CN111796104A - Exosome detection and typing microfluidic chip and exosome detection and typing method - Google Patents

Abstract

The invention relates to an exosome detection and typing microfluidic chip and an exosome detection and typing method. The microfluidic chip comprises an antibody strip chip and a sample injection chip, wherein the antibody strip chip is coupled with CD63 and CD81 antibodies, and the coupling method comprises the steps of firstly manufacturing a glass slide with a zinc oxide nano array, then performing MPS modification, GMBS modification and G protein modification, and finally connecting CD63 and CD81 antibodies; the upper part of the main channel of the sample injection chip is provided with a fishbone-shaped microstructure, and the height of the channel is 20 mu m. The exosome detection typing method is completed by adopting the microfluidic chip, and the sample injection speed is 1.8-2.2 mu L/min. The micro-fluidic chip can capture relevant exosomes to the maximum extent, and has high sensitivity.

Description

Exosome detection and typing microfluidic chip and exosome detection and typing method

Technical Field

The invention belongs to the field of biochips, and particularly relates to an exosome detection typing microfluidic chip and an exosome detection typing method.

Background

The microfluidic chip technology integrates basic operation units of sample preparation, reaction, separation, detection and the like in the processes of biological, chemical and medical analysis on a micron-scale chip, and automatically completes the whole analysis process. Due to its great potential in the fields of biology, chemistry, medicine and the like, the method has been developed into a new research field crossing the disciplines of biology, chemistry, medicine, fluid, electronics, materials, machinery and the like. The microfluidic chip is a main platform for realizing the microfluidic technology, and the device is mainly characterized in that the effective structure (channels, reaction chambers and other functional parts) for containing the fluid is in a micron scale at least in one latitude. Because of the micron-scale structure, the fluid exhibits and develops specific properties therein that differ from the macro-scale, thus developing unique analytical-generated properties. The microfluidic chip has the characteristics and the development advantages that: the micro-fluidic chip has the characteristics of controllable liquid flow, extremely less consumption of samples and reagents, ten-fold or hundred-fold improvement of analysis speed and the like, can simultaneously analyze hundreds of samples in a few minutes or even shorter time, and can realize the whole processes of pretreatment and analysis of the samples on line. The purpose of its resulting application is to achieve the ultimate goal of a micro total analysis system-lab-on-a-chip. The key application area of current work development is the life science area.

Exosomes are a subset of extracellular vesicles secreted by mammalian cells, typically ranging in size from 30-150nm, and are involved in many biological processes, including immune responses, tumorigenesis, and metastasis. Thus, circulating exosomes are becoming a new paradigm for fluid biopsies for the diagnosis of non-invasive cancers. However, tumor-associated exosomes are very rare in biological fluids at the early stages of disease development. Therefore, the development of new biosensing capabilities is imperative for the hypersensitivity and specificity analysis of disease-associated exosome subtypes in a normal cell-derived vesicle context. The current gold standard methods for exosome capture and identification, such as ultracentrifugation and western blot, have the problems of low separation efficiency, low sensitivity, long time consumption and large sample consumption. Microfluidics has recently been used to exploit exosome analysis, but progress in improving sensitivity, speed and diversity is quite limited.

Patent document CN111505309A, published japanese patent No. 2020.08.07, discloses a chip for simultaneously detecting exosome surface protein and internal miRNA, the preparation method of the chip is as follows: preparing a Y-shaped array chip by adopting a soft lithography method according to the drawn mask, sequentially introducing a biotinylated gelatin solution and an avidin solution into the chip, and assembling the chip layer by layer; and adding the avidin modified nano particles and the capture antibody CD63 to the surface of the assembled chip to obtain the microfluidic chip.

Patent document CN111208297A, published japanese 2020.05.29, discloses a kit for detecting exosome GPC1 protein with a microfluidic chip, the kit includes a microfluidic chip, the microfluidic chip includes a base layer and a top layer located on the base layer, the upper and lower layers are communicated; the basal layer and the top layer are both provided with a micro-flow channel and a hole, the micro-flow channel is communicated with the hole, and the bottom layer and the top layer are communicated through a mixing flow channel; the basal layer and the top layer are respectively provided with a sample adding hole, a reaction hole, a detection hole, a quality control hole and a waste liquid discharge hole; the reaction hole of the basal layer is coated with a monoclonal antibody of galactose coacervate-1 or an IgG antibody of GPC 1; spraying polyethylene glycol (PEG) 6000 polymer on the sampling holes and the reaction holes of the substrate layer; the reaction hole of the base layer is immobilized with capture nano magnetic beads coupled with quantum dot labeled exosome anti-GPC 1 specific antibody.

In the above two patent documents, CN111505309A does not disclose specific detection sensitivity, and CN111208297A discloses that the method is used for detecting 45 serum exosome GPC1 proteins pathologically diagnosed in pancreatic cancer patients, and 44 serum exosome GPC1 has an increased detection result, and the detection rate reaches 97.78%, the sensitivity is 97%, and the method is more accurate than the Elisa method.

However, it is still necessary to develop other microfluidic chips with high exosome capture efficiency and high detection sensitivity.

Disclosure of Invention

The invention aims to provide a novel exosome detection and typing microfluidic chip aiming at the defects in the prior art.

Still another object of the present invention is to provide an exosome detection typing method.

In order to achieve the first object, the invention adopts the technical scheme that:

a micro-fluidic chip for exosome detection and typing comprises an antibody strip chip and a sample injection chip, wherein the antibody strip chip and the sample injection chip are bonded together; the antibody strip chip is coupled with CD63 and CD81 antibodies.

As a preferred example of the present invention, the specific method for coupling CD63 and CD81 antibodies on the antibody strip chip is as follows: preparing a glass slide with a zinc oxide nano array; introducing 0.5% -2% MPS absolute ethyl alcohol solution into the zinc oxide channel of the glass slide with the zinc oxide nano array, incubating for 0.5-1.5h at room temperature, and washing with absolute ethyl alcohol for 1-5 times; then introducing a DMSO solution of 0.2-0.3mM GMBS into a zinc oxide channel for incubation for 0.5-1.5h, and washing 1-5 times with PBS; introducing PBS solution containing 9-11 μ g/mLG protein into zinc oxide channel, incubating for 0.5-1.5h, and washing with PBS for 1-5 times; and finally, introducing the CD63 and CD81 mixed monoclonal antibodies with the antibody content of 9-11 mu g/mL into a zinc oxide channel, and incubating at 4 ℃ for 0.5-1.5 h.

More preferably, the preparation method of the glass slide with the zinc oxide nano array comprises the following specific steps:

(1) manufacturing a PDMS chip;

(2) preparing a zinc oxide seed layer on a glass slide;

(3) bonding a PDMS chip to a glass slide with a zinc oxide seed layer;

(4) and continuously growing zinc oxide on the zinc oxide seed layer to form a zinc oxide nano array.

As another preferred example of the present invention, the sample injection chip has a fishbone-shaped microstructure above the main channel.

As another preferred example of the present invention, the height of the channel of the sample introduction chip is 20 μm.

As another preferred example of the invention, the channel width of the sample injection chip is 1.3mm, and the length is 1.3 cm.

As another preferred example of the present invention, the antibody strip chip has a width of 200 μm per channel, a length of 2cm and a height of 75 μm per channel.

In order to achieve the second object, the invention adopts the technical scheme that:

an exosome detection typing method, which employs a microfluidic chip as described in any one of the above.

As a preferred example of the invention, the exosome detection typing method comprises the following steps:

(1) sample introduction: taking an exosome sample, adding the exosome sample into a sample inlet hole, and pumping the sample by using an injection pump at the speed of 1.8-2.2 mu L/min;

(2) washing the channel with PBS;

(3) fluorescent dye infusion: introducing 48-52 μ M DiO working solution into the injection pump at a speed of 1.8-2.2 μ L/min, and incubating at room temperature for 0.5-1.5 h;

(4) washing the channel with PBS;

(5) and (5) observing the fluorescence intensity by placing the probe in a fluorescence microscope, judging whether the exosomes are captured or not, and obtaining a typing result.

The invention has the advantages that:

the invention discloses an exosome detection parting microfluidic chip. The antibody strip chip of the microfluidic chip is coupled with CD63 and CD81 antibodies, and the coupling method comprises the steps of firstly manufacturing a glass slide with a zinc oxide nano array, then performing MPS modification, GMBS modification and G protein modification, and finally connecting CD63 and CD81 antibodies. In the whole process, the structural parameters of the channel are reasonable, the adding amount of the antibody is proper, the aim of efficiently fixing the antibody is fulfilled, and the biological activity of the antibody is well maintained. The sample injection chip of the microfluidic chip is designed by adopting fishbone, has reasonable structural parameters and can promote the capture of target protein on exosome. Further, the invention optimizes the sample injection flow rate in the detection step. The final result shows that the microfluidic chip can capture relevant exosomes to the maximum extent and has high sensitivity.

Drawings

FIG. 1: a, ZnO growth chips, wherein each growth channel is 200 microns in width, 2cm in length and 75 microns in height. B. The top of the sample injection chip is carved with a fishbone structure, the width of the channel is 1.3mm, the length is 1.3cm, and the height is 20 mu m.

FIG. 2: A.ZnO nanoarray SEM. B. The nano-array captures exosomes SEM under the effect of antibodies.

FIG. 3: schematic representation of covalent modification of nanoarrays.

FIG. 4: A. and (3) screening the chip sample at the optimal flow rate, and obtaining the highest fluorescence intensity under the condition of 2 mul/min. B. And (4) screening the height of the sample injection chip channel, wherein 20 mu m is the optimal height. C. The nano-array surface is coupled with antibody species screening, and the capture antibody selection of CD63+ CD81 provides high exosome capture efficiency.

Detailed Description

The following description will further describe embodiments of the present invention with reference to the accompanying drawings.

EXAMPLE 1 microfluidic chip production method

First, antibody strip chip manufacturing

1. PDMS chip fabrication

(1) Drawing a graph of a chip by using CAD software, drawing a photoetching mask on a photographic negative film by using a high-resolution laser phototypesetter, then spin-coating an SU-8 photoresist layer on a silicon wafer, carrying out high-temperature curing treatment on the photoresist layer, covering the photoetching mask, exposing the cured SU-8 photoresist on the photoetching machine, and finally developing in a developing solution to obtain the silicon wafer male mold template.

(2) Mixing the PDMS prepolymer and the PDMS curing agent according to a ratio of 10:1, pumping air in vacuum, pouring the mixture on a manufactured male mold template, curing at high temperature, peeling to obtain a chip with a microstructure, and punching holes (an antibody filling hole and an antibody outflow hole) according to needs.

2. Preparing a zinc oxide seed layer on a glass slide

(1) Preparation of seed solution (preparation on the same day):

firstly, in the experimental process, a dehumidifier is started to prevent zinc acetate from hydrolyzing and prevent the influence of humidity on a ZnO nano structure;

② rinsing the 50ml volumetric flask with absolute ethyl alcohol (analytically pure) for 3 times;

③ adding 0.11g of zinc acetate into a 50ml volumetric flask, and quantifying absolute ethyl alcohol;

and fourthly, putting the volumetric flask with the constant volume into an ultrasonic cleaning machine, setting the temperature to be 10 ℃, carrying out ultrasonic treatment for 10 minutes, wherein the temperature rises in the ultrasonic treatment process, and cooling to the room temperature after the ultrasonic treatment is finished.

(2) Capping of a zinc oxide seed layer

Cleaning a slide: sequentially cleaning the glass slide with acetone, isopropanol and deionized water by ultrasonic15 minutes in combination with N₂Drying the glass slide;

secondly, washing the slide by plasma: vacuumizing for 3min, and cleaning for 1min30 s;

③ the spin coating process of the seed solution: forwarding for 1000r, 3 s; go back to 1800 + 2000r, 60 s. Spin coating for 3 times;

heat treatment: putting the spin-coated glass slide on a heating table at 300 ℃, and heating for 1-2 hours;

fifthly, the annealed Seed layer is put into a drying oven for standby (the effective period: 1 week).

3. Bonding a PDMS chip to a glass slide with a zinc oxide seed layer

And (3) placing the ZnO growth chip in a plasma cleaning machine, vacuumizing for 3min, adjusting the plasma mode to be ' Mid ', clockwise rotating an air inlet knob to 4 o ' clock, treating for 3min, closing the air inlet knob and the ' Mid ' mode, slowly deflating, taking out the chip, naturally attaching the glass slide with the zinc oxide seed layer and the exposed surface of the PDMS chip, and placing at 4 ℃ until the chip is used.

4. Continuing to grow zinc oxide on the zinc oxide seed layer

(1) Preparation of growth solution (preparation on the day):

0.4g of a 50% by mass PEI solution was weighed. A50 ml volumetric flask was added. (reagent 1)

② 0.372g of zinc nitrate hexahydrate powder is weighed. A50 ml volumetric flask was added. (reagent 2)

③ 0.0875g of hexamethylene tetramine powder is weighed. A50 ml volumetric flask was added. (reagent 3)

And fourthly, 40ml of deionized water is used for dissolving the substances.

Fifthly, adding 1.25ml of ammonia water.

Sixthly, deionized water is used for fixing the volume to 50 ml.

(2) Nanowire growth

Firstly, the bonded ZnO growth chip is placed on a 90 ℃ hot bench and connected with an injection pump. Adding the growth solution into 4 sample injection holes, setting an injection pumping mode, and pumping the growth solution at a flow rate of 4.8 mul/min;

② adding the growth solution into the sample feeding hole at regular time, the growth time is 2 hours. Finally forming the zinc oxide nano array.

5. Chemical modification of ZnO nano-array

See figure 3 for a method of covalent modification of a nanoarray.

(1) Diluting the antibody stock solution: diluting CD63 antibody stock solution by 500 mu g/mL by 50 times; CD81 antibody stock solution 500. mu.g/mL was diluted 50-fold. And (5) storing at 4 degrees.

(2) Chip preparation: the zinc oxide chip, previously prepared, was removed from the dry environment and a lower channel was wetted with deionized water drawn in by a syringe pump at a rate of 4.8 μ L/min.

(3) Coupling chip antibodies: first, a solution of 1% MPS in absolute ethanol, 0.25mM MBS in DMSO, 10. mu.g/mL G protein in PBS was prepared. Then, 1% MPS absolute ethyl alcohol solution is introduced into a zinc oxide channel to incubate for 1h at room temperature, and absolute ethyl alcohol is washed for 3 times. A0.25 mM GMBS solution in DMSO was then passed through the zinc oxide channel for incubation for 1h, and washed 3 times with PBS. Then, 10. mu.g/mL of protein G in PBS was passed through the zinc oxide channel and incubated for 1h, and the PBS was washed 3 times. Then, the mixed monoclonal antibody of CD63+ CD81 with 10. mu.g/mL antibody was passed into the zinc oxide channel and incubated at 4 ℃ for 1 h.

(4) The 4 zinc oxide channels were blocked with 5% BSA solution. The blocking time was 1 h.

(5) After the antibody strip chip is manufactured, the PDMS chip is removed, the antibody strip on the glass slide is formed, and the antibody strip is stored at 4 degrees until the antibody strip chip is used.

In FIG. 1, A is a zinc oxide growth chip, which is 4 parallel channels, each growth channel has a width of 200 μm, a length of 2cm and a height of 75 μm. In FIG. 2, A is the electron micrograph of ZnO nano-array.

Secondly, sample introduction chip manufacturing

The specific method is the same as the ZnO growth chip manufacturing method, and only different photoetching masks are used.

In fig. 1, B is a sample injection chip, and a fishbone structure is engraved on the top of the chip to break the laminar flow of the liquid in the microchannel, so that the liquid becomes turbulent flow and the exosome capturing efficiency is increased. The channel width was 1.3mm, length 1.3cm and height 20 μm.

Third, the antibody strip chip is bonded with the sample injection chip

Placing the sample introduction chip in a plasma cleaning machine, vacuumizing for 3min, adjusting the plasma mode to ' Mid ', clockwise rotating an air inlet knob to 4 o ' clock, closing the air inlet knob and ' Mid ' mode after treating for 3min, slowly deflating, taking out the chip, aligning and bonding the mark on the chip and the cross mark made on the surface of the antibody slide in advance, and crosswise attaching the longitudinal sample introduction chip and the transverse antibody bar code to form a cross point which is a ' pixel point '.

Example 2 use of microfluidic chips for exosome detection typing

1. Sample introduction

50 μ L of HOS cell-derived exosome suspension was taken, added to the inlet well, and the sample was withdrawn at 2 μ L/min by a syringe pump, and the effluent was collected at the outlet.

2. The channel was washed with PBS.

3. Fluorescent dye infusion:

the DiO dye is a lipophilic membrane dye, and the membrane of exosome is phospholipid bilayer like the cell membrane, so the DiO dye can be rapidly bonded to the membrane surface of exosome, so that exosome emits green fluorescence for observation under a fluorescence microscope.

Preparing DiO: 0.001g of DiO powder was weighed on an analytical balance, dissolved in 226. mu.l of DMSO to prepare a 5mM DiO stock solution, and the stock solution was stored in a-20 ℃ refrigerator. Mu.l of DiO dye stock solution was put into an EP tube, and 99. mu.l of PBS was added to prepare 50. mu.M DiO working solution.

100 mul DiO working solution was pumped in at 2 mul/min by syringe pump and incubated for 1h at ambient temperature.

4. The channel was washed with PBS.

5. And (5) placing the sample in a fluorescence microscope for observation.

6. And simultaneously sending an electron microscope for detection, and the result is shown as B in figure 2.

Example 3 microfluidic chip Structure and detection Condition optimization

Some parameters of the structure and detection conditions of the microfluidic chip were changed, and the remaining parameters were the same as in examples 1 and 2 to compare the exosome capture efficiency under different structures and detection conditions.

First, chip sample introduction flow velocity screening

1. And (4) preparing a zinc oxide chip.

2. Zinc oxide surface modified CD63+ CD81 antibody.

3. A total of 6 prepared and modified CD63+ CD81 zinc oxide chips were placed on a table, 50. mu.l of HOS cell line exosome suspension was added, and the injection pump was turned on to draw samples at 0, 1, 2, 4, 8, 10. mu.l/min.

4. After the above procedure was completed, the channel was washed 3 times with PBS.

5. Add 50. mu.M DiO solution and incubate for 50min at room temperature.

6. PBS was washed 3 times.

7. And (5) observing under a fluorescence microscope, and counting the fluorescence intensity. The results are shown in FIG. 4A. 2 mul/min is the optimal sample injection speed of the chip, and the effect is obviously superior to other sample injection speeds.

Second, chip channel height screening

1. And (4) preparing a zinc oxide chip.

2. By means of alignment, different rotation speeds are used to prepare different sample chip templates with different thicknesses, including different sample chips with 10, 20, 30 and 40 μm height.

3. The prepared and decorated zinc oxide chip of CD63+ CD81 is placed on a table top and covered with sample injection chips of different heights. 50. mu.l of HOS cell line exosome suspension was added, and the syringe pump was turned on to draw samples at 2. mu.l/min.

4. After the above procedure was completed, the channel was washed 3 times with PBS.

5. Add 50. mu.M DiO solution and incubate for 50min at room temperature.

6. PBS was washed 3 times.

7. And (5) observing under a fluorescence microscope, and counting the fluorescence intensity. The results are shown in FIG. 4B. The sample injection chip with a height of 10 μm was clogged. 20 μm is the optimum channel height, and the effect is significantly better than other channel heights.

Three, nano array surface coupling antibody species screening

1. And (4) preparing a zinc oxide chip.

2. Zinc oxide surface modification of different kinds of antibodies: 10 μ g/ml each of monoclonal antibodies to CD9, CD63 and/or CD 81.

3. The prepared and modified antibody zinc oxide chip is placed on a table top and covered with a sample injection chip with the height of 20 mu m. 50. mu.l of HOS cell line exosome suspension was added, and the syringe pump was turned on to draw samples at 2. mu.l/min.

4. After the above procedure was completed, the channel was washed 3 times with PBS.

5. Add 50. mu.M DiO solution and incubate for 50min at room temperature.

6. PBS was washed 3 times.

7. And (5) observing under a fluorescence microscope, and counting the fluorescence intensity. The results are shown in FIG. 4C. The capture antibody selection of CD63+ CD81 provided high exosome capture efficiency with a significant advantage over other antibodies or antibody combinations.

Example 4 sensitivity detection of microfluidic chip

Performing gradient dilution on exosome samples from HOS cells by using PBS (phosphate buffer solution) to obtain dilution times of 1, 10, 100 and 10³、10⁴、10⁵、10⁶、10⁷The sensitivity of the microfluidic chip was tested according to the methods of examples 1 and 2. As a result, the micro-fluidic chip of the invention can detect that the dilution multiple is 10⁷Whereas the conventional Elisa method only detects exosome samples with a maximum dilution factor of 100.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and additions can be made without departing from the principle of the present invention, and these should also be considered as the protection scope of the present invention.

Claims (9)

1. The microfluidic chip for exosome detection and typing is characterized by comprising an antibody strip chip and a sample injection chip, wherein the antibody strip chip and the sample injection chip are bonded together; the antibody strip chip is coupled with CD63 and CD81 antibodies.

2. The microfluidic chip according to claim 1, wherein the specific method for coupling the CD63 and CD81 antibodies on the antibody strip chip is as follows: preparing a glass slide with a zinc oxide nano array; introducing 0.5% -2% MPS absolute ethyl alcohol solution into the zinc oxide channel of the glass slide with the zinc oxide nano array, incubating for 0.5-1.5h at room temperature, and washing with absolute ethyl alcohol for 1-5 times; then introducing a DMSO solution of 0.2-0.3mM GMBS into a zinc oxide channel for incubation for 0.5-1.5h, and washing 1-5 times with PBS; introducing PBS solution containing 9-11 μ G/mL G protein into zinc oxide channel, incubating for 0.5-1.5h, and washing with PBS for 1-5 times; and finally, introducing the CD63 and CD81 mixed monoclonal antibodies with the antibody content of 9-11 mu g/mL into a zinc oxide channel, and incubating at 4 ℃ for 0.5-1.5 h.

3. The microfluidic chip according to claim 2, wherein the glass slide with the zinc oxide nano-array is prepared by the following specific method:

(1) manufacturing a PDMS chip;

(2) preparing a zinc oxide seed layer on a glass slide;

(3) bonding a PDMS chip to a glass slide with a zinc oxide seed layer;

(4) and continuously growing zinc oxide on the zinc oxide seed layer to form a zinc oxide nano array.

4. The microfluidic chip according to claim 1, wherein the sample introduction chip has a fishbone-shaped microstructure above the main channel.

5. The microfluidic chip according to claim 1, wherein the height of the channel of the sample feeding chip is 20 μm.

6. The microfluidic chip according to claim 1, wherein the channel width of the sample feeding chip is 1.3mm and the length is 1.3 cm.

7. The microfluidic chip according to claim 1, wherein each channel of the antibody strip chip has a width of 200 μm, a length of 2cm, and a height of 75 μm.

8. An exosome detection typing method, characterized in that the microfluidic chip according to any one of claims 1 to 5 is applied.

9. The method of claim 1, comprising the steps of:

(1) sample introduction: taking an exosome sample, adding the exosome sample into a sample inlet hole, and pumping the sample by using an injection pump at the speed of 1.8-2.2 mu L/min;

(2) washing the channel with PBS;

(3) fluorescent dye infusion: introducing 48-52 μ M DiO working solution into the injection pump at a speed of 1.8-2.2 μ L/min, and incubating at room temperature for 0.5-1.5 h;

(4) washing the channel with PBS;

(5) and (5) observing the fluorescence intensity by placing the probe in a fluorescence microscope, judging whether the exosomes are captured or not, and obtaining a typing result.

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