Peroxisome separation and enrichment method based on micro-fluidic chip

Abstract

The invention discloses a peroxisome separation and enrichment method based on a micro-fluidic chip, and belongs to the technical field of organelle separation.The micro-fluidic chip is provided with a micro-channel comprising a cracking module, an incubation module and a sorting module which are sequentially communicated, and the cracking module is provided with a plurality of necks; the cell suspension enters the lysis module, is split when flowing through the necking, and is mixed with the suspension of the separation system in the incubation module, the surface of the magnetic bead is combined with an antigen, an antibody or a secondary antibody, the antigen, the antibody or the secondary antibody can generate specific immunoreaction with substances in the cell lysis solution, and then the magnetic bead and the substances combined on the surface of the magnetic bead are separated through a magnet in the separation module; and competitive elution is carried out on the magnetic beads through biotin, so that the magnetic beads are separated from substances in the cell lysis buffer, and separation and enrichment of the peroxisome are realized. The method is simple, rapid, high in accuracy and sensitivity, strong in specificity and small in reagent consumption, and can be used for separating a small amount of cell peroxisome.

Description

Technical Field

[0001] The invention belongs to the technical field of organelle separation, and particularly relates to a peroxisome separation and enrichment method based on a microfluidic chip. Background Art

[0002] Peroxisomes were first discovered by J. Rhodin (1954) in rat renal tubular epithelial cells. They are heterogeneous organelles that vary in different organisms and developmental stages. They are about 0.2-1.5 μm in diameter, usually 0.5 μm, and are round, oval or dummy-shaped, surrounded by a single membrane. They contain one or more flavin-dependent oxidases and catalases (marker enzymes). More than 40 oxidases have been discovered, such as L-amino acid oxidase, D-amino acid oxidase, etc. The content of urate oxidase is so high that it forms the core of enzyme crystals in some species. In scientific research, the study of peroxisomes and their trace substances usually requires separation from the cytoplasm for detailed study. At present, the main principle of peroxisome separation technology is to achieve it through ultracentrifugation.

[0003] Although the existing separation method based on ultracentrifugation can separate most peroxisomes, it requires a large number of cells and cell membrane disruption before ultracentrifugation, which is time-consuming and has low precision. The peroxisome separation kit has complicated steps and is relatively expensive. Summary of the invention

[0004] The purpose of the present invention is to provide a peroxisome separation and enrichment method based on a microfluidic chip. The method is simple, rapid, highly accurate and sensitive, highly specific, and consumes little reagent, and can be used to separate a small amount of cell peroxisomes.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: A microfluidic chip comprises a chip body, wherein the chip body is provided with a microfluidic channel, and the microfluidic channel comprises a lysis module, an incubation module and a sorting module which are connected in sequence; The lysis module is provided with a first inlet for the cell suspension to enter; the lysis module is provided with a first flow channel for connecting the first inlet with the incubation module, and a plurality of constrictions are provided inside the first flow channel; The starting end of the incubation module is provided with a second inlet for injecting magnetic beads; the surface of the magnetic beads is coated with antibodies, antigens or secondary antibodies; The sorting module is provided with a first outlet and a second outlet for discharging liquid; the sorting module is provided with a magnet for adsorbing magnetic beads, and the magnet is arranged on a side of the sorting module away from the first outlet.

[0006] Furthermore, the first outlet and the second outlet are distributed in a Y shape.

[0007] Furthermore, a plurality of constrictions are arranged in an array in the first flow channel, and the size of the constriction near the first inlet is larger than the size of the constriction near the incubation module.

[0008] Furthermore, a plurality of first flow channels are arranged in parallel.

[0009] Furthermore, the incubation module includes a second flow channel, and the second flow channel is bent.

[0010] Preferably, the chip body includes a bottom plate and a cover plate, the bottom plate is a glass slide, the cover plate is a PDMS layer, and the microchannel is arranged inside the cover plate; the first inlet, the second inlet, the first outlet, and the second outlet are all connected to the outside.

[0011] Preferably, a plurality of first flow channels are arranged in parallel in the lysis module, the size of the internal constrictions of the first flow channels ranges from 1.5 to 30 μm, and the sizes of the constrictions arranged along the flow direction of the cell suspension gradually decrease.

[0012] The peroxisome separation and enrichment method based on a microfluidic chip adopts the above-mentioned microfluidic chip and comprises the following steps: S1. Sample addition: the cell suspension enters the microfluidic channel through the first inlet, and the cells are lysed when the cell suspension flows through several constrictions in the first flow channel; the separation system suspension enters the microfluidic channel through the second inlet, and the separation system suspension contains magnetic beads coated with antigens, antibodies or secondary antibodies; S2. Reaction: The cell lysate entering the microchannel from the first flow channel and the separation system suspension entering the microchannel from the second inlet merge in the incubation module to form a mixed solution, and the antigen, antibody or secondary antibody on the surface of the magnetic beads reacts specifically with the substance in the cell lysate; S3. Sorting: The mixed solution enters the sorting module, and the magnetic beads and the binding substances attached to the surface thereof flow out through the second outlet under the action of the magnet, and the remaining components in the mixed solution flow out through the first outlet; S4. Elution: Collect the magnetic beads obtained from the second outlet and use biotin to competitively elute the magnetic beads to separate the magnetic beads from the substances in the cell lysate.

[0013] Preferably, in step S1, the magnetic beads are superparamagnetic particles made of ferric oxide and / or ferrosoferric oxide; the particle size of the magnetic beads is 30-150 μm.

[0014] Preferably, the surface of the magnetic beads is bound to PMP70 antibodies.

[0015] Preferably, the preparation of magnetic beads with PMP70 antibodies bound to the surface comprises the following steps: Strep-tag II binds to anti-PMP70 antibody, and then to PMP70 antibody to form a Strep-tag II-anti-antibody-antibody complex; Strep-Tactin coated magnetic beads were used as solid substrates to undergo protein coupling reaction with Strep-tag II-antibody-antibody complexes to obtain magnetic beads with PMP70 antibodies bound to their surfaces.

[0016] Preferably, in step S1, the cell density in the cell suspension is 1×10 6 -1×10 8 cells / mL.

[0017] Preferably, in step S1, the separation system suspension includes buffer solution I, the concentration range of magnetic beads in the separation system suspension is 5-20% (v / v), the buffer solution I uses PBS as a solvent and contains PMSF 0.5-2mM, DTT 0.5-2mM, and EDTA 3-8mM; The amount of suspension in the separation system is 1×10 7 cells / 1-10ml.

[0018] Preferably, in step S3, the concentration of biotin is 1-2.5 mM; and the volume ratio of the separation system suspension to biotin is 1:1-5.

[0019] According to one aspect of the present invention, a buffer solution is provided, which uses PBS as a solvent and contains PMSF.

[0020] Preferably, in the above buffer solution, the concentration of PMSF is 0.5-2 mM.

[0021] Preferably, the buffer solution contains DTT and EDTA.

[0022] Preferably, in the above solution, the concentration of DTT is 0.5-2 mM, and the concentration of EDTA is 3-8 mM.

[0023] Compared with the prior art, the present invention has the following beneficial effects: 1. The microfluidic chip is used to separate and enrich intracellular peroxisomes. The method is simple to operate and has rapid separation. In addition, the magnetic beads with PMP70 antibodies bound to the surface are used to separate and enrich peroxisomes through the binding of PMP70 antibodies to antigens on the peroxisome membrane. The method has high specificity and can ensure accuracy and sensitivity in the peroxisome separation process.

[0024] 2. Using buffer solution I to treat the magnetic beads helps maintain the integrity of the peroxidase enzyme and reduce the degree of damage to the peroxidase enzyme, thereby ensuring its subsequent binding effect with the PMP70 antibody and improving the separation and enrichment effect.

[0025] 3. The microfluidic chip is used to separate and enrich intracellular peroxisomes. It is suitable for the separation of a small amount of cell peroxisomes, with low reagent consumption. In addition, the chip can achieve cell lysis through the shearing effect of the shrinkage, and can also achieve the purpose of sterile separation. It has potential application value for subsequent research on sterile requirements. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 This is a schematic diagram of the structure of a microfluidic chip according to Example 1 of the present invention; Figure 2 for Figure 1 Schematic diagram of the partially enlarged structure of part A in the middle.

[0026] Reference numerals: lysis module 10; first flow channel 11; constriction 12; first inlet 13; second inlet 14; incubation module 20; second flow channel 21; sorting module 30; first outlet 31; second outlet 32; magnet 33. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0028] Example 1 Microfluidic Chip Reference Figure 1-2 , this embodiment provides a microfluidic chip, including a chip body, the chip body is provided with a microfluidic channel, the microfluidic channel includes a lysis module 10, an incubation module 20 and a sorting module 30 connected in sequence; The lysis module 10 is provided with a first inlet 13 for the cell suspension to enter; the lysis module 10 is provided with a first flow channel 11 for connecting the first inlet 13 with the incubation module 20, and a plurality of constrictions 12 are provided inside the first flow channel 11; The starting end of the incubation module 20 is provided with a second inlet 14 for injecting magnetic beads; the surface of the magnetic beads is coated with antibodies, antigens or secondary antibodies; The sorting module 30 is provided with a first outlet 31 and a second outlet 32 ​​for discharging liquid; the sorting module 30 is provided with a magnet 33 for adsorbing magnetic beads, and the magnet 33 is arranged on a side of the sorting module 30 away from the first outlet 31 .

[0029] Furthermore, the first outlet 31 and the second outlet 32 ​​are distributed in a Y shape.

[0030] Furthermore, a plurality of constrictions are arranged in an array in the first flow channel 11 , and the size of the constriction 12 close to the first inlet 13 is larger than the size of the constriction 12 close to the incubation module 20 .

[0031] Furthermore, a plurality of first flow channels 11 are arranged in parallel.

[0032] Furthermore, the incubation module 20 includes a second flow channel 21, and the second flow channel 21 is bent.

[0033] Specifically, the chip body is 123 mm long and 51 mm wide; it includes a base plate and a cover plate, the base plate is a glass slide with a thickness of 1 mm, the cover plate is a PDMS layer with a thickness of 4 mm, and the microchannel is arranged inside the cover plate; the first inlet 13, the second inlet 14, the first outlet 31, and the second outlet 32 ​​are all connected to the outside.

[0034] Specifically, two first flow channels 11 symmetrically arranged in the upper and lower parts are provided in the lysis module 10, and the interior of the first flow channel 11 includes 30 constrictions 12 arranged in an array along the flow direction of the cell suspension; the constrictions 12 arranged in sequence along the flow direction of the cell suspension form a unit of every 10 constrictions 12; among them, the size of the 10 constrictions 12 close to the first inlet 13 is 20 μm, the size of the 10 constrictions 12 close to the incubation module 20 is 5 μm, and the size of the 10 constrictions 12 located between the two parts of the array is 10 μm.

[0035] Example 2 Microfluidic Chip The microfluidic chip provided in this embodiment is different from that in Embodiment 1 in that: The chip body is 120 mm long and 50 mm wide; the bottom plate is a 2 mm thick glass slide, and the cover plate is a 6 mm thick PDMS layer.

[0036] The interior of the first flow channel 11 includes 48 constrictions 12 arranged in an array along the flow direction of the cell suspension; wherein, the constrictions 12 arranged in sequence along the flow direction of the cell suspension form a unit of every 8 constrictions 12; and the sizes of the 6 constriction units arranged in sequence along the flow direction of the cell suspension are 30μm, 15μm, 8μm, 4μm, 2μm, and 1.5μm, respectively.

[0037] Example 3 Preparation of magnetic beads with PMP70 antibody bound to the surface The use of magnetic beads to capture peroxisomes in cells is based on the double antibody sandwich method in non-competitive binding assays, as well as the competitive method for determining antibodies or antigens. Generally, this method includes the following steps: 1) Connecting specific antibodies to solid phase carriers to form solid phase antibodies, and washing away unbound antibodies and impurities. 2) Adding the sample to be tested and incubating for reaction. The antigen in the specimen binds to the solid phase antibody to form a solid phase antigen-antibody complex, and other unbound substances are washed away. 3) Adding enzyme-labeled antibodies and incubating for reaction. The antigen on the solid phase antigen-antibody complex binds to the enzyme-labeled antibody, and the unbound enzyme-labeled antibody is washed away. At this time, the amount of enzyme carried on the solid phase carrier is related to the amount of the antigen to be tested in the specimen. 4) Adding substrate for color development. The enzyme on the solid phase catalyzes the substrate into a colored product, and the amount of antigen in the specimen is measured by colorimetry.

[0038] In this example, Strep-Tag II protein purification magnetic beads purchased from Beaver Nanotechnology (Suzhou) Co., Ltd. were used. The suspension concentration was 10% (v / v) magnetic bead suspension, the magnetic bead particle size was 30~150 μm, the ligand content was: ~6 mg Strep-Tactin / mLGel; the fusion protein binding amount was: ~7 mg Strep-tag II protein / mL Gel. The specific steps include: S(1): Take 1 mL of the above magnetic bead suspension, add 1 mL of 1:1000 diluted IgG, where IgG is mouse anti-PMP70 antibody, and the diluent is buffer solution I (buffer solution I uses PBS as solvent and contains 1 mM PMSF, 1 mM DTT, and 5 mM EDTA); vortex mixer at 150 r / min for 15 s to resuspend the magnetic beads, and incubate at 37°C for 30 min to allow the Strep-tag II protein to bind to the IgG.

[0039] Then, the above mixture was placed on a magnetic separator, and after the solution was clarified, the supernatant was removed, and then 2 mL of buffer solution I was added, and mixed on a vortex mixer at 150 r / min for 1 min. The above mixture was placed on a magnetic separator, and after the solution was clarified, the supernatant was removed. Wash twice in this way to remove unbound IgG.

[0040] S(2): Add 1 mL of 1:1000 diluted PMP70 antibody to the magnetic beads obtained in S(1), wherein the PMP70 antibody is from mouse and the diluent is buffer solution I; vortex on a vortex mixer at 150 rpm for 15 s to resuspend the magnetic beads, and incubate at 37°C for 30 min to allow the PMP70 antibody to be directionally linked to the Fab domain of IgG, thereby forming a magnetic bead-antibody-antibody complex with the PMP70 antibody bound to the surface.

[0041] Then, the mixed solution was placed on a magnetic separator, and after the solution was clarified, the supernatant was removed, and then 2 mL of buffer solution I was added, and the mixture was mixed on a vortex mixer at 150 r / min for 1 min, and then the mixed solution was placed on a magnetic separator, and after the solution was clarified, the supernatant was removed, and the unbound PMP70 antibody was removed by washing twice. Then, 2 mL of buffer solution I was added, and the mixture was mixed on a vortex mixer at 150 r / min for 10 min to obtain a magnetic bead complex suspension with PMP70 antibody bound to the surface.

[0042] Example 4 Peroxisome Isolation Methods The microfluidic chip in Example 1 is used to sort peroxisomes in HepG2 cells; the steps include: S1. Preparation of cell suspension: HepG2 cells purchased from Guangzhou Aidi Gene Technology Co., Ltd. were recovered and subcultured, and then digested to a single cell suspension in a clean bench. After mixing evenly by pipetting, the cells were counted. Then, the cells were centrifuged at 1100 rpm and 25°C for 4 min, the supernatant was discarded, and the cells were resuspended in 1-2 mL of 4°C pre-cooled cryopreservation solution, and then the cryopreservation solution was added to adjust the density to 1×10 7 Prepare a cell suspension of 10 cells / mL for later use.

[0043] S2. Sample addition: Use an electronic pump to pump the cell suspension obtained in S1 into the microfluidic chip through the first inlet 13 at a flow rate of 1 mL / min and an injection volume of 2 mL; pump the separation system suspension into the microfluidic chip through the second inlet 14 at a flow rate of 1 mL / min and an injection volume of 2 mL.

[0044] The separation system suspension is a magnetic bead complex suspension configured in Example 3 and having PMP70 antibodies bound to its surface, wherein the magnetic beads have PMP70 antibodies bound to their surfaces.

[0045] After the HepG2 cell suspension enters the lysis module 10 through the first inlet 13, it is ruptured under the action of shear force when passing through the constriction 12 inside the first flow channel 11, thereby releasing the peroxisomes therein.

[0046] S3. Reaction: The cell lysate entering the microchannel from the first flow channel 11 and the separation system suspension entering the microchannel from the second inlet 14 merge in the incubation module 20 to form a mixed solution. The peroxisomes in the cell lysate can be strongly bound to the PMP70 antibodies on the surface of the magnetic beads in the separation system suspension through the antigens on the peroxisome membrane, thereby binding to the surface of the magnetic beads.

[0047] S4. Sorting: The mixed solution in S3 enters the sorting module 30 , and the magnetic beads and the binding substances attached to the surface thereof flow out through the second outlet 32 ​​under the action of the magnet 33 , and the remaining components in the mixed solution flow out through the first outlet 31 .

[0048] S5. Elution: collect the mixed solution from the second outlet 32, place the mixed solution on a magnetic separator, remove the supernatant after the solution is clarified, then add 2 mL of buffer solution I, mix on a vortex mixer at 150 r / min for 1 min, place the mixed solution on a magnetic separator, remove the supernatant after the solution is clarified, wash twice, add 2.5 mM biotin, and the volume ratio of the separation system suspension to the biotin solution is 1:5; mix on a vortex mixer at 150 r / min for 10 min to perform competitive elution; then place the mixed solution on a magnetic separator, perform magnetic separation, collect the supernatant, and achieve peroxisome enrichment.

[0049] In this case, a microfluidic chip was used to separate and enrich peroxisomes using magnetic beads with PMP70 antibodies bound to the surface. The operation process is simple, no ultracentrifugation treatment is required, and it has strong specificity and convenience. It is suitable for cell peroxisome separation and can specifically separate small amounts of cells. The purpose of sterile separation can also be achieved in the chip, which has potential application value for subsequent research on sterile requirements.

[0050] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.

[0051] The above-described embodiments provide a detailed description of the technical solution of the present invention. It should be understood that the above-described embodiments are only specific embodiments of the present invention and are not intended to limit the present invention. Any person skilled in the art who is familiar with the present technical field can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A microfluidic chip, characterized in that: It comprises a chip body, the chip body is provided with a microfluidic channel, the microfluidic channel comprises a lysis module (10), an incubation module (20) and a sorting module (30) which are connected in sequence; The lysis module (10) is provided with a first inlet (13) for the inlet of a cell suspension; the lysis module (10) is provided with a first flow channel (11) for connecting the first inlet (13) and the incubation module (20); a plurality of constrictions (12) are provided inside the first flow channel (11); The starting end of the incubation module (20) is provided with a second inlet (14) for injecting magnetic bead particles; The sorting module (30) is provided with a first outlet (31) and a second outlet (32) for discharging liquid; the sorting module (30) is provided with a magnet (33) for adsorbing magnetic beads, and the magnet (33) is arranged on a side of the sorting module (30) away from the first outlet (31); The size of the constriction (12) ranges from 1.5 to 30 μm, and the sizes of the plurality of constrictions (12) arranged along the flow direction of the cell suspension gradually decrease.

2. A method for separation and enrichment of peroxisomes based on a microfluidic chip, characterized in that: The microfluidic chip according to claim 1 comprises the following steps: S1. Sample addition: the cell suspension enters the lysis module (10) through the first inlet (13), and the cells are lysed when the cell suspension flows through the plurality of constrictions (12) in the first flow channel (11); the separation system suspension enters the microchannel through the second inlet (14), and the separation system suspension contains magnetic beads coated with antigens, antibodies or secondary antibodies; S2. Reaction: The cell lysate entering the microchannel from the first flow channel (11) and the separation system suspension entering the microchannel from the second inlet (14) merge in the incubation module (20) to form a mixed solution, and the antigen, antibody or secondary antibody on the surface of the magnetic beads reacts specifically with the substance in the cell lysate; S3. Sorting: The mixed solution enters the sorting module (30), and the magnetic beads and the binding substances attached to the surface thereof flow out through the second outlet (32) under the action of the magnet (33), and the remaining components in the mixed solution flow out through the first outlet (31); S4. Elution: Collect the magnetic beads obtained from the second outlet (32), and use biotin to competitively elute the magnetic beads therein to separate the magnetic beads from the substances in the cell lysate.

3. The method for separation and enrichment of peroxisomes based on a microfluidic chip according to claim 2, characterized in that: In step S1, the magnetic beads are superparamagnetic particles made of ferric oxide and / or ferrosoferric oxide; the particle size of the magnetic beads is 30-150 μm.

4. The method for separation and enrichment of peroxisomes based on a microfluidic chip according to claim 2, characterized in that: The surface of the magnetic beads is bound to PMP70 antibodies.

5. The method for separation and enrichment of peroxisomes based on a microfluidic chip according to claim 4, characterized in that: The preparation of magnetic beads with PMP70 antibody bound to the surface comprises the following steps: Strep-tag II binds to anti-PMP70 antibody, and then to PMP70 antibody to form a Strep-tag II-anti-antibody-antibody complex; Strep-Tactin coated magnetic beads were used as solid substrates to undergo protein coupling reaction with Strep-tag II-antibody-antibody complexes to obtain magnetic beads with PMP70 antibodies bound to their surfaces.

6. The method for separation and enrichment of peroxisomes based on a microfluidic chip according to claim 2, characterized in that: In step S1, the cell density in the cell suspension is 1×10 6 -1×10 8 cells / mL.

7. The method for separation and enrichment of peroxisomes based on a microfluidic chip according to claim 2, characterized in that: In step S1, the separation system suspension includes a buffer solution I, the concentration of magnetic beads in the separation system suspension ranges from 5% to 20% (v / v), and the buffer solution I uses PBS as a solvent and contains PMSF; The amount of the separation system suspension is 1×10 7 cells / 1-10ml.

8. The method for separation and enrichment of peroxisomes based on a microfluidic chip according to claim 7, characterized in that: In the buffer solution I, the concentration of PMSF is 0.5-2 mM.

9. The method for separation and enrichment of peroxisomes based on a microfluidic chip according to claim 7, characterized in that: The buffer solution I also contains 0.5-2 mM DTT and 3-8 mM EDTA.

10. The method for separation and enrichment of peroxisomes based on a microfluidic chip according to claim 7, characterized in that: In step S3, the concentration of biotin is 1-2.5 mM; the volume ratio of the separation system suspension to biotin is 1:1-5.

Images