°C and 250W for 5 minutes to lyse the extracellular vesicles adsorbed on the inner wall of the open tubular column micro-reactor; then heat the sample in a water bath at 90°C for 5 minutes to quickly realize denaturation, reduction and alkylation of the vesicle proteins, and then mark the sample position again (mark the position of the two ends of the solution in the micro-reactor, the same below).

[0070] 4. Using high-precision syringe pump (Harvard) combined with syringe to inject 300 μL volume of 50% methanol aqueous solution into the open column to clean and remove residual surfactants and other interfering substances in the column, and then inject 300 μL of 50 mmol / L ammonium bicarbonate solution (pH=8) into the open column for cleaning; through high-precision syringe pump (Harvard) combined with syringe to inject 900 nL of protease solution (0.005 ng / nL Trypsin protease and 0.005 ng / nL Lys-C protease mixed enzyme solution, solvent is 0.05 mol / L ammonium bicarbonate solution) into the open column to completely cover the sample labeling position, and then plug the two ends of the open column with rubber plugs, and place in a water bath pot for 12 hours of enzymolysis at 37°C. Take out the enzymolysis product and store it at -80°C or directly perform liquid chromatography-mass spectrometry analysis.

Claims

1. A method for preparing an extracellular vesicle in-situ microreactor, characterized by: 1) activating and exposing the silanol groups on the inner surface of the capillary by alkali-acid treatment, and then performing silanization treatment, using a silane reagent with a carbon-carbon double bond in its molecular structure to perform a silanization bonding reaction with the silanol groups on the inner surface, to bond the double bond to the inner surface of the capillary; 2) subsequently modifying epoxy groups and amphiphilic molecules on the inner wall of the capillary by free radical polymerization, to modify the epoxy groups and amphiphilic molecules on the inner surface of the capillary to form an inner wall coating, thereby preparing a capillary open tubular column; and 3) sequentially modifying polyethyleneimine and iodoacetic acid N-hydroxysuccinimidyl ester to the sites on the inner wall of the capillary where the epoxy groups are located by covalent bonding, to finally prepare an amphiphilic solid-phase alkylated capillary microreactor.

2. The method according to claim 1, characterized in that: in step 1), the inner diameter of the capillary used ranges from 100 μm to 200 μm; the activation steps sequentially use sodium hydroxide solution (concentration of 1 mol / L to 3 mol / L, preferably 1 mol / L to 1.5 mol / L), hydrochloric acid (concentration of 1 mol / L to 3 mol / L, preferably 1 mol / L to 1.5 mol / L), and methanol to perform alkali washing, acid washing, and alcohol washing on the capillary; and the silane reagent used in the silanization treatment is two or one of 3-(methacryloyloxy)propyltrimethoxysilane (γ-MAPS) or vinyltrimethoxysilane (VMTS).

3. The method according to claim 1, characterized in that: in step 2), the epoxy groups and amphiphilic molecules are modified on the inner wall of the capillary by free radical polymerization; the monomer for modifying the epoxy groups is two or one of glycidyl methacrylate (GMA) or glycidyl acrylate, the monomer for modifying the amphiphilic molecules is [3-(methacryloylamino)propyl]dimethyl(3-sulfopropyl)ammonium hydroxide (SPP), the initiator is two or one of azobisisobutyronitrile (AIBN) or azobisisoheptyl nitrile (ABVN), the porogen is one or more than two of ethylene glycol, n-propanol, butanediol, or n-dodecanol, the mass ratio of the initiator, the epoxy group monomer, and the amphiphilic molecule monomer ranges from 1:30:70 to 1:50:50 (preferably 1:40:60 to 1:50:50), the ratio of the total mass of the monomers (the epoxy group monomer and the amphiphilic molecule monomer) to the mass of the porogen ranges from 1:5 to 1:10 (preferably 1:7 to 1:9), the reaction temperature ranges from 50°C to 70°C (preferably 60°C to 70°C), and the reaction time ranges from 5 hours to 7 hours (preferably 6 hours to 7 hours).

4. The method according to claim 1, characterized in that: ​ ​ ​ ​ ​ ​ ​ ​ In step 3), after the modification of the epoxy groups and the amphiphilic molecules on the inner surface of the microreactor, polyethyleneimine (PEI) is further introduced, and then N-hydroxysuccinimidyl iodoacetate is modified by covalent bonding, wherein the final concentration of the PEI aqueous solution used is in the range of 10 mg / mL to 20 mg / mL (preferably 10 mg / mL to 15 mg / mL); the reaction temperature is in the range of 50°C to 60°C (preferably 50°C to 55°C), and the reaction time is in the range of 6 hours to 8 hours (preferably 6 hours to 7 hours); N-hydroxysuccinimidyl iodoacetate (TCI) is dissolved in a solvent system composed of phosphate buffer and methanol, and the volume ratio of phosphate buffer to methanol is in the range of 1:3 to 1:6 (preferably 1:4 to 1:5); the final concentration of N-hydroxysuccinimidyl iodoacetate is in the range of 10 mg / mL to 20 mg / mL (preferably 10 mg / mL to 15 mg / mL), the concentration of phosphate buffer is in the range of 0.01 mol / L to 0.1 mol / L (preferably 0.05 mol / L to 0.1 mol / L), and the pH value is in the range of 7 to 9 (preferably 8 to 9); the reaction temperature is in the range of 40°C to 60°C (preferably 40°C to 45°C), and the reaction time is in the range of 24 hours to 26 hours.

5. A micro-extracellular vesicle in-situ microreactor prepared by the preparation method of any one of claims 1 to 4.

6. Use of the micro-extracellular vesicle in-situ microreactor of claim 5, characterized in that: It can be used for the pretreatment of one or more than two vesicle protein samples in cell culture medium, body fluid, lavage fluid, etc.

7. The use according to claim 6, characterized in that: It is an amphiphilic solid-phase alkylated capillary microreactor, which sequentially performs the coordination of extracellular vesicles by amphiphilic molecules and the covalent bonding of proteins by thiol alkylation reaction through the modification of iodine acetic groups on the inner surface of the capillary microreactor, and is based on this for the processing of extracellular vesicle proteome samples.

8. Use of the micro-extracellular vesicle in-situ microreactor according to claim 6 or 7, characterized in that: (1) The micro-extracellular vesicle in-situ microreactor with a length of 6 cm to 8 cm is cut, 0.5 μL to 1 μL of sample solution containing extracellular vesicles is introduced into the microreactor, and incubated at room temperature for 12 hours to 15 hours; after the incubation is completed, the positions of the two ends of the sample solution are marked, the sample solution is pushed out, a lysis solution is introduced into the microreactor to completely cover the original sample position, and the two ends of the microreactor are plugged during the incubation; Sodium dodecyl sulfate (0.0005 g / mL to 0.0015 g / mL) and reducing agent tris(2-carboxyethyl) phosphine (50 mmol / L to 75 mmol / L) are configured as the lysis solution; (2) The above microreactor is placed in an ultrasonic disruptor, and ultrasonic waves are used to lyse the extracellular vesicles adsorbed on the inner wall of the microreactor; then the sample is heated in a water bath (90°C to 95°C) for reaction, quickly realizing the denaturation, reduction and alkylation of the proteins in the vesicles, and after the reaction is completed, the positions of the two ends of the solution in the microreactor are marked again; (3) The microreactor is washed with a 30% to 60% methanol aqueous solution to remove residual interfering substances in the column, and then washed with an ammonium bicarbonate buffer solution. Finally, according to the marked sample position, a protease solution (0.005 ng / nL Trypsin protease and 0.005 ng / nL Lys-C protease mixed enzyme solution, solvent: 50 mmol / L ammonium bicarbonate solution) is introduced, the two ends of the microreactor are blocked, and enzymolysis is performed at a temperature of 35°C to 40°C for 12 to 15 hours.

9. Use of the micro-in situ microreactor for extracellular vesicles according to any one of claims 6 to 8, characterized in that: The enzymolysis product can be stored at low temperature (-60°C to -80°C) or directly subjected to liquid chromatography-mass spectrometry analysis. For proteomic analysis of micro-extracellular vesicles, a liquid chromatography-tandem mass spectrometry system is used for analysis; the mass spectrometry measurement adopts a data-independent acquisition mode; the mass spectrometry detection system is combined with FAIMS, the total flow rate of the carrier gas is set to 3 L / min to 5 L / min, the compensation voltage is -45 V and -65 V, the normalized collision fragmentation energy range is 20% to 50%, and the resolution setting range for MS / MS scanning is 20,000 to 40,000.

10. Use of the micro-in situ microreactor for extracellular vesicles according to any one of claims 6 to 8, characterized in that: The vesicle enrichment process of the sample solution containing extracellular vesicles is as follows: after the cell growth density reaches 80% to 90%, the cell culture solution is collected. The cell culture solution is subjected to first centrifugation (at a speed of 500 g to 700 g for 10 to 20 minutes) to remove cells in the culture solution; and then subjected to second centrifugation (at a speed of 4000 g to 4500 g for 20 to 30 minutes) to remove cell fragments in the culture solution. Finally, the cell culture solution subjected to the above two centrifugations is subjected to ultracentrifugation enrichment, first ultracentrifugation (at a speed of 10,000 g to 12,000 g for 60 to 90 minutes) to remove proteins in the cell culture solution; and then second ultracentrifugation (at a speed of 100,000 g to 120,000 g for 90 to 120 minutes) to enrich extracellular vesicles.

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