CN111796104B - Exosome detection typing micro-fluidic chip and exosome detection typing method - Google Patents

Abstract

The invention relates to an exosome detection typing micro-fluidic chip and an exosome detection 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 modifying by MPS, GMBS and G protein, and finally connecting the CD63 and CD81 antibodies; the sample injection chip has a fish bone-shaped microstructure above the main channel, and the height of the channel is 20 mu m. The exosome detection and typing method is completed by adopting the microfluidic chip, and the sample injection speed is 1.8-2.2 mu L/min. The microfluidic chip provided by the invention can capture related exosomes to the greatest extent, and has high sensitivity.

Description

Exosome detection typing micro-fluidic chip and exosome detection typing method

Technical Field

The invention belongs to the field of biological chips, and particularly relates to an exosome detection typing micro-fluidic chip and an exosome detection typing method.

Background

The microfluidic chip technology (Microfluidics) integrates basic operation units such as sample preparation, reaction, separation, detection and the like in biological, chemical and medical analysis processes onto a micron-scale chip, and automatically completes the whole analysis process. Because of its great potential in biological, chemical, medical and other fields, it has been developed into a new research field where the disciplines of biology, chemistry, medicine, fluids, electronics, materials, machinery and the like are crossed. Microfluidic chips are the main platform for microfluidic technology implementation, and their device features that their effective structures (channels, reaction chambers, and some other functional components) that hold fluids are at least on a micrometer scale in one latitude. Due to the micro-scale structure, the fluid exhibits and produces therein specific properties that differ from those of the macro-scale, thus developing unique analytical properties. The characteristics and development advantages of the microfluidic chip are as follows: the microfluidic chip has the characteristics of controllable liquid flow, extremely small consumption of samples and reagents, ten times or hundreds times higher analysis speed and the like, can simultaneously analyze hundreds of samples in a few minutes or even shorter, and can realize the whole pretreatment and analysis processes 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 field of current work development is the life science field.

Exosomes are a subset of extracellular vesicles secreted by mammalian cells, typically ranging in size from 30-150nm, involved in a number of biological processes including immune responses, tumorigenesis and metastasis. Thus, circulating exosomes are becoming a new paradigm for liquid biopsies for the diagnosis of non-invasive cancers. However, in early stages of disease progression, tumor-associated exosomes in biological fluids are very rare. Therefore, the development of new biosensing capabilities is imperative for the hypersensitive and specific analysis of disease-related exosome subtypes in the context of normal cell-derived vesicles. The existing 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 utilize exosome assays, but advances in improving sensitivity, speed and diversity are quite limited.

Patent document CN111505309a, publication date 2020.08.07, discloses a chip for simultaneously detecting exosome surface protein and internal miRNA, and 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 avidin modified nano particles and a capture antibody CD63 on the surface of the assembled chip to obtain the microfluidic chip.

Patent document CN111208297A, publication date 2020.05.29, discloses a kit for detecting exosome GPC1 protein by a microfluidic chip, the kit comprises a microfluidic chip, the microfluidic chip comprises a basal layer and a top layer positioned on the basal layer, and the upper layer and the lower layer are communicated; the substrate layer and the top layer are respectively provided with a micro-flow channel and a hole, the micro-flow channels are communicated with the holes, 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 well of the basal layer is coated with a monoclonal antibody of galactose coacervate-1 or an IgG antibody against GPC 1; the sample adding holes and the reaction holes of the basal layer are sprayed with polyethylene glycol PEG6000 polymer; the reaction Kong Guhua of the basal layer had captured nanomagnetic beads that were coupled to quantum dot labeled exosome anti-GPC 1 specific antibodies.

In the above two patent documents, CN111505309a does not disclose specific detection sensitivity, CN111208297a discloses that for detecting 45 cases of serum exosome GPC1 proteins of patients with pancreatic cancer through pathological diagnosis, the detection result of 44 cases of serum exosome GPC1 is increased, the detection rate reaches 97.78%, the sensitivity is 97%, and compared with the Elisa method, the detection method is more accurate.

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 at overcoming the defects in the prior art and provides a novel exosome detection typing micro-fluidic chip.

It is still another object of the present invention to provide a method for detecting and typing exosomes.

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

a microfluidic chip for exosome detection typing, 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 band 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 a 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 flushing with PBS for 1-5 times; introducing PBS solution containing 9-11 mug/mL G protein into a zinc oxide channel for incubation for 0.5-1.5h, and flushing with PBS for 1-5 times; and finally, introducing the mixed monoclonal antibodies of the CD63 and the CD81 with the antibody content of 9-11 mug/mL into a zinc oxide channel, and incubating for 0.5-1.5h at 4 ℃.

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 having 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 preferable example of the invention, the sample injection chip has a fish bone-shaped microstructure above the main channel.

As another preferable example of the invention, the height of the channel of the sample injection chip is 20 μm.

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

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

In order to achieve the second purpose, the invention adopts the following technical scheme:

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

As a preferred embodiment of the present invention, the exosome detection typing method comprises the steps of:

(1) And (3) sample injection: taking an exosome sample, adding a sample injection hole, and extracting the sample at a speed of 1.8-2.2 mu L/min by a syringe pump;

(2) The channels were rinsed with PBS;

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

(4) The channels were rinsed with PBS;

(5) And (5) placing the sample in a fluorescence microscope to observe the fluorescence intensity, and judging whether the exosomes are captured or not to obtain a typing result.

The invention has the advantages that:

the invention discloses an exosome detection parting micro-fluidic 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 modifying by MPS, GMBS and G proteins, and finally connecting the CD63 and the CD81 antibodies. In the whole process, the structural parameters of the channel are reasonable, the addition amount of the antibody is proper, the aim of high-efficiency fixation of the antibody is fulfilled, and the biological activity of the antibody is well maintained. The sample injection chip of the microfluidic chip adopts a fishbone design, has reasonable structural parameters, and can promote the capture of target proteins on exosomes. Furthermore, the invention optimizes the sample injection flow rate of the detection step. The final result shows that the microfluidic chip provided by the invention can capture related exosomes to the greatest extent, and has high sensitivity.

Drawings

Fig. 1: A.ZnO growing chip, each growing channel has a width of 200 μm, a length of 2cm and a height of 75 μm. B. The sample injection chip is characterized in that a fishbone structure is carved on the top of the chip, the width of a channel is 1.3mm, the length is 1.3cm, and the height is 20 mu m.

Fig. 2: znO nanoarray SEM. B. The nanoarrays capture exosomes SEM under antibody.

Fig. 3: schematic of covalent modification of nanoarrays.

Fig. 4: A. and (3) carrying out sample injection on the chip, and screening at an optimal flow rate, wherein the highest fluorescence intensity is obtained under the condition of 2 mu l/min. B. And the sample injection chip channel is subjected to high screening, and the optimal height is 20 mu m. C. The nano-array surface coupled antibody species screening, the cd63+cd81 capture antibody selection provides high exosome capture efficiency.

Detailed Description

The following description of the embodiments of the present invention will be given with reference to the accompanying drawings.

Example 1 microfluidic chip fabrication method

1. Antibody strip chip fabrication

1. PDMS chip manufacture

(1) Drawing the pattern of the chip by CAD software, drawing a photoetching mask on a photographic negative by a high-resolution laser photo-printer, spin-coating an SU-8 photoresist layer on a silicon wafer, performing 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 die template.

(2) Mixing PDMS prepolymer and PDMS curing agent according to the proportion of 10:1, pouring the mixture on a manufactured male die template after vacuum pumping, stripping the mixture after high-temperature curing to obtain a chip with a microstructure, and punching (antibody pouring holes and antibody outflow holes) according to requirements.

2. Preparation of Zinc oxide seed layer on glass slide

(1) Seed solution formulation (day formulation):

(1) in the experimental process, firstly, a dehumidifier is opened to prevent zinc acetate from hydrolysis and prevent the influence of humidity on the ZnO nano structure;

(2) absolute ethanol (analytically pure) rinse 50ml volumetric flask 3 times;

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

(4) and (3) placing the volumetric flask with the fixed volume into an ultrasonic cleaner, 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 completed.

(2) Coverage of zinc oxide seed layer

(1) Cleaning a slide: sequentially washing slides with acetone, isopropanol, deionized water, and ultrasound for 15 minutes, and with N ₂ Blow-drying the glass slide;

(2) plasma wash slide: vacuumizing for 3min, and cleaning for 1min for 30s;

(3) seed solution spin coating process: forward 1000r,3s; the back rotation is 1800-2000r,60s. Spin-coating for 3 times;

(4) and (3) heat treatment: placing the spin-coated slide on a heating table at 300 ℃ and heating for 1-2 hours;

(5) the annealed Seed layer was put in a dry box for use (expiration date: 1 week).

3. Bonding PDMS chips to slides with zinc oxide seed layer

Placing the ZnO growing chip in a plasma cleaning machine, vacuumizing for 3min, adjusting the plasma mode to ' Mid ', rotating the air inlet knob clockwise to 4 o ' clock, after treating for 3min, closing the air inlet knob and the ' Mid ' mode, slowly deflating, taking out the chip, naturally attaching the slide with the zinc oxide seed layer to the exposed surface of the PDMS chip, and placing at 4 ℃ until the use.

4. Growing zinc oxide on zinc oxide seed layer

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

(1) 0.4g of PEI solution with a mass fraction of 50% was weighed. Add 50ml volumetric flask. (reagent 1)

(2) 0.372g of zinc nitrate hexahydrate powder was weighed. Add 50ml volumetric flask. (reagent 2)

(3) 0.0875g of cyclohexamethylenetetramine powder was weighed. Add 50ml volumetric flask. (reagent 3)

(4) The above materials were dissolved in 40ml of deionized water.

(5) Then, 1.25ml of ammonia water was added.

(6) Finally, deionized water is used for volume determination to 50ml.

(2) Nanowire growth

(1) And placing the bonded ZnO growing chip on a 90 ℃ hot table, and connecting a syringe pump. Adding the growth solution into 4 sample injection holes, setting a pumping mode of injection pump, and pumping the growth solution at a flow rate of 4.8 mu l/min;

(2) and adding a growth solution into the sample injection hole at regular time, wherein the growth time is 2 hours. Finally, the zinc oxide nano array is formed.

5. Chemical modification of ZnO nano-arrays

The method of covalent modification of the nanoarrays is shown in figure 3.

(1) Diluting the antibody stock solution: CD63 antibody stock solution 500 μg/mL, 50-fold dilution; CD81 antibody stock solution 500. Mu.g/mL was diluted 50-fold. 4-degree preservation.

(2) Chip preparation: the previously prepared zinc oxide chip was removed from the dry environment and the channel was wetted with deionized water by pumping at a rate of 4.8 μl/min using a syringe pump.

(3) Chip antibody coupling: first, a solution was prepared, 1% MPS absolute ethanol solution, 0.25mM MgMBS in DMSO, 10. Mu.g/mL G protein PBS. Then, 1% mps absolute ethanol solution was introduced into the zinc oxide channel and incubated for 1h at room temperature, and the absolute ethanol was rinsed 3 times. Then, a DMSO solution of 0.25mM GMBS was passed into the zinc oxide channel for incubation for 1h, and washed 3 times with PBS. Then, 10. Mu.g/mL of G protein PBS solution was passed into the zinc oxide channel for incubation for 1h, and PBS was rinsed 3 times. The CD63+CD81 mixed monoclonal antibodies, each 10. Mu.g/mL, were then passed into the zinc oxide channel and incubated for 1h at 4 ℃.

(4) The 5% bsa solution blocked 4 zinc oxide channels. The closing time was 1h.

(5) After the antibody band chip is manufactured, the PDMS chip is removed, the antibody band on the slide is formed, and the slide is preserved at 4 degrees until the slide 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 an electron micrograph of the ZnO nanoarray.

2. Sample injection chip manufacturing

The specific method is the same as the method for manufacturing the ZnO growing chip, and only different photoetching mask plates are used.

In fig. 1, B is a sample injection chip, and a fishbone structure is engraved on the top of the chip, so as to break the laminar flow of the liquid in the micro-channel, make it become turbulent, and increase the capturing efficiency of the exosome. The channel has a width of 1.3mm, a length of 1.3cm and a height of 20. Mu.m.

3. Bonding of antibody strip chip and sample injection chip

Placing the sample injection chip in a plasma cleaning machine, extracting vacuum for 3min, regulating a plasma mode to be ' Mid ', rotating an air inlet knob clockwise to be 4 o ' clock, after treating for 3min, closing the air inlet knob and the ' Mid ' mode, slowly deflating, taking out the chip, aligning and bonding a mark on the chip with a cross mark which is made on the surface of an antibody glass in advance, and cross-bonding the longitudinal sample injection chip and a transverse antibody bar code, wherein a formed cross point is a ' pixel point '.

Example 2 exosome detection typing Using microfluidic chips

1. Sample injection

Taking 50 mu.l of HOS cell-derived exosome suspension, adding into a sample injection hole, taking out a sample at a speed of 2 mu L/min by a syringe pump, and collecting effluent from a sample outlet.

2. The channels were rinsed with PBS.

3. Fluorescent dye infusion:

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

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

The syringe pump was fed with 100. Mu.l of DiO working solution at a rate of 2. Mu.l/min and incubated for 1h at ambient temperature.

4. The channels were rinsed with PBS.

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

6. And meanwhile, the detection of the transmitting mirror is carried out, and the result is shown as B in fig. 2.

Example 3 microfluidic chip Structure and detection Condition optimization

Part of parameters of the microfluidic chip structure and detection conditions were changed, and the rest of the parameters were the same as examples 1 and 2 to compare exosome capturing efficiency under different structures and detection conditions.

1. Chip sample injection flow rate screening

1. And (5) preparing a zinc oxide chip.

2. Zinc oxide surface modifies CD63+CD81 antibodies.

3. 6 zinc oxide chips which are prepared and modified by CD63+CD81 are placed on a table top, 50 mu l of HOS cell line exosome suspension is added respectively, and a syringe pump is started to extract samples at the speed of 0, 1, 2, 4, 8 and 10 mu l/min respectively.

4. After the end of the above procedure, the channels were rinsed 3 times with PBS.

5. 50. Mu.M DiO solution was added and incubated at room temperature for 50min.

6. The 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 mu l/min is the optimal sample injection speed of the chip, and the effect is obviously superior to other sample injection speeds.

2. Chip channel height screening

1. And (5) preparing a zinc oxide chip.

2. Different sample injection chip templates with different thicknesses are prepared by using different rotating speeds in an alignment mode, and different sample injection chips with heights of 10, 20, 30 and 40 mu m are respectively arranged.

3. And placing the prepared zinc oxide chip with the modified CD63+CD81 on a desktop, and covering sample injection chips with different heights. 50 μl of HOS cell line exosome suspension was added, and the syringe pumps were turned on to withdraw samples at a rate of 2 μl/min, respectively.

4. After the end of the above procedure, the channels were rinsed 3 times with PBS.

5. 50. Mu.M DiO solution was added and incubated at room temperature for 50min.

6. The 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 the height of 10 μm is blocked. The 20 mu m is the optimal channel height, and the effect is obviously superior to other channel heights.

3. Nanoarray surface-coupled antibody species screening

1. And (5) preparing a zinc oxide chip.

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

3. And placing the zinc oxide chip prepared and modified with the antibody on a desktop, and covering a sample injection chip with the height of 20 mu m. 50 μl of HOS cell line exosome suspension was added, and the syringe pumps were turned on to withdraw samples at a rate of 2 μl/min, respectively.

4. After the end of the above procedure, the channels were rinsed 3 times with PBS.

5. 50. Mu.M DiO solution was added and incubated at room temperature for 50min.

6. The 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 cd63+cd81 capture antibody selection provides high exosome capture efficiency with significantly better efficacy than other antibodies or antibody combinations.

Example 4 sensitivity detection of microfluidic chips

Subjecting HOS cell-derived exosome sample to gradient dilution with PBS to obtain dilution factors of 1, 10, 100, and 10 respectively ³ 、10 ⁴ 、10 ⁵ 、10 ⁶ 、10 ⁷ The sensitivity of the microfluidic chip was measured as in examples 1 and 2. As a result, the microfluidic chip of the present invention can detect a dilution ratio of 10 ⁷ Whereas the conventional Elisa method can only detect exosome samples with a maximum dilution of 100 fold.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and additions to the present invention may be made by those skilled in the art without departing from the principles of the present invention and such modifications and additions are to be considered as well as within the scope of the present invention.

Claims (4)

1. The microfluidic chip for detecting and typing the exosome 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 a glass slide coupled with CD63 and CD81 antibodies, and each channel is 200 mu m in width, 2cm in length and 75 mu m in height; the sample injection chip is a PDMS chip with a fishbone microstructure above the main channel; the height of the channel of the sample injection chip is 20 mu m, the width of the channel is 1.3mm, and the length of the channel is 1.3cm; 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 a 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 DMSO solution of 0.2-0.3mM MgMBS into a zinc oxide channel for incubation for 0.5-1.5h, and flushing with PBS for 1-5 times; introducing PBS solution containing 9-11 mug/mLG protein into a zinc oxide channel for incubation for 0.5-1.5h, and flushing with PBS for 1-5 times; finally, the mixed monoclonal antibodies of CD63 and CD81 with the antibody content of 9-11 mug/mL are introduced into a zinc oxide channel, and incubated for 0.5-1.5h at 4 ℃.

2. The microfluidic chip according to claim 1, wherein the preparation of the glass slide with zinc oxide nano-arrays 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 having a zinc oxide seed layer;

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

3. An exosome detection typing method, wherein the method employs the microfluidic chip according to any one of claims 1 to 2.

4. A method according to claim 3, comprising the steps of:

(1) And (3) sample injection: taking an exosome sample, adding a sample injection hole, and extracting the sample at a speed of 1.8-2.2 mu L/min by a syringe pump;

(2) The channels were rinsed with PBS;

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

(4) The channels were rinsed with PBS;

(5) And (5) placing the sample in a fluorescence microscope to observe the fluorescence intensity, and judging whether the exosomes are captured or not to obtain a typing result.

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