A microscale cell in-situ microreactor and its preparation and application

Abstract

The present invention relates to the preparation and application of a micro-cell in-situ microreactor. By subjecting the inner wall of a capillary to silanization treatment and modifying epoxy groups on its surface through free radical polymerization reaction, an open-tubular capillary column is prepared. Then, polyethyleneimine and iodoacetic acid-N-succinimidyl ester are successively modified by covalent bonding to prepare a solid-phase alkylated capillary microreactor. The advantages of the present invention are that proteomic sample processing is carried out in-situ, which is conducive to reducing sample loss caused by multiple transfers during traditional sample processing. Therefore, this solid-phase alkylated open-tubular column is suitable for proteomic pretreatment of trace samples. In addition, this method is tolerant to various surfactants and strong lysis reagents and has good sample compatibility.

Description

Technical Field

[0001] The present invention relates to a microcellular in-situ microreactor, which can be applied to the efficient pretreatment of proteins in 1-100 cells, trace body fluids and tissue samples. Background Art

[0002] Traditional bottom-up proteomics sample processing procedures usually include protein extraction, denaturation, reduction, alkylation, desalting, and enzymatic digestion, etc. These operations are usually completed under offline conditions and require multiple transfers, making it difficult to avoid protein sample loss. In addition, most proteomics sample processing methods use detergents or urea to lyse cells (J Proteome Res, 2015, 14: 3403-3408). Since these chemicals are incompatible with mass spectrometry, they need to be removed before LC-MS analysis. To solve the above problems, Wisniewski et al. developed the filter-aided sample preparation (FASP) method, which performs sample preparation in a single-container ultrafiltration tube and removes surfactants and other low-molecular-weight contaminating interfering substances by centrifugation. They were able to identify 905 proteins from 500 HeLa cells by using FASP (J Proteome Res, 2011, 10: 3040-3049). However, the recovery rate of this method is usually only 50%-80%, thus limiting its application in trace sample processing.

[0003] To solve the above problems, we prepared a microcellular in-situ microreactor for in-situ pretreatment of proteomic samples in an open tubular column, while achieving rapid removal of other interfering reagents (such as small molecules like lysis reagents, surfactants, reducing agents, etc.). The confinement effect of the open tubular column is also beneficial to improving the reaction efficiency of proteins, providing an important means for the efficient processing and analysis of microcellular proteomes. Summary of the Invention

[0004] The present invention relates to the preparation and application of a micro-cell in-situ microreactor. By subjecting the inner wall of a capillary to silanization treatment and modifying epoxy groups on its surface through free radical polymerization reaction, an open tubular capillary column is prepared. Then, polyethyleneimine and iodoacetic acid N-succinimide ester are sequentially modified by covalent bonding to prepare a solid-phase alkylated capillary microreactor. This microreactor is used for sample treatment of trace proteome in 1 - 100 cells. The specific treatment process is as follows: First, the cell lysate and cell suspension are sequentially introduced into the microreactor to form a "lysate - cell - lysate" sandwich structure. Then, the cells in the microreactor are lysed by ultrasonic disruption; meanwhile, under high-temperature conditions, the denaturation, reduction, and alkylation reactions of proteins are rapidly achieved, 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 enzymatic digestion is carried out at an appropriate temperature. The enzymatic digestion products can be directly subjected to liquid chromatography - mass spectrometry analysis. The advantages of the present invention are that proteome sample treatment is carried out in-situ, which is beneficial to reducing sample loss caused by multiple transfers in the traditional sample treatment process. Therefore, this solid-phase alkylated open tubular column is suitable for pre-treatment of trace samples of proteome. In addition, this method is tolerant to various surfactants and strong lysis reagents and has good sample compatibility.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] (1) A quartz capillary with an inner diameter of 100 - 200 μm and a polyacetimide coating is used. First, the capillary is activated to expose the hydroxyl groups on its inner wall, and the capillary is subjected to alkali washing, acid washing, and alcohol washing with sodium hydroxide, hydrochloric acid, and methanol in sequence. The concentrations of sodium hydroxide and hydrochloric acid are both 1 - 2 M.

[0007] (2) The above capillary is subjected to silanization treatment to introduce double bonds on its inner surface and then copolymerize with methacrylic acid monomers. The silanization reagent used is 3-(methacryloyloxy)propyltrimethoxysilane (γ-MAPS) or vinyltrimethoxysilane (VMTS), and the concentration volume ratio is 50% - 100%.

[0008] (3) Modify epoxy groups on the inner wall of the capillary through free radical polymerization reaction: Uniformly disperse the initiator, monomer, and porogen in the solution, degas, and introduce it into the silanized capillary. Initiate polymerization through the initiator, and react at 50 - 60 °C for 5 - 7 hours to form a coating structure on the inner surface of the capillary. The mass ratio of the initiator to the monomer in the polymerization solvent is 1:100 - 1:200, and the mass ratio of the monomer to the porogen is 1:5 - 1:20. The monomer used is an allyl monomer containing epoxy groups, such as glycidyl methacrylate (GMA); the initiators used for initiating the polymerization reaction include one of azobisisobutyronitrile (AIBN) and azobisisoheptonitrile (ABVN); the porogen includes one or more of n-propanol, butanediol, and n-dodecanol.

[0009] (4) Introduce the dendritic hydrophilic compound polyethyleneimine (PEI) on the surface of the open-tubular capillary column, and further modify iodoacetic acid-N-succinimide ester by covalent bonding. The specific reaction process is as follows: Pass the PEI solution into the open-tubular capillary column and react at 50 - 60 °C for 4 - 6 hours. After the reaction is completed, remove the residual PEI in the open-tubular column and wash it with water until neutral. Then dissolve iodoacetic acid-N-succinimide ester in a mixed system of methanol and phosphate buffer solution, add the open-tubular capillary column modified with PEI, and react at 25 - 40 °C for 24 hours (supplement it every 4 - 6 hours). Finally, rinse with methanol and dry it by passing nitrogen to obtain a microcellular in-situ microreactor. The final concentration of PEI is 10 - 100 mg / mL; the final concentration of the iodoacetic acid-N-succinimide ester solution is 10 - 100 mg / mL, and the volume ratio of the phosphate buffer solution to methanol is 1:5 - 1:20, where the concentration of the phosphate buffer solution is 0.01 - 0.1 M and the pH value is 7 - 9.

[0010] (5) Apply the above microcellular in-situ microreactor to the pretreatment of microcellular proteome samples. The specific process is as follows: Sequentially pass the lysis solution, cell solution, and lysis solution into the microreactor, and the volume of each is 50 - 200 nL. First, wash the cells three times with PBS buffer, and adjust the cell density to 10 4 -10 6cells / mL. The lysis buffer contains surfactants such as sodium dodecyl sulfate (SDS) and the reducing agent tris(2-carboxyethyl)phosphine (TCEP). The mass-volume ratio of SDS is 0.01%-0.5%, and the concentration of TCEP is 25-100 mM. The cells are completely lysed by ultrasonic treatment at 4°C for 5-15 minutes, and then reacted at 90-95°C in a water bath for 5-10 minutes to rapidly achieve protein denaturation, reduction, and alkylation. Then, it is washed with 50% methanol or acetonitrile to remove the residual lysis buffer and reducing agent in the microreactor and replaced with a weakly alkaline buffer system such as ammonium bicarbonate (ABC) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), with a concentration of 25-100 mM and a pH value of 7-9. Finally, a protease such as trypsin, Staphylococcus aureus V8 protease (Glu-C), lysyl endopeptidase (Lys-C), or more than one of them is introduced into the microreactor. The addition amount of the above protease is 10-100 ng, and enzymatic hydrolysis is carried out at 37°C for 6-12 hours. The enzymatic hydrolysis product is stored at -80°C or directly subjected to liquid chromatography-mass spectrometry analysis.

[0011] The present invention has the following advantages:

[0012] 1. A capillary open tubular column is prepared by radical polymerization reaction, and iodoacetic acid N-succinimidyl ester is modified on its surface. This preparation process is easy to control and has good repeatability;

[0013] 2. The prepared microscale in-situ cell microreactor is used for sample treatment, which can be compatible with various surfactants, improve the extraction efficiency of microscale cell proteins, avoid the loss of microscale samples caused by multi-step transfer, and improve the recovery rate of microscale sample treatment;

[0014] 3. The microscale in-situ cell microreactor can realize the simultaneous progress of multi-step reactions (including protein denaturation, reduction, and alkylation). Due to the confinement effect of the capillary open tubular column, reducing the reaction volume (nanoliter) is beneficial to improving the efficiency of sample treatment. Description of the Drawings

[0015] Figure 1 Schematic diagram of the preparation of the microscale in-situ cell microreactor;

[0016] Figure 2 Scanning electron microscope characterization diagram of the microscale in-situ cell microreactor;

[0017] Figure 3 Flow chart of in-situ protein pretreatment based on the microscale in-situ cell microreactor;

[0018] Figure 4 Mass spectrometry diagram obtained by proteomics analysis of 100 HeLa cells using the microscale in-situ cell microreactor;

[0019] Figure 5 Mass spectrometry graph obtained from proteomic analysis of 10 HeLa cells using a microfluidic in-situ microreactor;

[0020] Figure 6 Mass spectrometry graph obtained from proteomic analysis of 1 HeLa cell using a microfluidic in-situ microreactor;

[0021] Figure 7 Number of proteins and peptides identified by LC-MS / MS analysis of proteomics of 100 HeLa cells using a microfluidic in-situ microreactor at different reaction times Detailed implementation mode

[0022] Example 1

[0023] Preparation of a microfluidic in-situ microreactor

[0024] 1. First, cut a 3 m long quartz capillary with a polyimide coating (inner diameter 200 μm, outer diameter 360 μm), flush it with 1 M NaOH (8 μL / min) for 3 hours, and then flush it with water until neutral; then flush it with 1 M HCl (8 μL / min) for 2 hours, and flush it with water until neutral; finally, flush it with methanol (8 μL / min) for 2 hours, and blow it dry completely with nitrogen.

[0025] 2. Introduce a methanol solution of 3-(methacryloyloxy)propyltrimethoxysilane (concentration volume ratio 50%) into the above-activated capillary. After filling, plug both open ends of the capillary with rubber stoppers, place it in an oven at 50 °C and react for 24 hours. Then, flush it with methanol for 1 hour, and blow it dry completely with nitrogen.

[0026] 3. Mix and disperse azobisisobutyronitrile (AIBN), glycidyl methacrylate (GMA), and n-propanol (mass ratio 1:100:900) evenly, remove the oxygen in it by purging with nitrogen. Introduce the above polymerization solution into the silanized capillary. After filling, plug both open ends of the capillary with rubber stoppers, place it in a water bath at 60 °C and react for 5 hours to form a coating structure on the inner surface of the capillary. After the reaction is completed, flush it with methanol for 1 hour, and blow it dry completely with nitrogen.

[0027] 4. Introduce a polyethyleneimine (PEI) solution (10 mg / mL) into the open capillary column. After filling, plug both open ends of the capillary with rubber stoppers, place it in a water bath at 60 °C and react for 5 hours. After the reaction is completed, remove the residual PEI in the open column, flush it with water until neutral, and then flush it with methanol.

[0028] 5. Prepare an iodacetate N-succinimide ester (IAA-NHS) solution (10 mg / mL) under light protection. Weigh IAA-NHS (10 mg) and dissolve it in a mixed system of methanol (1 mL) and phosphate buffer solution (200 μL, pH = 8). Pass it into the open capillary column modified with PEI. After filling, plug both open ends of the capillary with rubber stoppers. React at 40 °C for 24 hours. Add the iodacetate N-succinimide ester solution (10 mg / mL) every 5 hours. After filling, plug both open ends of the capillary with rubber stoppers. Finally, rinse with methanol for 1 hour and blow dry with nitrogen to obtain a microcellular in-situ microreactor.

[0029] Example 2

[0030] The microcellular in-situ microreactor is used for the pretreatment of proteome samples of 100 HeLa cells

[0031] 1. Culture Hela cells in a 75 cm 2 culture flask. When the cell growth density reaches 80%-90% (about 5×10 6 cells), add 0.5 mL of trypsin and digest for 2 min, then collect the cells. Add 5 mL of PBS buffer solution and wash three times. Centrifuge for 5 min to collect the cell pellet. Resuspend the Hela cells in PBS buffer solution and finally adjust the cell density to 5×10 5 cells / mL.

[0032] 2. Prepare a cell lysate containing sodium dodecyl sulfate (mass-volume (mg / ml) ratio of 0.05%) and a reducing agent tris(2-carboxyethyl)phosphine (50 mM). Cut a 5-cm-long microcellular in-situ microreactor of Example 1. Pass 100 nL of lysate, 200 nL of cell solution (5×10 5 cells / mL), and 100 nL of lysate into the microreactor in sequence through a high-precision syringe pump (Harvard). The total volume is 400 nL. Avoid introducing air bubbles during the sampling process. Plug both ends of the microreactor with rubber stoppers.

[0033] 3. Place the above microreactor in a micro-sample ultrasonic crusher and ultrasonicate at 4 °C for 5 minutes to completely lyse the cells; then heat the sample in a water bath at 90 °C for 5 minutes to achieve rapid denaturation, reduction, and alkylation of proteins.

[0034] 4. Methanol with a volume concentration of 50% was introduced into the microreactor (50 μL) to wash away interfering substances such as residual surfactants, and then a 50 mM ammonium bicarbonate solution (50 μL, pH = 8) was introduced for washing; finally, a mixed solution of Trypsin and Lys-C (50 - 80 ng, mass ratio 1:1) was introduced, and both ends of the microreactor were plugged with rubber stoppers and placed in a water bath at 37 °C for enzymatic digestion for 12 hours. The enzymatic digestion products were stored at -80 °C or directly subjected to liquid chromatography - mass spectrometry analysis. As Figure 7 shown, the microreactor for in situ trace cells can achieve protein denaturation, reduction, and alkylation within 5 minutes, and 735 proteins can be identified from 100 HeLa cells.

[0035] Example 3

[0036] Pretreatment of proteome samples of 10 HeLa cells using the microreactor for in situ trace cells

[0037] 1. Hela cells were cultured in a 75 cm 2 culture flask. When the cell growth density reached 80% - 90% (about 5×10 6 cells), 0.5 mL of trypsin was added for digestion for 2 min, and then the cells were collected. Then, 5 mL of PBS buffer was added for washing three times, and the cells were collected by centrifugation for 5 min to obtain a cell pellet. The Hela cells were resuspended in PBS buffer, and finally the cell density was adjusted to 1×10 5 cells / mL.

[0038] 2. A cell lysate containing sodium dodecyl sulfate (mass - volume (mg / ml) ratio of 0.05%) and the reducing agent tris(2 - carboxyethyl)phosphine (50 mM) was prepared. A 5 - cm - long microreactor of Example 1 was intercepted, and 100 nL of lysate, 100 nL of cell solution (1×10 5 cells / mL), and 100 nL of lysate were successively introduced into the microreactor through a high - precision syringe pump (Harvard). The total volume was 300 nL. Bubbles were avoided during the sampling process. Both ends of the microreactor were plugged with rubber stoppers.

[0039] 3. The above - mentioned microreactor was placed in a micro - sample ultrasonic crusher, and ultrasonicated at 4 °C for 5 minutes to completely lyse the cells; then the sample was heated in a water bath at 90 °C for 5 minutes to achieve rapid protein denaturation, reduction, and alkylation.

[0040] 4. Methanol with a volume concentration of 50% was introduced into the microreactor (50 μL) to wash away residual interfering substances such as surfactants, and then 50 mM ammonium bicarbonate solution (50 μL, pH = 8) was introduced for washing; finally, a mixed solution of Trypsin and Lys-C (40 - 60 ng, mass ratio 1:1) was introduced. Both ends of the microreactor were sealed with rubber stoppers and placed in a water bath at 37 °C for enzymatic digestion for 12 hours. The enzymatic digestion product was directly subjected to liquid chromatography - mass spectrometry analysis, and the obtained mass spectrum was as shown in Figure 5 shown.

[0041] Example 4

[0042] Micro - cell in - situ microreactor for pretreatment of proteome samples of 1 HeLa cell

[0043] 1. Hela cells were cultured in a 75 cm 2 culture flask. When the cell growth density reached 80% - 90% (about 5×10 6 cells), 0.5 mL of trypsin was added for digestion for 2 min, and then the cells were collected. Then, 5 mL of PBS buffer was added for washing three times, and the cell pellet was collected by centrifugation for 5 min. The Hela cells were resuspended in PBS buffer, and finally the cell density was adjusted to 1×10 3 cells / mL.

[0044] 2. Prepare sodium dodecyl sulfate (mass - to - volume (mg / ml) ratio 0.05%) and the reducing agent tris(2 - carboxyethyl)phosphine (50 mM) as the cell lysis solution. Cut a 5 - cm - long micro - cell in - situ microreactor of Example 1. Through a high - precision syringe pump (Harvard), 50 nL of the lysis solution was introduced into the microreactor, then 1 Hela cell was aspirated under a microscope, and finally another 50 nL of the lysis solution was aspirated. The total volume was 120 - 300 nL. Bubbles were avoided during the sampling process. Both ends of the microreactor were sealed with rubber stoppers.

[0045] 3. The above - mentioned microreactor was placed in a micro - sample ultrasonic crusher and ultrasonically treated at 4 °C for 5 minutes to completely lyse the cells; then the sample was heated in a water bath at 90 °C for 5 minutes to achieve rapid protein denaturation, reduction, and alkylation.

[0046] 4. Methanol with a volume concentration of 50% was introduced into the microreactor (50 μL) to wash away residual interfering substances such as surfactants, and then 50 mM ammonium bicarbonate solution (50 μL, pH = 8) was introduced for washing; finally, a mixed solution of Trypsin and Lys-C (20 - 50 ng, mass ratio 1:1) was introduced. Both ends of the microreactor were sealed with rubber stoppers and placed in a water bath at 37 °C for enzymatic digestion for 12 hours. The enzymatic digestion product was directly subjected to liquid chromatography - mass spectrometry analysis, and the obtained mass spectrum was as shown in Figure 6 shown.

Claims

1. A method for preparing a microreactor for trace cells in situ, characterized in that: Before the radical polymerization reaction is carried out on the inner wall of the capillary, the capillary is first activated to expose the hydroxyl groups on its surface, and then silanization treatment is carried out to introduce double bonds on the inner surface of the capillary, and then copolymerize with methacrylic acid monomers. Epoxy groups are modified on the surface through radical polymerization reaction to prepare a capillary open tubular column; among them, the inner diameter of the capillary used is 100-200 μm; the monomer is glycidyl methacrylate, and the initiator is two or one of azobisisobutyronitrile and azodiisooctanenitrile. The porogen is one or more of n-propanol, butanediol, and n-dodecanol. The mass ratio of the initiator to the monomer is 1:100-1:200, and the mass ratio of the monomer to the porogen is 1:5-1:

20. The reaction temperature is 50-60 °C, and the reaction time is 5-7 hours; then polyethyleneimine and iodoacetic acid-N-succinimide ester are sequentially modified by covalent bonding to prepare a solid-phase alkylated capillary microreactor; through the selective reaction of the sulfhydryl group of the protein, the protein is fixed in the solid-phase alkylated capillary microreactor, thereby performing in-situ pretreatment of trace protein samples; the microreactor for trace cells in-situ is used for processing protein samples of 1-100 cells.

2. The preparation method of the microcellular in-situ microreactor according to claim 1, characterized in that: In the capillary activation process, the capillary is sequentially washed with sodium hydroxide, hydrochloric acid and methanol for alkali washing, acid washing and alcohol washing. The concentrations of sodium hydroxide and hydrochloric acid are both 1-2 M; the types of silanization reagents used are two or one of 3-(methacryloyloxy)propyltrimethoxysilane or vinyltrimethoxysilane, and the concentration volume ratio is 50%-100%.

3. The preparation method of the micro cell in-situ microreactor according to claim 1, wherein: After the inner surface of the microreactor is modified with epoxy groups, dendritic hydrophilic compound polyethyleneimine is further introduced, and then iodoacetic acid-N-succinimide ester is modified by covalent bonding. Among them, the final concentration of the polyethyleneimine solution used is 10-100 mg / mL; iodoacetic acid-N-succinimide ester is dissolved in a mixed system of methanol and phosphate buffer solution, and the volume ratio of phosphate buffer solution to methanol is 1:5-1:20; the final concentration of iodoacetic acid-N-succinimide ester is 10-100 mg / mL, the concentration of phosphate buffer solution is 0.01-0.1 M, and the pH value is 7-9; the reaction temperature for modifying iodoacetic acid-N-succinimide ester is 25-40 °C, and the reaction time is 12-24 hours.

4. A microreactor for trace cells in-situ obtained by the preparation method according to any one of claims 1-3.

5. Use of the microcellular in-situ microreactor according to claim 4, characterized in that: Applied to the pretreatment of one or more protein samples in trace cells, body fluids, and tissues.

6. The application of the micro cell in-situ microreactor according to claim 5, characterized in that: The lysis solution and the cell suspension are sequentially introduced into the microreactor to form a "lysis solution-cell-lysis solution" sandwich structure; then, the cells in the microreactor are lysed by ultrasonic disruption; at the same time, protein denaturation, reduction and alkylation reactions are quickly realized at 90-95 °C for 5-10 minutes, so that the protein is covalently fixed on the inner surface of the microreactor; finally, the microreactor is washed with one or more solvents of methanol, acetonitrile and ammonium bicarbonate to remove surfactant, lipid and sugar interfering substances; protease is added for enzymatic hydrolysis; the enzymatic hydrolysis product is directly subjected to liquid chromatography-mass spectrometry analysis.

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