CN116851047A - Microfluidic biochip, magnetic bead and kit - Google Patents
Microfluidic biochip, magnetic bead and kit Download PDFInfo
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- 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
- B01L3/502761—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 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G—PHYSICS
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- B—PERFORMING OPERATIONS; TRANSPORTING
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Abstract
The invention discloses a microfluidic biochip, magnetic beads and a kit, and relates to the technical field of biomedical engineering. The microfluidic biochip comprises a chip body, wherein the chip body is provided with a separation channel, a cracking zone and a detection zone; the microfluidic biochip integrates a plurality of functional areas, has an integrated detection function, and can be used for separation, detection, screening and the like of outer vesicles and contents thereof.
Description
Technical Field
The invention relates to the technical field of biomedical engineering, in particular to a microfluidic biochip, magnetic beads and a kit.
Background
The protein chips on the market at present mostly depend on large detection equipment (unit price is more than 100 ten thousand) and matched expensive chip-kit consumable materials (unit price is more than 5000 yuan), and mostly focus on the detection of limited types of free proteins with higher abundance, and no small equipment specially aiming at the separation-detection integration of the outer vesicles exists, so that the detection field aiming at important pathological monitoring indexes (neuron proteins and post-protein modification components) in the outer vesicles is more blank. The domestic protein chip industry is mainly focused on the middle-low end field, mainly introduces the outsourcing service of third-party equipment, and no mature pure domestic protein chip is sold in the market. In addition, the detection of the protein of the neuron-derived outer vesicle depends on targeted acquisition of the neuron-derived outer vesicle of a single population, and the products are not produced at home and abroad. The independent research and development production of chips for external vesicle proteomics and post-modification histology is still blank.
In view of this, the present invention has been proposed.
Disclosure of Invention
The invention aims to provide a microfluidic biochip, magnetic beads and kit, which can be used for separating and detecting outer vesicles and contents thereof (such as proteins, mRNA/miRNA, DNA, lipids and the like).
The invention is realized in the following way:
the invention provides a microfluidic biochip for detecting outer vesicles or the contents thereof (such as proteins, mRNA/miRNA, DNA, lipid, etc.), comprising: the chip body, the chip body is provided with the following functional area of intercommunication in proper order: a separation channel for separation of outer vesicles, a lysis zone in communication with the separation channel, and a detection zone in communication with the lysis zone.
One end of the separation channel is provided with a sample input port and a magnetic bead input port, when magnetic beads enter from the magnetic bead input port, the magnetic beads can be specifically combined with outer vesicles entering from the sample input port to form outer vesicle-magnetic bead complexes, and the outer vesicle-magnetic bead complexes are separated from a sample and enriched in the cracking zone under the action of external force;
the detection area is fixed with a capture object capable of specifically combining with a molecule to be detected; the molecule to be detected is derived from the outer vesicle.
The microfluidic biochip provided by the invention can realize enrichment and pyrolysis of external vesicles by arranging the pyrolysis zone, the pyrolysis zone can enrich the external vesicle-magnetic bead complex under the action of an external magnetic field force, and the external vesicles can be cracked after pyrolysis liquid is added into the zone, so that the external vesicles release contents (such as protein, mRNA/miRNA, DNA, lipid and the like) and the like. The contents flow into the detection zone, and the corresponding molecules to be detected (i.e., the contents generated by the cleavage of the outer vesicles) are captured by the capture object. Then adding corresponding reaction reagent to generate signal reflecting whether the molecule to be detected exists or not or the content of the molecule to be detected.
The type of capture object for the molecule to be detected can be reasonably selected by a person skilled in the art according to the molecule to be detected.
For example, when the molecule to be detected is a protein, the capture object may be an antibody or ligand to the protein; when the molecule to be detected is a DNA or RNA fragment, the capture can be a nucleic acid probe complementary to the DNA or RNA fragment.
Alternatively, in some embodiments, the molecule to be detected is selected from a protein, a nucleic acid, or a combination thereof.
Alternatively, in some embodiments, the nucleic acid is DNA, RNA, or a combination thereof.
Alternatively, in some embodiments, the capture is selected from an antibody, a ligand, a nucleic acid probe complementary to the nucleic acid, or a combination thereof.
Optionally, in some embodiments, the magnetic bead surface is modified with an antibody or ligand that specifically binds to the outer vesicle surface membrane protein.
The antibody modified by the magnetic beads can be selected according to the specific characteristics of the surface membrane proteins of the outer vesicles, wherein the surface membrane proteins of the outer vesicles from different tissues have certain heterogeneity, for example, the outer vesicles from neurons, and the specific membrane proteins comprise L1CAM, LRRK2, TREM-2 and the like. Also can be cancer tissue derived outer vesicle, and its specific membrane protein includes PDL1, FASL or TRAIL, etc.
It should be noted that, the selection of the type of the antibody or ligand binding to the surface membrane protein of the outer vesicle can be reasonably selected according to the protein object to be bound, which is easy to be realized by those skilled in the art, and any modification of the protein or ligand falls within the scope of the present invention.
It should be noted that, the antibodies or ligands modified by the magnetic beads may be labeled membrane proteins, such as CD63, which are specific to the outer vesicles; it should be noted that, in a specific application scenario or practice, the type of the antibody or ligand modified by the magnetic beads can be reasonably selected by those skilled in the art according to the detection requirement.
The above ligand is selected similarly to an antibody, as long as it can specifically bind to the membrane protein of the outer vesicle of interest, and any ligand falls within the scope of the present invention.
Neurodegenerative diseases are a general term for a group of diseases resulting from chronic progressive degenerative changes of central nervous tissue, and are associated with aging. Neurodegenerative diseases generally occur through key processes such as protein misfolding and aggregation, neuroinflammation, or altered cell signaling. The brain neurons release extracellular vesicles into the blood circulation, which directly reflect brain cell secretory activity, from which it is possible to screen for high value biomarkers directly associated with parkinson's disease. The ultra-high-sensitivity high-flux protein chip can analyze the content of various proteins by collecting trace blood samples and screen out protein biomarkers related to diseases. Thereby judging whether the organism has diseases or at which stage of the diseases, and realizing the accurate health management of the tested person. At present, an external vesicle protein chip system is not utilized, the parkinsonism secretion activity is studied from the perspective of neuron extracellular vesicles, and high-precision markers are searched, so that the technology is a new technological progress representing accurate medical treatment from the aspect of biochip working performance and pathological diagnosis. However, up to now the market has not been specific to biochips for analysis of exovesicle proteomics. The embodiment of the invention provides a microfluidic biochip for screening biomarker proteins from neuronal extracellular vesicles, which has great practical significance and urgency for early prevention and intervention of neurodegenerative diseases (such as Parkinson's disease), and effectively fills the technical gap that the biochip does not have a professional aspect for the field of proteomics detection in the outer vesicles at present.
Optionally, in some embodiments, the detection zone comprises one or more reaction chambers; the capture object is fixed in the reaction chamber.
The synchronous detection of different molecules to be detected can be realized by arranging a plurality of reaction chambers, and the detection flux and the efficiency are improved.
Optionally, in some embodiments, a sensor for collecting and transmitting detection signals is disposed within the reaction chamber.
Optionally, in some embodiments, the sensor is selected from the group consisting of an electrochemical signal sensor, an electrochemiluminescent signal sensor, an optical sensor, and the like.
It should be noted that the sensor is not limited to the electrochemical signal sensor and the electrochemical luminescence signal sensor, but any other signal sensor, no matter what sensor, is within the protection scope of the present invention.
Optionally, in some embodiments, a valve is disposed between the detection zone and the lysing zone for controlling the flow of liquid: when the valve is in an open state, the liquid in the cracking zone can flow to the detection zone; when the valve is in a closed state, the liquid in the cracking zone can be blocked from flowing to the detection zone.
It should be noted that in some embodiments, the valve may or may not be provided, and is reasonably selected according to the requirements of the detection area.
Optionally, in some embodiments, the surface of the magnetic beads is further modified with a layer of anti-fouling molecules.
The blood components are complex, the modification of the anti-fouling molecular layer can greatly shield the non-specific protein adsorption of the outer vesicle analysis in the blood, reduce false positive signals generated by background noise, and can be used for researching the fingerprint biological information of the target outer vesicle in a targeted and reliable way, thereby effectively improving the proteomics detection depth.
The anti-pollution capability of the magnetic beads can be improved through modification of the anti-pollution molecular layer, and the combination of nonspecific protein adsorption is avoided.
The components of the anti-fouling molecular layer are hydrophilic high molecular materials, zwitterionic polymers and polysaccharide high molecules. Optionally, in some embodiments, the component of the anti-fouling molecular layer is selected from one or a combination of several of APPC, PEG (Polyethylene glycol), PMPC (poly (2-methacryloyloxyethyl phosphorylcholine), PSBMA (poly (sulfobetaine methacrylate)), and PCBMA (poly (carboxybetaine methacrylate)).
It should be noted that any other polymer that can reduce nonspecific binding with anti-fouling can be used in the present invention, and all the polymers fall within the scope of the present invention.
Alternatively, in some embodiments, the anti-fouling molecular layer is formed by polymerization of a compound selected from the group consisting of: PSBMA and PMPC.
Stable targeted extraction and external vesicle purification in complex sample matrices (e.g., serum) is achieved by anti-fouling modification using bi-component amphiphilic molecules.
Optionally, in some embodiments, the chip body includes a base layer at the bottom and a chip layer attached to the base layer, the separation channel and the cleavage area are disposed on a side of the chip away from the base layer, and the detection area is disposed on a side of the chip layer close to the base layer.
Alternatively, in some embodiments, the sensor may be disposed on the chip layer or on the substrate layer, so long as it is capable of collecting and detecting the reaction signal of the detection zone. Optionally, in some embodiments, the substrate layer is made of a material selected from the group consisting of paper, glass, PDMS (polydimethylsiloxane), PMMA (polymethyl methacrylate), COC (cyclic olefin copolymer), PC (polycarbonate), and PS (polystyrene).
Optionally, in some embodiments, the material of the chip layer is selected from paper, glass, PDMS, PMMA, COC, PC, and PS.
It should be noted that, the chip of the present invention manufactured by using other high polymer materials also belongs to the protection scope of the present invention.
In another aspect, the invention provides a magnetic bead, the surface of which is modified with an antibody or ligand that specifically binds to an outer vesicle membrane protein, and an anti-fouling molecular layer.
The components of the anti-fouling molecular layer are hydrophilic high molecular materials, zwitterionic polymers and polysaccharide high molecules. Optionally, in some embodiments, the anti-contaminant polymer is selected from one or a combination of several of APPC, PEG, PMPC, PSBMA, and PCBMA.
In another aspect, the invention provides a kit for detection of an outer vesicle or its content, comprising a microfluidic biochip according to any one of the above and/or a magnetic bead according to any one of the above.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 is a schematic structural diagram of a microfluidic biochip for detecting or screening an outer vesicle protein provided in example 1.
Fig. 2 is a schematic view of the chip layer structure of the microfluidic biochip of example 1 (the dimensions of the functional regions in the drawing are only exemplary, and may be set as needed in other examples).
Fig. 3 is a schematic structural diagram of a microfluidic biochip in example 2.
Fig. 4 is a schematic diagram of a detection flow of the microfluidic biochip in example 2.
Fig. 5 is a photograph of a physical image of the microfluidic biochip of example 2.
FIG. 6 shows the result of SDS polyacrylamide gel electrophoresis of the anti-fouling magnetic bead conjugate in the example.
FIG. 7 is a schematic diagram of the anti-fouling modification of magnetic beads in the examples.
FIG. 8 shows the results of the detection of the anti-fouling ability of magnetic beads by APPC modification at different concentrations in example 4.
FIG. 9 shows Western blot analysis of the expression of proteins (CD 9 protein, syntenin protein, L1 cell adhesion molecule protein (L1 CAM)) in extracellular vesicles in examples.
Fig. 10 is a TEM image of extracellular vesicles in the example.
FIG. 11 is a nanotrajectory analysis (NTA) of the size distribution of extracellular vesicles in the examples.
FIG. 12 shows the sensitivity results of the microfluidic biochip in the example for detecting L1CAM proteins.
Fig. 13 is an electrochemical sensor result determination of the microfluidic biochip in the example.
FIG. 14 shows the results of specific detection of the microfluidic biochip in the examples.
The labels in fig. 1-3 are respectively: 100. 200-chip body, 110-separation channel, 111-sample input port, 112-immunomagnetic bead input port, 120-cleavage zone, 130-detection zone, 131-reaction chamber, 140-chip layer, 150-substrate layer, 160-valve, 170-electrochemical signal sensor.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
The present embodiment provides a microfluidic biochip having a structure as shown in fig. 1 and 2:
the chip comprises a chip body 100, wherein the chip body 100 comprises a basal layer 150 at the bottom and a chip layer 140 attached to the basal layer 150. The materials of substrate layer 150 and chip layer 140 may be suitably selected based on materials well known to those skilled in the art.
The surface of the chip layer 140 has the following functional areas: a separation channel 110 for separation of outer vesicles, a lysis zone 120 in communication with the outer separation channel 110, a detection zone 130 in communication with the lysis zone 120. In this embodiment, the detection zone 130 has one reaction chamber, and in other embodiments may be a plurality of reaction chambers. A valve 160 is provided between the detection zone 130 and the lysis zone 120 for controlling the flow of liquid: when the valve is in an open state, the liquid in the cracking zone can flow to the detection zone; when the valve is in a closed state, the liquid in the cracking zone can be blocked from flowing to the detection zone.
The separation channel 110 has a sample input port 111 and an immunomagnetic bead input port 112. The magnetic beads are modified with capture antibodies (e.g., capture antibodies that specifically bind to membrane proteins of neuronal outer vesicles) that specifically bind to membrane proteins of outer vesicles of a specific origin. The shape of the separation channel 120 may be set according to the actual situation, and is set in a serpentine shape (or clip shape) in this embodiment. The sample input port 111 and the immunomagnetic bead input port 112 have a Y-shaped structure.
The detection region 130 is immobilized with an immobilized antibody (i.e., a capture object, a specific antibody type may be reasonably selected according to the target protein to be detected) that can specifically bind to the target protein (i.e., the molecule to be detected, the protein released after cleavage of the outer vesicle), and in other embodiments, the capture object may be a ligand that specifically binds to the molecule to be detected, or when the detection molecule is a nucleic acid, the capture object may be a probe complementary to the molecule to be detected. An electrochemical signal sensor 170 is disposed within the detection zone 130. The electrochemical signal sensor can be connected and identified by an interface of the detection system, so that the acquisition and the reading of target signals in a detection area are realized, and the detection of molecules to be detected is further realized.
The separation channel 120 and the cleavage region 120 are disposed on a side of the chip layer 140 away from the substrate layer, the detection region 130 is disposed on a side of the chip layer 140 close to the substrate layer 10, and the sensor is disposed on the substrate layer and is capable of detecting and collecting detection signals of molecules to be detected in the reaction chamber.
In an embodiment, the functional area may be configured as a groove structure.
In this embodiment, the material of the chip layer 140 is PDMS, and the material of the base layer 150 may be glass. In other embodiments, the materials may be reasonably selected according to actual needs.
Example 2
The present embodiment provides a microfluidic biochip having a structure as shown in fig. 3, which is substantially the same as that of the microfluidic biochip of embodiment 1, except that in the present embodiment, the detection region 130 has a plurality of reaction chambers 131; each reaction chamber 131 communicates with the cracking zone 120. Each reaction chamber 131 is modified with an immobilized antibody. The immobilized antibodies modified in each reaction chamber 131 may be the same or different, and each reaction chamber is provided with a sensor.
By arranging a plurality of reaction chambers, the detection of a plurality of different target markers can be realized synchronously.
The method of screening the extracellular vesicle protein of the neuron is exemplified by the method of screening the extracellular vesicle protein of the above-mentioned example (see FIG. 4):
serum sample (or other liquid sample) is pushed from the sample input port by the external precise injection pump, and enters the separation channel, the immunomagnetic beads are pushed by the external precise injection pump to advance from the immunomagnetic bead input port, and the separation channel is contacted with and combined with the outer vesicle (the antibodies modified by the immunomagnetic beads specifically combine with membrane proteins on the outer vesicle), and after passing through the separation channel, the outer vesicle is separated from the sample (such as serum). The magnetic beads combined with the outer vesicles (outer vesicle-magnetic bead complexes) flow to the cracking zone, the outer vesicle-magnetic bead complexes are enriched in the cracking zone under the action of external force of a magnetic field, and accordingly, the outer vesicles are enriched in the zone, and the rest liquid components flow out and are collected and removed. When the bead enrichment step is completed, a lysis solution, such as 1% Triton X-100, is added to the lysis zone and the outer vesicles are lysed to release the internal proteins. And applying pressure through a pump, opening a valve, allowing the protein released by the outer vesicle to flow into a detection area, specifically binding and fixing the protein by a corresponding fixed antibody, and then adding an enzyme-linked detection antibody (such as horseradish oxidase-detection antibody) to form a sandwich structure of the fixed antibody, target protein and the enzyme-linked detection antibody. And then a substrate Lu Miluo and hydrogen peroxide are added to catalyze the horseradish oxidase, the product of the enzymatic reaction can emit light (namely, a mark can be detected, the luminous intensity can be collected by an electrochemical signal sensor and can be transmitted to a detector to be detected), and the luminous intensity is directly positively related to the protein content of the target outer vesicle. The ELISA antibody for target protein is fixed in the detection area, and the expression level (namely luminous intensity) of the proteins in the disease group and the healthy group is compared, so that the screening of the outer vesicle protein with high expression difference is realized.
The chip provided by the embodiment can further screen out highly relevant markers of diseases and construct a composite biomarker (alpha-Synuclein+X), so that the diagnosis precision is improved. For example, screening for biomarker proteins from extraneuronal vesicles, for early prevention and intervention in neurodegenerative diseases such as parkinson's disease, and the like.
Example 3
The reference preparation method of the microfluidic biochip provided by the embodiment comprises the following steps:
and (3) preparing a mould:
the first step is the preparation of the chip substrate, which requires cleaning and drying. Firstly, taking out a silicon wafer, putting the silicon wafer into a cleaning solution (concentrated sulfuric acid: hydrogen peroxide=3:1), and lightly shaking the dish every 2-3min to fully clean the silicon wafer; then, the silicon wafer is quickly cleaned by ultrapure water, and the surface of the silicon wafer is not required to be dried in the cleaning process; then, respectively treating the silicon wafer for 1min according to the sequence of ethanol and acetone to clean organic matters on the surface of the silicon wafer; cleaning the silicon wafer with ultrapure water again, placing the silicon wafer above an electric heating plate, placing the silicon wafer in the center of the electric heating plate for heating and dehydrating (200 ℃ for 10 min) after the moisture on the surface of the silicon wafer is completely evaporated, and rotating the silicon wafer during the period to uniformly heat the silicon wafer; after the heating is finished, the silicon wafer is cooled to the room temperature, and the silicon wafer is exposed to hexamethyldisilazane vapor for 5min, so that the adhesion of the photoresist on the surface of the silicon wafer substrate can be effectively enhanced.
And the second step is to spin-coat photoresist on the surface of the silicon wafer. Firstly, uniformly coating the photoresist at a low speed (500 rpm) for 15 seconds to spread the photoresist; then, the photoresist was homogenized at a high speed (4000 rpm) for 75 seconds to obtain a flat photoresist layer.
The third step is pre-baking to remove excess organic solvent from the photoresist and to release some of the photoresist stress. Setting parameters and keeping the baking sequence at 65 ℃ for 1min; maintaining at 95deg.C for 5min; kept at 65℃for 1min.
And fourthly, using a high-power ultraviolet light source and a mask containing chip design patterns to carry out ultraviolet exposure on the photoresist, wherein the exposure time is 90s.
Fifthly, post-baking the exposed silicon wafer (good adhesion between photoresist and the silicon wafer can be ensured during development) is required, and the temperature is kept at 65 ℃ for 1min; maintaining at 95deg.C for 5min; kept at 65℃for 1min. After the post-bake is completed, the design pattern can be slightly observed on the surface of the silicon wafer.
The sixth step is a development process. And (3) placing the silicon wafer subjected to post-baking in a dish, cooling to room temperature, slowly adding a developing solution until the silicon wafer is completely soaked, and alternately developing by shaking and standing to clearly see that the uncured photoresist on the silicon wafer is eluted by the developing solution in the process, so as to leave a cured structure. Through development for about 1-3min, the uncured photoresist can be completely removed, and then the developing solution on the surface of the silicon wafer is blown dry by a low-pressure air gun.
The seventh step is hard baking (also called hard mould) to remove residual solvent and reinforcing structure, and the parameters are set to 150 ℃ and kept for 5min.
And finally, placing the silicon wafer under a microscope for examination, marking, evaluating and storing.
And (3) preparing a chip:
the first step is the pretreatment of the mold. In order to effectively increase the adhesion between PDMS and the mold, a high-fidelity microfluidic chip is formed. Firstly, the prepared silicon wafer mould is stuck to the center of a culture dish (110 mm) by using a double-sided adhesive tape, then the silicon wafer mould is transferred into a vacuum dryer, 3-5 drops of trimethylchlorosilane are dripped into the dish beside the dish, and steam fumigation is carried out for 5min.
The second step is the weighing of PDMS prepolymer and chip curing. 25g (g can be adjusted according to the required chip height) of PDMS prepolymer (mass ratio A: B=10:1) is weighed; the prepolymer was thoroughly mixed by manual stirring, after which the prepolymer was slowly added to a dish with a mold; after pouring the adhesive, scraping the wall of the cup by using a tool, placing the dish in a vacuum dryer, and pumping bubbles under negative pressure; after the bubbles were completely withdrawn, the dish was placed in an oven and the chip was held at 80℃for 30min.
The third step is chip separation and assembly. Taking out the PDMS chip after solidification from the oven, cooling to room temperature, cutting the PDMS layer along the edge of the silicon wafer by using a specific cutter, and slowly separating the PDMS layer from the silicon wafer; and then, separating the PDMS chip from the whole into single chips according to a planned blank area by using a specific cutter, sorting and placing the chips according to different types, making relevant marks, and completing the preparation work of the microfluidic chip, wherein a prepared chip physical photo is shown in fig. 5.
Example 4
Capture capability detection of anti-pollution magnetic beads
The method comprises the following specific steps:
1. NHS modified magnetic bead with diameter of 1 micrometer
2. Preparing an antibody (such as anti-CD 63 antibody) solution (concentration of 0.1-2 mg/mL);
3. washing NHS-magnetic beads once, and activating carboxyl;
4. the supernatant was discarded and the antibody solution was added. Suspending vertically at room temperature, and incubating for 1-2 h;
5. and retaining the flow-through liquid. Washing the beads 4 times with M-Water;
6. removing the supernatant, adding APPC (4-aminophenylphosphocholine) with the same concentration as the antibody solution, and incubating for 1-2 h at room temperature under vertical suspension and in dark;
7. the supernatant was discarded, washed once with M-Water and 2 times with PBS;
8. adding preservation solution (0.1% proclin-300 PBS), and preserving at 4deg.C; anti-CD 63 antibodies and anti-fouling polymer modified magnetic beads (CD 63-MB) were obtained.
Detecting the ability of the magnetic beads to capture the outer vesicles:
(1) Taking CD63-MBs, and incubating with serum (obtained from blood of a patient by gradient centrifugation) at room temperature overnight; discarding the supernatant, collecting the magnetic beads, adding a lysate (0.1% Triton-X100), and lysing the outer vesicles; collecting supernatant, measuring protein concentration of BSA, subpackaging, and storing at-20deg.C for use.
(2) CD63-MBs were taken and incubated with serum. The unadsorbed material was removed and the bound material was eluted with 0.1% Triton-X100 solution. All samples were resuspended in loading buffer and separated by SDS polyacrylamide gel electrophoresis. Western blot analysis was performed with horseradish enzyme secondary antibody. The location of the molecular weight markers (in kilodaltons) is shown on the right. Lanes 1,2 and lanes 3,4 from different patients. By detecting the marker protein CD63 of the outer vesicle, it is determined whether the substance captured by CD63-MB is an outer vesicle sample. CD63 protein molecular weight 26kDa, western blot in FIG. 6 matches expected CD63 protein molecular weight. It can be concluded that the captured substance is present in the target protein CD 63.
In other embodiments, the method of this embodiment can be referenced to modify the corresponding antibodies to the magnetic beads according to any of their target proteins of interest to specifically bind to the outer vesicles, as would be readily apparent to one of skill in the art.
Example 5
Influence of APPC modification of different concentrations on anti-fouling capability of magnetic beads
1. The antifouling magnetic beads were prepared and different experimental groups were set up:
1) Washing the magnetic beads with a washing liquid three times;
2) Adding a protein (1 mg/mL, BSA protein modified with fluorescent molecules) solution, and incubating for 3 hours at room temperature;
3) Washing with PBS solution three times;
4) APPC solution (concentration: 0. 0.167, 0.33, 0.66, 1.32, 2.64 mg/mL) in the dark, incubating for 3h at room temperature;
5) Positive control group was set: ethanolamine (3 m, ph=9) was used as a blocking solution.
6) Washing with PBS three times;
7) The suspension is resuspended to a suitable concentration for later use.
2. Characterization of anti-fouling magnetic beads:
1) The beads were incubated with BSA-FITC for 1 hour at room temperature in the dark. BSA-FITC concentration: 1mg/mL;
2) PBS wash 3 times, maintaining bead concentration: 10mg/mL, care to avoid light;
3) Diluting the concentration of the magnetic beads to 1mg/mL, adding a lightproof 384-well plate, and keeping out of light with tin paper;
4) The fluorescence values at 495-525 mm were immediately tested on-machine.
The results are shown in fig. 7 and 8:
FIG. 7 shows the principle of anti-fouling modification of magnetic beads, avoiding adsorption of specific proteins by modification of anti-fouling polymers, and FIG. 8 shows that the fluorescence value of BSA-FITC increases with increasing APPC concentration until the concentration reaches 2.63mg/mL. The anti-fouling rate of APPC to the magnetic beads is gradually increased until 2.63mg/mL, and the anti-fouling rate is reduced, perhaps related to the long chain structure of the APPC. And APPC is used as an anti-fouling agent, and the effect is better than that of positive control ethanolamine.
Example 6
The embodiment provides a kit for detecting or screening an outer vesicle protein, which comprises a microfluidic biochip and magnetic beads;
the structure of the microfluidic biochip was essentially the same as in example 1, and the magnetic beads were essentially the same as in example 5, except that the magnetic beads were modified with anti-L1 CAM, LRRK2, or TREM-2 antibodies, and with 1.32mg/ml APCC.
Example 7
Application of microfluidic biochip: the microfluidic chip successfully separated extracellular vesicles.
The expression of four transmembrane proteins CD9, syntenin and the neuronal extracellular vesicle-specific membrane protein L1CAM, which were expressed positively in the outer vesicles compared to healthy and blank controls, was further confirmed using the microfluidic chip of example 1 to isolate neuronal extracellular vesicles, followed by Western blot of the extracellular vesicles (fig. 9). Captured extracellular vesicles are typically cupped in morphology, which can be seen in TEM images (fig. 10). NTA showed that the average size of extracellular vesicles isolated from the chip of example 1 was 148.5.+ -. 4.3nm at a concentration of 2.25X10 8 ±1.51×10 7 Particles/ml (FIG. 11).
From the standpoint of biomarkers, morphology, particle size and distribution, the microchip of example 1 can be used exclusively for isolating neuronal extracellular vesicles for downstream detection.
Example 8
L1CAM protein sensitivity
Using the microfluidic chip of example 1, L1CAM standard proteins with different concentrations were added to the detection zone at 300, 250, 150, 75, 25, 5, 1, 0.5, 0pg/mL, respectively, and incubated with the sensor for detection, as shown in fig. 12, the electrochemical signal value outputted gradually decreased with decreasing standard antigen concentration until the electrochemical signal value tended to coincide with the background signal value at a concentration of 0.5 pg/mL. The sensitivity of the chip sensor of example 1 to detect L1CAM antigen was 1pg/mL.
Example 9
Specific detection
Using the microfluidic chip of example 1, electrochemical reaction verification was performed with the addition of proteins (L1 CAM, CD 81) of relevant extracellular vesicles and other protein components (IgG, BSA, IL-6) in blood, respectively, to confirm the specificity of the chip. As shown in FIGS. 13 and 14, the chip has no electrochemical reaction with the proteins which may form infection and has good specificity. Therefore, the chip can specifically detect the specific target protein L1CAM of the extracellular vesicles from neuron sources.
To sum up:
(1) The embodiment of the invention provides a novel anti-pollution magnetic bead; realizes a high-purity external vesicle subgroup purification system with good stability and good anti-pollution effect. Compared with the method reported in the literature, the method is simple and convenient and has wider universality.
(2) The micro-fluidic chip for detecting or screening the outer vesicle protein provided by the embodiment of the invention is integrated with three modules of magnetic bead extraction, cleavage and detection. In the separation-detection integrated micro-fluidic chip, the outer vesicles extracted by the magnetic beads release inner protein components after being cracked and are identified and combined by the antibodies on the chip, and then the antibodies are detected by the enzyme labels which are mixed in advance, so that a sandwich structure of primary antibody-antigen-secondary antibody is formed. Finally adding chemiluminescent reagent (such as lumino+H) 2 O 2 ) The detection of the target protein can be completed by connecting the chip to the detector. The content of the protein marker is directly reflected by the luminous intensity, so that different disease groups or proteins with large expression difference between the disease groups and the healthy control group are screened out. The detection area of the chip can customize the detection content individually according to the user requirement, but is not limited to a small numberAnd (3) detecting the target object.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A microfluidic biochip for detection of outer vesicles or their contents, comprising: the chip body, the chip body is provided with the following functional areas that communicate in proper order: a separation channel for separating outer vesicles, a cleavage zone communicated with the separation channel, and a detection zone communicated with the cleavage zone;
one end of the separation channel is provided with a sample input port and a magnetic bead input port, when magnetic beads enter from the magnetic bead input port, the magnetic beads can be specifically combined with outer vesicles entering from the sample input port to form outer vesicle-magnetic bead complexes, and the outer vesicle-magnetic bead complexes are separated from a sample and enriched in the cracking zone under the action of external force;
the detection area is fixed with a capture object capable of specifically combining with a molecule to be detected; the molecule to be detected is derived from the outer vesicle.
2. The microfluidic biochip of claim 1, wherein the magnetic bead surface is modified with antibodies or ligands that specifically bind to the outer vesicle surface membrane protein;
preferably, the molecule to be detected is selected from a protein, a nucleic acid, or a combination thereof;
preferably, the nucleic acid is DNA, RNA, or a combination thereof;
preferably, the capture object is selected from an antibody, a ligand, a nucleic acid probe complementary to the nucleic acid, or a combination thereof.
3. The microfluidic biochip of claim 2, wherein the detection zone comprises one or more reaction chambers; the capture object is fixed in the reaction chamber;
preferably, a sensor for collecting and transmitting detection signals is arranged in the reaction chamber;
preferably, the sensor is selected from the group consisting of an electrochemical signal sensor, an electrochemiluminescent signal sensor and an optical sensor.
4. The microfluidic biochip of claim 3, wherein the surface of the magnetic beads is further modified with an anti-fouling molecular layer; preferably, the components of the anti-fouling molecular layer are hydrophilic high molecular materials, zwitterionic polymers and polysaccharide high molecules;
preferably, the component of the anti-fouling molecular layer is selected from one or a combination of a plurality of APPC, PEG, PMPC, PSBMA and PCBMA.
5. The microfluidic biochip according to claim 1, wherein the chip body comprises a substrate layer at the bottom and a chip layer attached to the substrate layer, the separation channel and the cleavage area are disposed on a side of the chip away from the substrate layer, and the detection area is disposed on a side of the chip layer close to the substrate layer.
6. The microfluidic biochip according to claim 5, wherein the substrate layer is made of a material selected from the group consisting of paper, glass, PDMS, PMMA, COC, PC, and PS.
7. The microfluidic biochip according to claim 6, wherein the material of the chip layer is selected from the group consisting of paper, glass, PDMS, PMMA, COC, PC, and PS.
8. A magnetic bead, characterized in that the surface is modified with an antibody or ligand that specifically binds to an outer vesicle membrane protein, and an anti-fouling molecular layer.
9. The magnetic bead according to claim 8, wherein the components of the anti-fouling molecular layer are hydrophilic high molecular materials, zwitterionic polymers, and polysaccharide high molecular materials;
preferably, the anti-fouling molecular layer is one or a combination of a plurality of APPC, PEG, PMPC, PSBMA and PCBMA.
10. Kit for the detection of outer vesicles or their contents, characterized in that it comprises a microfluidic biochip according to any one of claims 1 to 7 and/or magnetic beads according to claim 8 or 9.
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