CN113499811B - Micro-fluidic chip based on surface growth ZnO nanowire glass microspheres and application - Google Patents

Micro-fluidic chip based on surface growth ZnO nanowire glass microspheres and application Download PDF

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CN113499811B
CN113499811B CN202110747516.2A CN202110747516A CN113499811B CN 113499811 B CN113499811 B CN 113499811B CN 202110747516 A CN202110747516 A CN 202110747516A CN 113499811 B CN113499811 B CN 113499811B
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glass
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CN113499811A (en
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杜鲁涛
黄晓文
王传新
杨雪梅
李培龙
张太毅
陈嘉词
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Qilu University of Technology
Second Hospital of Shandong University
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Second Hospital of Shandong University
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Abstract

The invention belongs to the technical field of biochips, and relates to a glass microsphere microfluidic chip based on ZnO nanowires grown on the surface and application of the glass microsphere microfluidic chip in exosome capture. According to the invention, the exosome-specific membrane protein antibody modification is carried out on the ZnO nanowires grown in situ on the glass microspheres, exosomes in a specific capture sample are specifically recognized through antigen-antibody immunity, and the microspheres and the microfluidic chip are systematically integrated, so that the microfluidic chip based on the glass microspheres with the ZnO nanowires grown on the surfaces is invented, and has the advantages of less sample consumption, high accuracy, high capture efficiency, high expandability and simplicity and convenience in operation.

Description

Micro-fluidic chip based on ZnO nanowire glass microspheres grown on surface and application
Technical Field
The invention belongs to the technical field of biochips, and relates to a glass microsphere microfluidic chip based on ZnO nanowires growing on the surface and application thereof in exosome capture.
Background
Exosomes are double-layer membrane vesicle bodies with the diameter of 30-150 nm, and are mostly secreted into various body fluids such as blood, urine, breast milk, cerebrospinal fluid and the like by cells through the processes of endocytosis, fusion and efflux. Exosomes carry a variety of bioinformatic molecules, such as proteins, lipids and nucleic acids, and control the biological behavior of recipient cells by targeted delivery of these bioinformatic molecules to the recipient cells, playing an important role in intercellular information transfer. Research proves that almost all types of cells used by a human body can generate exosomes, and the exosomes secreted by different cells have different components and functions, so that the exosomes are expected to become novel biomarkers for clinical disease diagnosis, disease monitoring and prognosis judgment.
In recent years, nanotechnology and microfluidic technology have gradually become the most promising technologies for exosome capture detection due to their controllability in micro-nano scale, good biocompatibility, high detection flux, integratability, and the like. Due to its unique properties, nanomaterials have been widely used in the fields of research such as optics, electrochemistry, and biomedicine. Common one-dimensional nanostructures include carbon nanotubes, silicon nanowires, and ZnO nanowires. The ZnO as a wide bandgap semiconductor material has high length-diameter ratio, large specific surface area, good mechanical and chemical stability and abundant nanometer morphology, shows more excellent performance than bulk phase materials in the aspects of ultraviolet lasers, nanometer/gas sensors, field electron emission devices, molecular level nanometer photoelectronic devices and the like, and is widely applied to the fields of biochemical sensors and the like. Meanwhile, the surface of the ZnO nanowire is rich in various functional groups, and biomolecules with specific recognition capability can be grafted on the surface of the ZnO nanowire, so that the ZnO nanowire is considered to be one of the best tools for improving the capture specificity of exosomes.
The micro-fluidic chip adopts a micro-electro-mechanical processing technology similar to a semiconductor to construct a micro-channel system on the chip, transfers the experiment and analysis processes to a chip structure consisting of a path and a small liquid-phase chamber which are mutually connected, and adopts a micro-mechanical pump and other methods to drive the flow of a buffer solution in the chip and form a micro-channel after loading a biological sample and a reaction liquid, thereby finally realizing the automatic and continuous reaction on the chip. In recent years, the technology of exosome capture analysis based on microfluidic chips has attracted attention. How to organically integrate the ZnO nanowire and the microfluidic chip and fully utilize the advantages of the ZnO nanowire and the microfluidic chip in exosome capture has become a hot point and a difficult point concerned in the medical field to realize high specificity and high-efficiency capture of exosomes in complex samples.
Disclosure of Invention
The invention provides a novel micro-fluidic chip based on ZnO nanowire glass microspheres grown on the surface and application thereof, aiming at the problems in the traditional chip exosome capture.
In order to achieve the purpose, the invention adopts the following technical scheme:
a micro-fluidic chip of glass microspheres with ZnO nanowires growing on the surface is characterized in that a glass slide is used as a substrate, polydimethylsiloxane is used as a main body, functional micro-channels are etched on the polydimethylsiloxane, micro-channels are constructed by methods such as a photoetching technology and plasma etching (not limited to the method), a micro-mixer formed by the glass microspheres is filled in the micro-channels, the ZnO nanowires growing in situ by a chemical bath deposition method are arranged on the surfaces of the glass microspheres, surface antibody modification is carried out on the nanowires, and exosomes are captured specifically. The detection sample can be cell culture solution supernatant or various body fluid samples of clinical patients.
Preferably, the diameter of the glass microsphere is 540-960nm; the glass microspheres are of a face-centered cubic structure.
Preferably, the ZnO nanowire has a length of 0.5-1um, a diameter of 50-100nm, and a length-diameter ratio of up to 20.
Preferably, the glass slide has a length of 6.5-7.6 cm, a width of 2.0-2.5 cm and a thickness of 2mm; the polydimethylsiloxane is 3.5-4.6 cm in length and 1.0-1.5 cm in width; the overall length of the micro-channel is 1.2-1.5 cm, the widest part of the micro-channel is about 0.3-0.5cm, and the narrowest part of the micro-channel is about 0.8-1.2mm; the diameters of the sample inlet hole and the sample outlet hole are 0.8-1.2 mm.
Preferably, the ZnO nanowire surface-modified antibody is any one of CD63, CD9, CD81, TSG101 and GPC-1.
The preparation method of the glass microsphere micro-fluidic chip based on the ZnO nano wire growing on the surface comprises the following steps:
(1) Preparing a glass slide and polydimethylsiloxane, and etching a sample introduction channel, a sample discharge channel and a micro-channel connected with the sample introduction channel and the sample discharge channel on the polydimethylsiloxane;
(2) Performing surface silanization modification on polydimethylsiloxane, and filling glass microspheres which are closely arranged into a face-centered cubic structure into a microchannel;
(3) Preparing ZnO nanowires growing in preferred orientation by using a chemical bath deposition method: a ZnO seed layer is prepared on the glass microspheres in advance by adopting a laser molecular beam epitaxy method, so that nucleation points are provided for the deposition of ZnO in the chemical bath deposition process, and the nucleation work is effectively reduced; the method comprises the following steps of utilizing a high molecular material with uniformly distributed polar groups to combine with a hexamethylenetetramine solution and a zinc salt solution to react, and enabling ZnO crystal nuclei to directionally grow ZnO nanowires with one-dimensional structures on a seed layer;
(4) The ZnO nanowire on the surface of the glass microsphere is modified with a specific antibody, and the antibody can be specific to exosomes of different diseases, such as CD63, CD9, CD81, TSG101, GPC-1 and the like.
Preferably, the step (3) is specifically operated as follows: ultrasonically cleaning glass microspheres for 2-3 times by using acetone, absolute ethyl alcohol and distilled water respectively, wherein each time is 5-10min, and then drying by using nitrogen; melting ZnO by using a laser molecular beam epitaxial deposition system, and conveying the ZnO to the surface of the glass microsphere in the form of plasma plume to finish the deposition preparation of the seed layer, wherein the deposition temperature is 150-300 ℃, and the deposition time is 20-40min; ultrasonically cleaning the seed layer with acetone, anhydrous ethanol and distilled water for 5-10min, and blow-drying with nitrogen gas; placing the prepared glass microsphere containing the seed layer in a container containing 3-5 mol/L polyethyleneimine and 0.05-1 mol/L Zn (NO) 3 ) 2 Mixing with 0.05-1 mol/L hexamethylenetetramine, incubating at 85-95 deg.C for 6-12 h, cleaning, air drying, and baking at 200-400 deg.C for 20-40min;
the specific operation of the step (4) is as follows: immersing the chip of the glass microsphere with the grown ZnO nanowire into a 3-mercaptopropyl trimethoxy silane solution, and reacting for 1 hour at room temperature; washing the excess 3-MPS with 70% ethanol for 3 times; then immersing the chip into N- (4-maleimide butyryl) succinimide solution, and reacting for 30 min at room temperature; after washing with PBS, the chip was immersed in an antibody solution of an antibody to an exosome-membrane-specific protein, reacted at room temperature for 1-2 h, and then the chip microchannel was washed with PBS at a flow rate of 1-2.5. Mu.l/min using a syringe pump for 40-60 min, thereby completing antibody coating.
Preferably, the cell culture supernatant or the body fluid sample is injected into the chip for exosome capture, and then eluted with Tris-HCl buffer at a flow rate of 1-2.5. Mu.l/min, and the eluate is collected.
Compared with the prior art, the invention has the advantages and positive effects that:
the invention discloses a micro-fluidic chip based on glass microspheres with ZnO nanowires grown on the surfaces, which is prepared by modifying ZnO nanowires grown in situ on the glass microspheres with exosome-specific membrane protein antibodies, capturing exosomes in samples through antigen-antibody immune recognition specificity, and systematically integrating the microspheres and the micro-fluidic chip, and has the following advantages:
1. the sample consumption is less: as the reaction is only carried out in the microchannel, and only 20 mul is needed for filling the microchannel with the sample, the dead volume of the sample generated by an external pipeline is avoided, and the sample loading amount of the sample is effectively reduced.
2. The accuracy is high: according to the invention, the glass microspheres are used for filling each micro mixer in the micro channel, and the glass microspheres have no magnetism, so that the chip overcomes the limitation that signals are interfered with each other due to agglomeration of the magnetic microspheres, and the detection accuracy is greatly improved.
3. The capture efficiency is high: according to the invention, the ZnO nanowires are grown on the surface of the glass microsphere in situ, so that the surface area of the glass microsphere is effectively increased, and the capture efficiency of exosomes is improved.
4. The expandability is high: the invention adopts the form of the microfluidic chip, so that the exosome capturing system can be integrated with the microfluidic chip for sample pretreatment and the like, and the miniaturization development of an exosome separation-detection-analysis system is promoted.
5. The operation is simple and convenient: the microfluidic chip of the invention can directly separate exosome from a sample by targeting exosome specific membrane protein such as CD9, CD63, CD81, GPC-1 and the like, and has simple and convenient operation steps and short time consumption compared with the current gold standard (ultracentrifugation method) for exosome separation.
Drawings
FIG. 1 is a diagram of an exosome-capturing microfluidic chip in real object.
Fig. 2-3 are schematic diagrams of exosome-capturing microfluidic chips.
FIG. 4 is a graph showing the particle size distribution of particles in the eluate.
FIG. 5 is a graph comparing the contents of substances in the initial solution and the eluate.
FIG. 6 is a Western Blot image of the eluate.
The figures are numbered: 1 glass slide, 2 polydimethylsiloxane, 3 sample inlet holes, 4 sample outlet holes, 5 micro-channels, 6 micro-mixers and 7 glass microspheres.
Detailed Description
In order that the above objects, features and advantages of the present invention may be more clearly understood, the present invention will be further described with reference to specific embodiments. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments of the present disclosure.
As shown in fig. 1 to 3, the microfluidic chip of the present invention is composed of three parts, including a lower glass slide 1 and an upper polydimethylsiloxane 2; a sample inlet hole 3, a sample outlet hole 4 and a micro-channel 5 connected with the sample inlet hole and the sample outlet hole are etched on the upper polydimethylsiloxane 2; a micro mixer 6 is arranged in the micro channel 5, a glass microsphere 7 in a face-centered cubic shape is arranged in the micro mixer, znO nanowires 8 grow on the surface of the glass microsphere 7 in situ, and specific recognition molecules are decorated and modified on the ZnO nanowires.
The volume of the micro-fluidic chip is 7.6cm 2.5cm 2mm, the length of the upper polydimethylsiloxane 2 is 4.6cm, the width of the upper polydimethylsiloxane is 1.5cm, and the diameters of the sample inlet hole 3 and the sample outlet hole 4 are 1.2mm; the overall length of the micro-channel 5 is 1.5cm, the widest part of the micro-channel is about 0.5cm, and the narrowest part of the micro-channel is about 1.2mm; the diameter of the glass microsphere 7 is 960 mm; the ZnO nanowire 8 has the length of 0.5-1 mu m, the diameter of 50-100nm and the length-diameter ratio of 20; the specific recognition molecule is one of antibodies which can specifically recognize the exosome membrane surface marker.
Example 1
1. The micro-fluidic chip of the glass microsphere with the ZnO nano-wire grown on the surface comprises:
(1) the chip is composed of a glass slide glass on the lower half part and polydimethylsiloxane on the upper half part. The method specifically comprises the following steps: the length of the glass slide is 6.5cm-7.6cm, the width is 2.0cm-2.5cm, and the thickness is about 2mm; the polydimethylsiloxane is 3.5cm-4.6cm in length and 1.0cm-1.5cm in width.
(2) And etching a sample inlet channel, a sample outlet channel and a micro-channel connected with the sample inlet channel and the sample outlet channel on the polydimethylsiloxane. The method specifically comprises the following steps: constructing a micro-channel by a photoetching technology and a plasma etching method (which can be but is not limited to the method), wherein the diameters of the sample inlet hole and the sample outlet hole are 0.8mm-1.2mm; the overall length of the micro-channel is 1.2cm-1.5cm, the widest part of the micro-channel is about 0.3cm-0.5cm, and the narrowest part of the micro-channel is about 0.8mm-1.2mm.
(3) The micro-channel is filled with glass microspheres which are tightly arranged into a face-centered cubic structure to form the micro-mixer.
(4) Growing ZnO nanowires on glass microspheres in situ, which comprises the following steps: firstly, ultrasonically cleaning glass microspheres for 2-3 times by using acetone, absolute ethyl alcohol and distilled water respectively, wherein each time is 5min-10min, and then drying by using nitrogen; then melting ZnO by using a laser molecular beam epitaxial deposition system, and conveying the ZnO to the surface of the glass microsphere in the form of plasma plume to finish the deposition preparation of the seed layer (the deposition temperature is 150-300 ℃, and the deposition time is 20-40 min); finally, the seed layer is ultrasonically cleaned by acetone, absolute ethyl alcohol and distilled water for 5min to 10min respectively, and is dried by nitrogen for standby. Secondly, placing the prepared glass microspheres containing the seed layer into a container containing 3mol/L-5mol/L Polyethyleneimine (PEI) and 0.05mol/L-1mol/L Zn (NO 3) 2 And 0.05mol/L-1mol/L hexamethylenetetramine, incubating for 6h-12 h at 85-95 ℃, cleaning, air drying and baking for 20min-40min at 200-400 ℃ after growth is finished, and keeping the mixture for later use.
(5) The modified antibody on the ZnO nanowire specifically comprises the following steps: immersing the chip of the glass microsphere for growing the ZnO nanowire into a 3-mercaptopropyl trimethoxy silane (3-MPS) (5 percent, absolute ethyl alcohol) solution, and reacting for about 1 hour at room temperature; washing the excess 3-MPS with 70% ethanol for 3 times; then immersing the chip in a solution of N- (4-maleimidobutyryl) succinimide (GMBS) (0.25 mg/ml, dimethyl sulfoxide) to react for about 30 min at room temperature; after washing with PBS, the chip was immersed in an antibody solution of an exosome-specific protein (20 ug/ml-100ug/ml, PBS) and reacted at room temperature for 1 h-2h, and then the chip microchannel was washed with PBS at a flow rate of 1 ul/min-2.5. Mu.l/min for 40 min-60min using a syringe pump, completing antibody coating.
2. The application of the glass microsphere micro-fluidic chip based on the ZnO nano-wire grown on the surface in the exosome capture is as follows:
the method for capturing the exosome by utilizing the micro-fluidic chip for modifying the specific antibody. The method comprises the following specific steps: the cell culture supernatant or body fluid sample is injected into the chip at a flow rate of 0.5ul/min-1 mul/min for exosome capture, then 10 mM Tris-HCl buffer (pH 8.8) is used for elution at a flow rate of 1ul/min-2.5 mul/min, and the eluate is collected.
Example 2
1. Preparing a micro-fluidic chip: and etching a sample inlet, a sample outlet and a micro-channel connected with the sample inlet and the sample outlet on the polydimethylsiloxane by utilizing a photoetching technology and a plasma etching method.
The specific process is as follows:
(1) preparing a mould: preparing a glass slide and a silicon wafer, cleaning the silicon wafer, drying, spin coating (SU-8 photoresist), spin coating, pre-baking, exposing, thick baking, developing and the like to manufacture a mold (manufacturing a micro-channel).
(2) According to the weight ratio of polydimethylsiloxane: uniformly mixing a curing agent (medical liquid silica gel pouring sealant RTV 615) =10 according to the mass ratio, removing bubbles in the mixture, pouring the mixture on a mold, heating and curing at 80-90 ℃ for 1 hour, and carefully peeling off the mold and a cured substance without cooling to obtain the upper polydimethylsiloxane of the microfluidic chip.
(3) Processing polydimethylsiloxane (with the pressure of-98 Kpa in an instrument and the radio-frequency power of an ultraviolet lamp of 90W for 15 s) on the upper layer of the microfluidic chip by using plasma, carrying out surface silanization modification on the polydimethylsiloxane on the upper layer of the microfluidic chip by using GPTMS (5 percent, absolute ethyl alcohol) absolute ethyl alcohol solution at room temperature for 1 h, filling glass microspheres which are tightly arranged into a face-centered cubic structure into a microchannel, and punching sample inlet holes and sample outlet holes on an upper cover plate by using punching needles.
2. The method for growing the ZnO nanowire on the surface of the glass microsphere comprises the following steps: and growing ZnO nanowires growing perpendicular to the c axis in situ on the surfaces of the glass microspheres by using a chemical bath deposition method.
The specific process is as follows:
(1) seed layer preparation: firstly, ultrasonically cleaning glass microspheres for 3 times by using acetone, absolute ethyl alcohol and distilled water respectively, wherein each time is 10min, and then drying the glass microspheres by using nitrogen; then, melting ZnO by using a laser molecular beam epitaxial deposition system, and conveying the ZnO to the surface of the glass microsphere in a plasma plume form to finish the deposition preparation of the seed layer (the deposition temperature is 200 ℃, and the deposition time is 25 min); finally, the seed layer is ultrasonically cleaned by acetone, absolute ethyl alcohol and distilled water for 10min respectively, and is dried by nitrogen for standby.
(2) Growing ZnO nanowires on the glass microspheres containing the seed layers: mixing PEI solution (5 mmol/L), zn (NO 3) 2 And uniformly mixing the solution (0.05 mol/L) and the hexamethylenetetramine solution (0.05 mol/L) in a beaker, putting the glass microspheres on which the seed layer is deposited in advance into the solution, incubating for 9 hours at 90 ℃, after the growth is finished, cleaning, air-drying, and baking for 25 minutes at 300 ℃ to remove organic residues on the surface of the ZnO nanowire.
3. Capture of exosomes in pancreatic cancer cell culture supernatant
The method for capturing the exosome by the microfluidic chip comprises the following specific steps:
(1) coating BSA or modifying specific GPC-1 antibody on ZnO nanowire: immersing the chip of the glass microsphere for growing the ZnO nanowire into a 3-mercaptopropyl trimethoxy silane (3-MPS) (5 percent, absolute ethyl alcohol) solution, reacting for about 1 hour at room temperature, and washing redundant 3-MPS for 3 times by using 70 percent ethyl alcohol; then immersing the chip in a solution of N- (4-maleimidobutyryl) succinimide (GMBS) (0.25 mg/ml, dimethyl sulfoxide) to react for about 30 min at room temperature; after washing with PBS, the chip was immersed in a GPC-1 antibody solution (100 ug/ml, PBS) and reacted at room temperature for 2h, followed by washing the chip microchannel with PBS at a flow rate of 2.5. Mu.l/min using a syringe pump for 40min to complete the GPC-1 antibody coating.
Correspondingly, a chip of the glass microsphere for growing the ZnO nanowire is immersed in a 3-mercaptopropyltrimethoxysilane (3-MPS) (5 percent, absolute ethyl alcohol) solution to react for about 1 h at room temperature, and the redundant 3-MPS is washed for 3 times by 70 percent of ethyl alcohol; then immersing the chip in a solution of N- (4-maleimidobutyryl) succinimide (GMBS) (0.25 mg/ml, dimethyl sulfoxide) to react for about 30 min at room temperature; after washing with PBS, the above chip was immersed in BSA solution (100 ug/ml, PBS) and reacted at room temperature for 2h, followed by washing the chip microchannel with PBS for 40min at a flow rate of 2.5. Mu.l/min using a syringe pump, thereby completing BSA coating.
(2) Sample exosome detection: the supernatants (original solutions) of pancreatic cancer PANC-1 cell line cultures were injected at a flow rate of 1. Mu.l/min into GPC-1 antibody-and BSA-coated chips, respectively, for exosome capture, followed by elution with 10 mM Tris-HCl buffer (pH 8.8) at a flow rate of 2.5. Mu.l/min, and the respective eluates were collected. The nanoparticle tracking technology is used for analyzing the exosome separation effect, the immunoblotting experiment is used for representing the exosomes in the eluent, the nanoparticle tracking technology is used for analyzing the number of particles in the original solution and the eluent of a GPC-1 coated group and a BSA coated group, and the chip capture efficiency is detected.
As can be seen from fig. 4, nanoparticle tracking analysis found that the average particle size of the particles in the eluate and their main peak were within the particle size range of the exosomes. As can be seen from FIG. 5, nanoparticle tracking analysis finds that the number of exosomes in the eluate of the GPC-1 antibody coating group accounts for about 90% of that of the initial solution, which indicates that the exosome capture efficiency can reach 90%, while the number of exosomes in the eluate of the BSA coating group is less than 5%, which indicates that the nonspecific capture rate of the chip is extremely low. FIG. 6 is a Western Blot image of the eluate, and the Western Blot result shows that the eluate contains the specific markers of exosomes such as GPC-1, CD9 and TSG101, thus proving that the particles separated and captured by using the chip are exosomes. The microfluidic chip comprises a glass slide, a micro-channel and polydimethylsiloxane, wherein a sample inlet channel and a sample outlet channel are etched on the polydimethylsiloxane, and the micro-channel is connected with the sample inlet channel and the sample outlet channel, a micro-mixer is arranged in the micro-channel and consists of glass microspheres which are tightly arranged into a face-centered cube, znO nanowires are grown on the surfaces of the glass microspheres by adopting a chemical bath deposition method, and an exosome-specific membrane protein antibody is further modified on the nanowires grown on the glass microspheres, so that the efficient capture of exosomes in cell culture supernatants or body fluid samples is realized. The invention has simple preparation, low cost and low energy consumption, and has wide application prospect in the aspects of basic research, clinical disease diagnosis, monitoring, prognosis judgment, treatment and the like.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention.

Claims (7)

1. A microfluidic chip based on glass microspheres with ZnO nanowires grown on the surface is characterized in that the chip takes a glass slide as a substrate, takes polydimethylsiloxane as a main body, functional microchannels are etched on the polydimethylsiloxane, a micro mixer composed of the glass microspheres is filled in the microchannels, the surfaces of the glass microspheres are provided with the ZnO nanowires grown in situ by a chemical bath deposition method, and the surfaces of the ZnO nanowires are subjected to antibody modification;
the diameter of the glass microsphere is 540-960nm; the glass microspheres are of a face-centered cubic structure;
the ZnO nanowire has the length of 0.5-1um, the diameter of 50-100nm and the length-diameter ratio of 20.
2. The microfluidic chip of glass microspheres with ZnO nanowires grown on the surface according to claim 1, wherein the glass slide has a length of 6.5-7.6 cm, a width of 2.0-2.5 cm and a thickness of 2mm; the polydimethylsiloxane is 3.5-4.6 cm in length and 1.0-1.5 cm in width; the total length of the micro-channel is 1.2-1.5 cm, the widest part of the micro-channel is 0.3-0.5cm, and the narrowest part of the micro-channel is 0.8-1.2mm; the diameters of the sample inlet hole and the sample outlet hole are 0.8-1.2 mm.
3. The microfluidic chip of glass microspheres with ZnO nanowires grown on the surface according to claim 1, wherein the ZnO nanowire surface-modified antibody is any one of CD63, CD9, CD81, TSG101 and GPC-1.
4. Use of the glass microsphere micro fluidic chip based on ZnO nanowires with surface growth of any one of claims 1 to 3 in exosome capture.
5. The preparation method of the glass microsphere based on the ZnO nanowire with the surface growth as claimed in any one of claims 1 to 3, which is characterized by comprising the following steps:
(1) Preparing a glass slide and polydimethylsiloxane, and etching a sample introduction channel, a sample discharge channel and a micro-channel connected with the sample introduction channel and the sample discharge channel on the polydimethylsiloxane;
(2) Performing surface silanization modification on polydimethylsiloxane, and filling glass microspheres which are tightly arranged into a face-centered cubic structure into a micro-channel;
(3) Preparing ZnO nanowires growing in preferred orientation by using a chemical bath deposition method: preparing a ZnO seed layer on the glass microspheres in advance by adopting a laser molecular beam epitaxy method; the method comprises the following steps of utilizing a high molecular material with uniformly distributed polar groups to combine with a hexamethylenetetramine solution and a zinc salt solution to react, and enabling ZnO crystal nuclei to directionally grow ZnO nanowires with one-dimensional structures on a seed layer;
(4) And modifying the antibody on the ZnO nanowire.
6. The preparation method of the micro-fluidic chip of the glass microsphere based on the surface growth ZnO nanowire disclosed by claim 5, wherein the step (3) is specifically operated as follows: ultrasonically cleaning glass microspheres with acetone, anhydrous ethanol and distilled water for 5-10min for 2-3 times respectively, and washing with distilled waterDrying by nitrogen; melting ZnO by using a laser molecular beam epitaxial deposition system, and conveying the ZnO to the surface of the glass microsphere in a plasma plume form to finish the deposition preparation of the seed layer, wherein the deposition temperature is 150-300 ℃, and the deposition time is 20-40min; ultrasonically cleaning the seed layer with acetone, anhydrous ethanol and distilled water for 5-10min, and blow-drying with nitrogen gas; placing the prepared glass microsphere containing the seed layer in a container containing 3-5 mol/L polyethyleneimine and 0.05-1 mol/L Zn (NO) 3 ) 2 Mixing with 0.05-1 mol/L hexamethylenetetramine, incubating at 85-95 deg.C for 6-12 h, cleaning, air drying, and baking at 200-400 deg.C for 20-40min;
the specific operation of the step (4) is as follows: immersing a chip of the glass microsphere for growing the ZnO nanowire into a 3-mercaptopropyl trimethoxy silane solution, and reacting for 1 h at room temperature; washing the excess 3-MPS with 70% ethanol for 3 times; then immersing the chip into N- (4-maleimide butyryl) succinimide solution, and reacting for 30 min at room temperature; after washing with PBS, the coated chip was immersed in an antibody solution of an exosome membrane-specific protein, reacted at room temperature for 1-2 h, and then the chip microchannel was washed with PBS at a flow rate of 1-2.5. Mu.l/min using a syringe pump for 40-60 min to complete antibody coating.
7. The method for capturing exosomes by using the microfluidic chip prepared by the method of claim 5, wherein a cell culture supernatant or a body fluid sample is injected into the chip for capturing exosomes, and then Tris-HCL buffer solution is used for eluting at a flow rate of 1-2.5 μ l/min, and an eluent is collected.
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