CN113413933A - Micro-fluidic chip based on glass microspheres and application thereof - Google Patents
Micro-fluidic chip based on glass microspheres and application thereof Download PDFInfo
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- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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Abstract
The invention discloses a micro-fluidic chip based on glass microspheres for exosome capture, which has wide application prospect in the aspects of basic research, clinical disease diagnosis, disease monitoring, prognosis judgment, treatment and the like. The micro-fluidic control chip consists of a glass slide, a micro-channel and polydimethylsiloxane, wherein a sample inlet channel, a sample outlet channel and the micro-channel connected with the sample inlet channel and the sample outlet channel are etched on the polydimethylsiloxane, a micro-mixer is formed by filling glass microspheres which are tightly arranged into a face-centered cubic structure in the micro-channel, and specific antibodies are modified on the glass microspheres to specifically capture exosomes. The micro-fluidic chip can realize high-efficiency capture of cell culture solution supernatant and exosomes in human body liquid due to unique micro-channel design and specific antibody recognition, and has the advantages of high integration level, convenience in operation, no pollution, low energy consumption and the like.
Description
Technical Field
The invention belongs to the technical field of biomedical equipment, and relates to a micro-fluidic chip based on glass microspheres and application thereof.
Background
Exosome is a tiny vesicle with the diameter of 30 nm-150 nm secreted by cells, and is widely present in body fluids such as human blood, urine, cerebrospinal fluid, hydrothorax, ascites and the like. The outer layer of the exosome is a phospholipid bimolecular membrane, and the exosome contains a large amount of biomolecules such as protein, nucleic acid and the like from parent cells and can reflect the metabolic and pathological states of the parent cells, so that the exosome has great potential as a novel biomarker for disease diagnosis, disease condition monitoring and prognosis judgment. At present, a plurality of methods for capturing and separating exosomes are available, and a density gradient centrifugation method, an ultracentrifugation method, an immunoaffinity capture method and a sedimentation method mediated by sedimentation agents such as polyethylene glycol are common, but the methods are difficult to meet the requirements of basic research and clinical detection due to the limitations of expensive instruments, complex operation, long time consumption, low purity and concentration of the obtained exosomes and the like. Therefore, the development of new technologies and devices for capturing exosomes with high efficiency and high quality has become an urgent problem and hotspot in the field of exosome research.
The microfluidic chip is called as a lab-on-a-chip (lab-on-a-chip) or a micro-integrated analytical chip (micro-analytical systems), is a main platform for realizing the microfluidic technology, and can integrate operation units related to an analysis process, such as sample preparation, reaction, separation, detection and the like, on a micron-scale chip through the technologies of microelectronics, micromachining and the like and automatically complete the whole analysis process. Because the structure for containing the fluid in the microfluidic chip is in a micron-scale (at least one dimension), the instrument using the chip not only has greatly reduced volume, but also has obviously improved analysis efficiency, and simultaneously the loss of the sample and the reagent is correspondingly reduced, thereby fully reflecting the development trend of miniaturization, integration and portability of the prior laboratory equipment, and being widely applied to the fields of biology, chemistry, medicine and the like. In recent years, the exosome separation analysis technology based on the microfluidic chip is receiving attention. Researchers at the university of kansas developed an integrated microfluidic chip for capturing exosomes through CD 63-labeled magnetic microspheres in 2014, but the aggregation phenomenon of the magnetic microspheres easily causes mutual interference of signals of the microspheres, and affects the accuracy of the detection result.
Disclosure of Invention
The invention provides a novel micro-fluidic chip based on glass microspheres and application thereof, aiming at the problems in the traditional exosome capture.
In order to achieve the purpose, the invention is realized by adopting the following technical scheme:
a micro-fluidic chip based on glass microspheres takes a glass slide as a substrate, takes polydimethylsiloxane as a main body, and etches a micro-channel on the polydimethylsiloxane, the micro-channel contains a plurality of glass microspheres which are of face-centered cubic structures and are used for antibody modification, and two sides of the micro-channel are respectively provided with a sample inlet and a sample outlet.
Preferably, the diameter of the glass microsphere is 540-960nm, and the diameters of the sample inlet hole and the sample outlet hole are 0.8-1.2 mm.
Preferably, the width of the middle part of the micro-channel is 0.3-0.5cm, and the micro-channel gradually narrows from the middle part to the width of the sample inlet hole and the sample outlet hole.
The glass microsphere-based microfluidic chip is applied to capture any exosome of cell culture solution supernatant or serum sample, whole blood sample, urine sample, cerebrospinal fluid sample and hydrothorax and ascites sample.
The preparation method of the micro-fluidic chip based on the glass microspheres comprises the following steps:
(1) etching a sample inlet hole, a sample outlet hole and a micro-channel connected with the sample inlet hole and the sample outlet hole on the polydimethylsiloxane by utilizing a photoetching technology and a plasma etching method;
(2) uniformly mixing dimethyl siloxane and a curing agent, removing bubbles in the mixture, pouring the mixture on a mold, heating and curing the mixture, and stripping the mold and a cured substance to obtain a polydimethylsiloxane layer;
(3) treating the polydimethylsiloxane layer by using plasma, carrying out surface silanization modification on the polydimethylsiloxane layer by using GPTMS absolute ethyl alcohol solution, and periodically arranging and filling glass microspheres which are tightly arranged into a face-centered cubic structure in a microchannel; and (3) carrying out irreversible bonding on the upper-layer polydimethylsiloxane and the lower-layer glass slide, and punching a sample inlet hole and a sample outlet hole on the upper-layer polydimethylsiloxane by using a punching needle to obtain the prepared microfluidic chip.
Preferably, the mass ratio of the polydimethylsiloxane to the curing agent in the step (2) is 10:1, and the curing temperature is 80-90 ℃.
The application method of the micro-fluidic chip based on the glass microspheres comprises the following steps:
A. PDA pretreatment of glass microspheres in a chip, washing a chip microchannel by using NaOH solution, injecting APTES solution, incubating, washing the microchannel by using absolute ethyl alcohol, drying, baking, injecting dopamine solution into the chip microchannel on a hot plate, and washing by using Tris-HCL buffer solution;
B. specific antibody and coating, performing antibody immobilization at room temperature in the chip microchannel modified by the antibody, and washing the chip microchannel by using reaction buffer solution; the specific recognition molecule is one of antibodies which are specifically recognized and marked on the surface of an exosome membrane;
C. and (3) sample exosome capture, injecting the original solution into the chip for exosome capture, then eluting with buffer solution, and collecting respective eluates.
Compared with the prior art, the invention has the advantages and positive effects that:
sample consumption is low: as the reaction is only carried out in the microchannel, and the sample is filled in the microchannel and only needs 20 mul, the dead volume of the sample generated by an external pipeline is avoided, and the sample loading amount of the sample is effectively reduced.
High accuracy: the glass microspheres are filled in the micro-channel to form the micro-mixer, and the glass microspheres have no magnetism, so that the chip overcomes the limitation that signals are mutually interfered due to agglomeration of the magnetic microspheres, and the detection accuracy is greatly improved.
High expandability: 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.
Fourthly, 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. 1-4 are schematic diagrams of chip structures.
FIG. 5 is a graph showing the particle size distribution of particles in the eluate.
FIG. 6 is a graph comparing the content of substances in the initial solution and the eluate.
FIG. 7 is a Western Blot image of the eluate.
The figures are numbered: 1. glass slide 2. polydimethylsiloxane; 3. sample inlet hole, 4. sample outlet hole, 5. microchannel; 6. micro mixer 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.
Example 1
This example provides a process for preparing and using glass microsphere-based microfluidic chips.
As shown in fig. 1 to 4, the microfluidic chip comprises 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; the micro-channel 5 is internally provided with a micro-mixer 6 filled with glass microspheres 7 which are closely arranged into a face-centered cubic structure.
1. Preparing a micro-fluidic chip based on glass microspheres:
preparing a glass slide and a silicon wafer, and manufacturing a mold (the mold is used for manufacturing a micro-channel) through the steps of cleaning the silicon wafer, drying, spin coating (SU-8 photoresist), spin coating, pre-drying, exposure, thick drying, developing and the like.
According to the weight ratio of polydimethylsiloxane: uniformly mixing a curing agent (medical liquid silica gel pouring sealant RTV 615) =10:1 in mass ratio, removing bubbles in the mixture, pouring the mixture on a mold, heating and curing at 80-90 ℃ for 1 hour, and carefully stripping the mold and a cured substance after curing is finished to obtain the polydimethylsiloxane on the upper layer of the microfluidic chip.
Treating polydimethylsiloxane on the upper layer of the microfluidic chip by using plasma (the pressure in the instrument is-98 Kpa, the radio frequency power of an ultraviolet lamp is 90W, the treatment time is 15 s), reacting GPTMS (5 percent, absolute ethyl alcohol) solution at room temperature for 1h, carrying out surface silanization modification on the polydimethylsiloxane on the upper layer of the microfluidic chip, and filling glass microspheres which are tightly arranged into a face-centered cubic structure into a microchannel. And (3) carrying out irreversible bonding on the upper polydimethylsiloxane and the lower glass slide of the chip (completed by using a Plasma cleaner instrument), and punching a sample inlet hole and a sample outlet hole on the upper polydimethylsiloxane by using a punching needle to obtain the prepared microfluidic chip.
The glass microspheres are pretreated by Polydopamine (PDA) modification, and the PDA-modified glass microspheres can be subjected to subsequent antibody solidification. The pretreatment scheme specifically comprises the following steps: washing the chip microchannel with 1M NaOH solution, then injecting 10% (v/v) 3-Aminopropyltriethoxysilane (APTES) solution 500ul, and incubating the reactor at 50 ℃ for 3 h for amine functionalization; after incubation, washing the chip microchannel treated by APTES with absolute ethyl alcohol, drying and baking for 1h at 125 ℃; PDA modification of glass microspheres in the chip microchannel was accomplished by injecting 1.5 ml of dopamine solution (1 mg/ml) into the chip microchannel on a hot plate at 50 ℃ at a flow rate of 2.5. mu.l/min, followed by washing with 10 mM Tris-HCl buffer (pH 8.8).
The chip of this example consists of a glass slide in the bottom half and polydimethylsiloxane in the top half. The method specifically comprises the following steps: the length of the glass slide is 6.5-7.6 cm, the width is 2.0-2.5 cm, and the thickness is about 2 mm; the polydimethylsiloxane has a length of 3.5-4.6 cm and a width of 1.0-1.5 cm.
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 layer. 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 0.8-1.2 mm; 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, the narrowest part of the micro-channel is about 0.8-1.2mm, and the diameter of the glass microsphere is 960 nm.
A plurality of micromixers (not limited to the structure, but also other arrangement modes) which are periodically arranged according to a zebra crossing are distributed in the microchannel, and each micromixer is formed by filling glass microspheres which are closely arranged into a face-centered cubic structure, as shown in fig. 1-4.
2. The application of the glass microsphere-based microfluidic chip in exosome capture is as follows:
coating capture antibody on the chip micro-channel glass microsphere. The method specifically comprises the following steps: injecting a certain amount (20 ug/ml-100 ug/ml) of antibody into the chip microchannel modified by PDA, preserving at room temperature for 4h-6 h for immobilization of the antibody, and then washing the chip microchannel with reaction buffer solution at a flow rate of 1-2.5 μ l/min by using a syringe pump for 40-60min to complete antibody immobilization.
② a method for capturing exosome by using the micro-fluidic chip for modifying specific antibody. The method specifically comprises the following steps: the cell culture supernatant or body fluid sample is injected into the chip at a flow rate of 0.5-1. mu.l/min for exosome capture, and then eluted with 10 mM Tris-HCl buffer (pH 8.8) at a flow rate of 1-2.5. mu.l/min, and the eluate is collected.
Example 2
The fabrication of the chip in this example was consistent with example 1.
The method for capturing exosomes in pancreatic cancer cell culture solution supernatant comprises the following specific steps:
firstly, PDA pretreatment of glass microspheres in a chip: the chip microchannels were washed with 1M NaOH solution, 10% (v/v) APTES solution was injected, the reactor was incubated at 50 ℃ for 3 h for amine functionalization, the chip microchannels were washed with absolute ethanol after incubation, dried and baked at 125 ℃ for 1h, 1.5 ml of dopamine solution (1 mg/ml) was injected into the chip microchannels at a flow rate of 2.5. mu.l/min on a 50 ℃ hotplate, followed by washing with 10 mM Tris-HCL buffer (pH 8.8).
Specific GPC-1 antibody and BSA coating: GPC-1 antibody (50. mu.g/ml) was injected into a PDA-modified chip microchannel, and the immobilization of the antibody was carried out by keeping the chip microchannel at room temperature for 6 hours, followed by washing the chip microchannel with a reaction buffer solution at a flow rate of 2.5. mu.l/min using a syringe pump for 40 minutes, thereby completing the immobilization of the antibody. Accordingly, BSA (50. mu.g/ml) was injected into another chip microchannel after PDA modification, and then immobilized by keeping at room temperature for 6 hours, followed by washing the chip microchannel with a reaction buffer at a flow rate of 2.5. mu.l/min using a syringe pump for 40 minutes, thereby completing BSA immobilization.
③ detecting the secretion of the sample: the supernatants (original solutions) of PANC-1 cell line culture for pancreatic cancer were injected into GPC-1 and BSA coated chips at a flow rate of 1. mu.l/min for exosome capture, and then eluted with 10 mM Tris-HCl buffer (pH 8.8) at a flow rate of 2.5. mu.l/min, and the eluates were collected. The nanoparticle tracking technology analyzes the exosome separation effect, the immunoblotting experiment characterizes exosomes in eluent, the nanoparticle tracking technology analyzes the number of particles in original solutions of a GPC-1 coated group and a BSA coated group and in eluent, and the capture efficiency of the chip is detected.
FIG. 5 is a graph showing the particle size distribution of particles in the eluate. As can be seen from the figure, nanoparticle tracking analysis found that the average particle size of the particles in the eluate and its main peak were within the particle size range of the exosomes.
FIG. 6 is a comparison graph of the content of substances in the initial solution and the eluent, and the nanoparticle tracking analysis finds that the number of exosome particles in the eluent of the GPC-1 antibody coating group is about 80% of that of the initial solution, which indicates that the exosome capture efficiency can reach 80%, and the number of exosome particles in the eluent of the BSA coating group is less than 5%, which indicates that the nonspecific capture rate of the chip is extremely low.
FIG. 7 is a Western Blot image of the eluate, and the Western Blot result shows that the eluate contains the specific markers of the exosomes such as GPC-1, CD9 and TSG101, thereby proving that the particles separated and captured by using the chip are the exosomes.
The above description is only a preferred embodiment of the present invention, and not intended to limit the present invention in other forms, and any person skilled in the art may apply the above modifications or changes to the equivalent embodiments with equivalent changes, without departing from the technical spirit of the present invention, and any simple modification, equivalent change and change made to the above embodiments according to the technical spirit of the present invention still belong to the protection scope of the technical spirit of the present invention.
Claims (7)
1. A micro-fluidic chip based on glass microspheres is characterized in that a glass slide is used as a substrate, polydimethylsiloxane is used as a main body, a micro-channel is etched on the polydimethylsiloxane, the micro-channel contains a plurality of glass microspheres with face-centered cubic structures and used for antibody modification, and a sample inlet hole and a sample outlet hole are respectively arranged on two sides of the micro-channel.
2. The glass microsphere-based microfluidic chip according to claim 1, wherein the diameter of the glass microsphere is 540-960nm, and the diameters of the sample inlet hole and the sample outlet hole are 0.8-1.2 mm.
3. The glass microsphere-based microfluidic chip according to claim 1, wherein the width of the middle part of the microchannel is 0.3-0.5cm, and the microchannel gradually narrows from the middle part to the width of the sample inlet and outlet holes.
4. The use of the glass microsphere based microfluidic chip according to any one of claims 1 to 3 in any exosome capture device selected from the group consisting of cell culture supernatant or serum sample, whole blood sample, urine sample, cerebrospinal fluid sample, pleural effusion sample and ascites sample.
5. The method for preparing the glass microsphere-based microfluidic chip according to any one of claims 1 to 3, which is characterized by comprising the following steps:
(1) etching a sample inlet hole, a sample outlet hole and a micro-channel connected with the sample inlet hole and the sample outlet hole on the polydimethylsiloxane by utilizing a photoetching technology and a plasma etching method;
(2) uniformly mixing dimethyl siloxane and a curing agent, removing bubbles in the mixture, pouring the mixture on a mold, heating and curing the mixture, and stripping the mold and a cured substance to obtain a polydimethylsiloxane layer;
(3) treating the polydimethylsiloxane layer by using plasma, carrying out surface silanization modification on the polydimethylsiloxane layer by using GPTMS absolute ethyl alcohol solution, and periodically arranging and filling glass microspheres which are tightly arranged into a face-centered cubic structure in a microchannel; and (3) carrying out irreversible bonding on the upper-layer polydimethylsiloxane and the lower-layer glass slide, and punching a sample inlet hole and a sample outlet hole on the upper-layer polydimethylsiloxane by using a punching needle to obtain the prepared microfluidic chip.
6. The use method of the micro-fluidic chip based on the glass microspheres as claimed in claim 5, which is characterized by comprising the following steps:
A. PDA pretreatment of glass microspheres in a chip: cleaning the chip microchannel with NaOH solution, injecting APTES solution, incubating, washing the microchannel with absolute ethanol, drying, baking, injecting dopamine solution into the chip microchannel on a hot plate, and washing with Tris-HCL buffer solution;
B. specific antibody and coating: carrying out antibody immobilization at room temperature in the chip microchannel modified by the antibody, and washing the chip microchannel by using a reaction buffer solution;
C. sample exosome capture: injecting the original solution into a chip for exosome capture, then eluting with a buffer solution, and collecting the eluates.
7. The preparation method of the micro-fluidic chip based on the glass microspheres as claimed in claim 5, wherein the mass ratio of the polydimethylsiloxane to the curing agent in the step (2) is 10:1, and the curing temperature is 80-90 ℃.
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