A microfluidic chip-based peroxisome separation and enrichment method

Abstract

The application 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-fluid channel comprising a lysis module, an incubation module and a separation module which are sequentially connected. The lysis module is provided with a plurality of constrictions. Cell suspension enters the lysis module, is lysed when flowing through the constrictions, and then is mixed with a suspension system in the incubation module. The surface of a magnetic bead is combined with an antigen, an antibody or a secondary antibody, and can specifically react with substances in a cell lysate. Then, the magnetic bead and the substances combined on the surface of the magnetic bead are separated in the separation module through a magnet. The magnetic bead in the cell lysate is competitively eluted through biotin, the magnetic bead is separated from the substances in the cell lysate, and the separation and enrichment of peroxisomes are realized. The method is simple, fast, accurate, sensitive, specific, and has a small reagent consumption, and can be used for the separation of peroxisomes of a small amount of cells.

Description

TECHNICAL FIELD

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

[0002] Peroxysomes were first discovered by J. Rhodin in 1954 in mouse kidney tubular epithelial cells, are a kind of organelles with heterogeneity, and are different in different organisms and different development stages; the diameter is about 0.2-1.5 microns, usually 0.5 microns, and is round, oval or dumbbell-shaped, and is surrounded by a single layer of membrane; the common feature is that one or more flavin-dependent oxidases and hydrogen peroxide enzymes (marker enzymes) are contained, and more than 40 kinds of oxidases have been found, such as L-amino acid oxidase, D-amino acid oxidase and the like, among which the content of uric acid oxidase is extremely high, so that in some species, enzyme crystals form a core. In scientific research, the peroxisome and the trace substances therein are usually separated from the cytoplasm for detailed research, and the current peroxisome separation technology mainly realizes separation through ultracentrifugation.

[0003] The existing separation method based on ultracentrifugation can separate most peroxisomes, but a large amount of cells and cell membrane crushing before ultracentrifugation are needed, the time consumption is long, and the precision is not high; the peroxisome separation kit has a complicated procedure and a high price. SUMMARY

[0004] The application aims to provide a peroxisome separation and enrichment method based on a microfluidic chip, which is simple, fast, high in accuracy and sensitivity, strong in specificity, small in reagent consumption, and can be used for separation of peroxisomes of a small amount of cells.

[0005] The technical scheme adopted by the application to achieve the above-mentioned purpose is as follows:

[0006] A microfluidic chip comprises a chip main body, and the chip main body is provided with a microfluid channel, and the microfluid channel comprises a lysis module, an incubation module and a sorting module which are sequentially connected;

[0007] The lysis module is provided with a first inlet for cell suspension liquid inlet; the lysis module is provided with a first flow channel for connecting the first inlet and the incubation module, and the inside of the first flow channel is provided with a plurality of constrictions;

[0008] The starting end of the incubation module is provided with a second inlet for sample injection of magnetic bead particles; the surface of the magnetic bead is coated with an antibody, an antigen or a secondary antibody;

[0009] The sorting module is configured with a first outlet and a second outlet for liquid outlet; the sorting module is configured with a magnet for adsorbing magnetic beads, and the magnet is arranged on the side of the sorting module away from the first outlet.

[0010] Further, the first outlet and the second outlet are distributed in a Y shape.

[0011] Further, a plurality of necked arrays are arranged in the first flow channel, and the size of the necked array close to the first inlet is larger than that close to the incubation module.

[0012] Further, a plurality of first flow channels are arranged side by side.

[0013] Further, the incubation module comprises a second flow channel, and the second flow channel is arranged in a curve.

[0014] Preferably, the chip body comprises a bottom plate and a cover plate, the bottom plate carries a glass slide, the cover plate is a PDMS layer, and the micro flow channel is arranged inside the cover plate; the first inlet, the second inlet, the first outlet and the second outlet are all in communication with the outside.

[0015] Preferably, a plurality of first flow channels are arranged side by side in the lysis module, the size of the necked array inside the first flow channel ranges from 1.5 to 30 μm, and the size of the necked array arranged along the flow direction of the cell suspension gradually decreases.

[0016] The peroxisome separation and enrichment method based on the microfluidic chip adopts the above-mentioned microfluidic chip, comprising the following steps:

[0017] S1. Sample loading: the cell suspension enters the micro flow channel through the first inlet, and the cells are lysed when the cell suspension flows through the necked arrays in the first flow channel; the separation system suspension enters the micro flow channel through the second inlet, and the magnetic beads coated with antigens, antibodies or secondary antibodies are contained in the separation system suspension;

[0018] S2. Reaction: the cell lysate from the first flow channel and the separation system suspension from the second inlet are mixed in the incubation module to form a mixed solution, and the antigens, antibodies or secondary antibodies on the surface of the magnetic beads specifically react with the substances in the cell lysate;

[0019] S3. Sorting: the mixed solution enters the sorting module, 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;

[0020] S4. Elution: the magnetic beads obtained from the second outlet are collected, and the magnetic beads are competitively eluted by biotin to separate the magnetic beads from the substances in the cell lysate.

[0021] Preferably, in step S1, the magnetic beads are superparamagnetic particles, and the material is ferroferric oxide and / or magnetite; the particle size of the magnetic beads is 30-150 μm.

[0022] Preferably, the magnetic beads have PMP70 antibodies bound to their surfaces.

[0023] Preferably, the preparation of the magnetic beads having PMP70 antibodies bound to their surfaces comprises the following steps:

[0024] The Strep-tag II binds to the anti-PMP70 antibody, and then the anti-PMP70 antibody binds to the Strep-tag II, forming a Strep-tag II-anti-antibody-antibody complex.

[0025] The Strep-tag II-anti-antibody-antibody complex is subjected to a protein coupling reaction with the coated Strep-Tactin magnetic beads as a solid substrate, and the magnetic beads having PMP70 antibodies bound to their surfaces are obtained.

[0026] Preferably, in step S1, the cell density in the cell suspension is 1×10 6 -1×10 8 cells / mL.

[0027] Preferably, in step S1, the separation system suspension comprises buffer solution I, and the concentration of the magnetic beads in the separation system suspension ranges from 5% to 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.

[0028] The amount of the separation system suspension is 1×10 7 cells / 1-10ml.

[0029] Preferably, in step S3, the concentration of biotin is 1-2.5mM, and the volume ratio of the separation system suspension to biotin is 1:1-5.

[0030] According to an aspect of the present application, a buffer solution is provided, which uses PBS as a solvent and contains PMSF.

[0031] Preferably, in the above buffer solution, the concentration of PMSF is 0.5-2mM.

[0032] Preferably, the above buffer solution contains DTT and EDTA.

[0033] Preferably, in the above solution, the concentration of DTT is 0.5-2mM, and the concentration of EDTA is 3-8mM.

[0034] Compared with the prior art, the present application has the following beneficial effects:

[0035] 1. The microfluidic chip is used to separate and enrich peroxisomes in cells, and the method is simple to operate and fast to separate, and through the use of magnetic beads with PMP70 antibody on the surface, the separation and enrichment of peroxisomes are realized by the combination of PMP70 antibody and the antigen on the peroxisome membrane, which is high in specificity and can ensure the accuracy and sensitivity of the peroxisome separation process.

[0036] 2. The buffer solution I is used to treat the magnetic beads, which helps to maintain the integrity of peroxisomes and reduce the damage degree of peroxisomes, so as to ensure the binding effect of peroxisomes with PMP70 antibody and improve the separation and enrichment effect.

[0037] 3. The microfluidic chip is used to separate and enrich peroxisomes in cells, which is suitable for the separation of peroxisomes in a small amount of cells, and the reagent consumption is small, and the shearing effect of the neck in the chip can also achieve the purpose of sterile separation, which has potential application value for subsequent sterile research. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 It is a structure schematic view of the microfluidic chip according to the embodiment 1 of the present application.

[0039] Figure 2 It is a structure schematic view of the microfluidic chip according to the embodiment 1 of the present application. Figure 1

[0040] The drawing reference: lysis module 10; first flow channel 11; neck 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

[0041] The technical solutions of the present application will be further described in combination with the specific embodiments and the drawings. It should be understood that the specific embodiments described herein are only used to explain the present application, and are not used to limit the present application.

[0042] Embodiment 1

[0043] Microfluidic chip

[0044] Referring to Figures 1-2 , the embodiment provides a microfluidic chip, which comprises a chip main body, and the chip main body is provided with a micro flow channel, and the micro flow channel comprises a lysis module 10, an incubation module 20 and a sorting module 30 which are sequentially communicated.

[0045] The lysis module 10 is provided with a first inlet 13 for cell suspension liquid; the lysis module 10 is provided with a first flow channel 11 for connecting the first inlet 13 and the incubation module 20, and the inside of the first flow channel 11 is provided with a plurality of necks 12.​

[0046] The starting end of the incubation module 20 is configured with a second inlet 14 for sample injection of magnetic bead particles; the magnetic bead surface of the magnetic beads is coated with antibodies, antigens or secondary antibodies;

[0047] The sorting module 30 is configured with a first outlet 31 and a second outlet 32 for liquid outlet; the sorting module 30 is configured with a magnet 33 for adsorbing magnetic beads, and the magnet 33 is arranged on the side of the sorting module 30 away from the first outlet 31.

[0048] Further, the first outlet 31 and the second outlet 32 are distributed in a Y shape.

[0049] Further, a plurality of necked arrays are arranged in the first flow channel 11, and the size of the neck 12 close to the first inlet 13 is larger than the size of the neck 12 close to the incubation module 20.

[0050] Further, a plurality of first flow channels 11 are arranged side by side.

[0051] Further, the incubation module 20 comprises a second flow channel 21, and the second flow channel 21 is arranged in a curved manner.

[0052] Specifically, the chip main body is 123 mm long and 51 mm wide; the chip main body comprises a bottom plate and a cover plate, the bottom 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 micro flow channel 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 in communication with the outside.

[0053] Specifically, the lysis module 10 is provided with two first flow channels 11 which are symmetrical upward and downward, and the inside of the first flow channel 11 comprises 30 necked arrays 12 arranged along the flow direction of the cell suspension; every 10 necked arrays 12 arranged along the flow direction of the cell suspension is a unit; wherein the size of the 10 necked arrays 12 close to the first inlet 13 is 20 μm, the size of the 10 necked arrays 12 close to the incubation module 20 is 5 μm, and the size of the 10 necked arrays 12 between the two arrays is 10 μm.

[0054] Embodiment 2

[0055] Microfluidic chip

[0056] The microfluidic chip provided in the embodiment is different from that in Embodiment 1 in that:

[0057] The chip main body is 120 mm long and 50 mm wide; the bottom plate is a glass slide with a thickness of 2 mm, and the cover plate is a PDMS layer with a thickness of 6 mm.

[0058] The inner part of the first flow channel 11 comprises 48 constrictions 12 arranged in an array along the flow direction of the cell suspension; wherein every 8 constrictions 12 arranged along the flow direction of the cell suspension is a unit; and the sizes of the 6 units of constrictions arranged along the flow direction of the cell suspension are 30 μm, 15 μm, 8 μm, 4 μm, 2 μm and 1.5 μm in turn.

[0059] Example 3

[0060] Preparation of magnetic beads with PMP70 antibody bound to the surface

[0061] The use of magnetic beads to capture peroxisomes in cells is based on a double antibody sandwich method in a non-competitive binding assay, and a competitive method for determining antibodies or antigens. Generally, this method comprises the following steps: 1) coupling a specific antibody with a solid carrier to form a solid antibody, and washing away unbound antibodies and impurities. 2) adding the test sample and incubating. The antigen in the specimen binds to the solid antibody to form a solid antigen-antibody complex, and other unbound substances are washed away. 3) adding an enzyme-labeled antibody and incubating. The antigen on the solid antigen-antibody complex binds to the enzyme-labeled antibody, and unbound enzyme-labeled antibody is washed away. At this time, the amount of enzyme carried on the solid carrier is related to the amount of the antigen to be tested in the specimen. 4) adding a substrate to develop color. The enzyme on the solid phase catalyzes the substrate to become a colored product, and the amount of antigen in the specimen is determined by colorimetric measurement.

[0062] In this example, Strep-Tag II protein purification magnetic beads purchased from Beaver Nanotech (Suzhou) Co., Ltd. are used, with a suspension concentration of 10% (v / v) magnetic bead suspension, a magnetic bead particle size of 30-150 μm, and a ligand content of ~6 mg Strep-Tactin / mL Gel; the fusion protein binding capacity is ~7 mg Strep-tag II protein / mL Gel. The specific steps include the following:

[0063] S(1): Take 1 mL of the above magnetic bead suspension, add 1:1000 diluted IgG 1 mL, where the IgG is a mouse-derived anti-PMP70 antibody, and the diluent is buffer solution I (buffer solution I uses PBS as a solvent and contains PMSF 1 mM, DTT 1 mM, and EDTA 5 mM); vortex on a vortex mixer at 150 r / min for 15 s to resuspend the magnetic beads, then incubate at 37°C for 30 min to allow the Strep-tag II protein to bind to the IgG.

[0064] Then, place the above mixture 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, then place the above mixture on a magnetic separator, remove the supernatant after the solution is clarified, and wash twice to remove unbound IgG.

[0065] S(2): In the magnetic beads obtained in S(1), 1 mL of 1:1000 diluted PMP70 antibody was added, wherein the PMP70 antibody was of mouse origin and the diluent was buffer solution I; the magnetic beads were resuspended by vortexing on a vortex mixer at 150 r / min for 15 s, and incubated at 37°C for 30 min to allow the PMP70 antibody to be oriented to the Fab domain of IgG to form a magnetic bead-anti-antibody-antibody complex with PMP70 antibody bound to the surface.

[0066] 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. After mixing at 150 r / min for 1 min on a vortex mixer, the above mixture was placed on a magnetic separator, and after the solution was clarified, the supernatant was removed. This washing was repeated twice to remove unbound PMP70 antibody. Then, 2 mL of buffer solution I was added, and after mixing at 150 r / min for 10 min on a vortex mixer, a magnetic bead complex suspension with PMP70 antibody bound to the surface was obtained.

[0067] Example 4

[0068] Peroxisome separation method

[0069] The peroxisomes in the HepG2 cells were sorted using the microfluidic chip in Example 1; the following steps were included:

[0070] S1. Prepare a cell suspension: HepG2 cells purchased from Guangzhou Aidgen Biotech Co., Ltd. were recovered and subcultured, then digested into a single-cell suspension in a clean bench, mixed uniformly by blowing, and then counted. Then, centrifuged at 1100 rpm and 25°C for 4 min, the supernatant was discarded, and the cells were resuspended with 1-2 mL of 4°C pre-cooled cryopreservation solution. Then, the density of the cell suspension was adjusted to 1×10 7 cells / mL with the cryopreservation solution, and was ready for use.

[0071] S2. Sample loading: the cell suspension obtained in S1 was pumped into the microfluidic chip through the first inlet 13 using an electronic pump at a flow rate of 1 mL / min, and the sample loading amount was 2 mL. The separation system suspension was pumped into the microfluidic chip through the second inlet 14 at a flow rate of 1 mL / min, and the sample loading amount was 2 mL.

[0072] The separation system suspension was the magnetic bead complex suspension with PMP70 antibody bound to the surface prepared in Example 3.

[0073] The HepG2 cell suspension enters the lysis module 10 through the first inlet 13, and is broken and releases peroxisomes therein under the action of shearing force when passing through the neck 12 inside the first flow channel 11.

[0074] S3. Reaction: The cell lysate entering the micro-flow channel from the first flow channel 11 and the separation system suspension entering the micro-flow channel from the second inlet 14 are combined in the incubation module 20 to form a mixed solution, and the peroxisomes in the cell lysate can be strongly combined with the PMP70 antibody on the surface of the magnetic beads in the separation system suspension through the antigen on the peroxisome membrane, so as to be combined on the surface of the magnetic beads.

[0075] S4. Sorting: The mixed solution in S3 enters the sorting module 30, and the magnetic beads and the combined 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.

[0076] S5. Elution: The mixed solution of the second outlet 32 is collected, and the above-mentioned mixed solution is placed on a magnetic separator, and after the solution is clarified, the supernatant is removed, then 2 mL of buffer solution I is added, mixed on a vortex mixer at 150 r / min for 1 min, and then the above-mentioned mixed solution is placed on a magnetic separator, and after the solution is clarified, the supernatant is removed, and after being washed for 2 times, 2.5 mM of biotin Biotin is added, and the volume ratio of the separation system suspension to the biotin solution is 1:5; vortex mixer, 150 r / min, mixed for 10 min, and then the mixed solution is placed on a magnetic separator, and the supernatant is collected to realize the enrichment of peroxisomes.

[0077] In this example, the magnetic beads with PMP70 antibody on the surface are used to realize the separation and enrichment of peroxisomes based on the micro-fluidic chip, the operation process is simple, no ultracentrifugation treatment is needed, and the method has strong specificity and convenience, is suitable for cell peroxisome separation, can also specifically separate a small amount of cells, and can also achieve the purpose of sterile separation in the chip, and has potential application value for subsequent sterile research.

[0078] The conventional operations in the operation steps of the present application are well known to those skilled in the art, and will not be described here.

[0079] The above-described embodiments of the present application have been described in detail, and it should be understood that the above-described embodiments are only specific embodiments of the present application and are not used to limit the present application, and any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

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

2. A microfluidic chip-based method for isolating and enriching peroxisomes, characterized by, The microfluidic chip of claim 1 comprises the following steps: S1. Sample loading: cell suspension enters the lysis module (10) through the first inlet (13), and the cells are lysed when the cell suspension flows through the constrictions (12) in the first flow channel (11); the separation system suspension enters the micro flow channel through the second inlet (14), and the magnetic beads coated with antigens, antibodies or secondary antibodies are contained in the separation system suspension; S2. Reaction: the cell lysate from the first flow channel (11) and the separation system suspension from the second inlet (14) are combined in the incubation module (20) to form a mixed solution, and the antigens, antibodies or secondary antibodies on the surface of the magnetic beads react specifically with the substances in the cell lysate; S3. Sorting: the mixed solution enters the sorting module (30), 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: the magnetic beads obtained from the second outlet (32) are collected, and the magnetic beads are competitively eluted by biotin to separate the magnetic beads from the substances in the cell lysate.

3. The microfluidic chip-based peroxisome separation and enrichment method of claim 2, wherein in step S1, the magnetic beads are superparamagnetic particles made of diiron trioxide and / or ferroferric oxide, and the particle size of the magnetic beads is 30-150 μm.

4. The microfluidic chip-based peroxisome separation and enrichment method of claim 2, wherein the surface of the magnetic beads is combined with PMP70 antibodies.

5. The microfluidic chip-based peroxisome separation and enrichment method of claim 4, wherein the preparation of the magnetic beads combined with PMP70 antibodies comprises the following steps: Strep-tag II is combined with anti-PMP70 antibodies, and then combined with PMP70 antibodies to form a Strep-tag II-anti-antibody-antibody complex. ​ ​ ​ The Strep-Tactin coated magnetic beads are used as solid substrate to react with the Strep-tag II-anti-antibody-antibody complex to obtain magnetic beads with PMP70 antibody on the surface. 6.The microfluidic chip-based peroxisome separation and enrichment method according to claim 2, characterized in that, In step S1, the cell density in the cell suspension is 1 x 10 6 -1 x 10 8 cells / mL. 7.The microfluidic chip-based peroxisome separation and enrichment method according to claim 2, characterized in that, In step S1, the separation system suspension comprises buffer solution I, the concentration of magnetic beads in the separation system suspension ranges from 5% to 20% (v / v), the buffer solution I uses PBS as solvent and contains PMSF; The amount of the separation system suspension used is 1 x 10 7 cells per 1-10 ml. 8.The microfluidic chip-based peroxisome separation and enrichment method according to claim 7, characterized in that, In the buffer solution I, the concentration of PMSF ranges from 0.5 mM to 2 mM. 9.The microfluidic chip-based peroxisome separation and enrichment method according to claim 7, characterized in that, In the buffer solution I, DTT ranges from 0.5 mM to 2 mM and EDTA ranges from 3 mM to 8 mM. 10.The microfluidic chip-based peroxisome separation and enrichment method according to claim 7, characterized in that, In step S3, the concentration of biotin ranges from 1 mM to 2.5 mM, and the volume ratio of the separation system suspension to biotin ranges from 1:1 to 1:5.

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