CN110339874B - Microfluidic device for exosome separation and surface protein detection and use method - Google Patents
Microfluidic device for exosome separation and surface protein detection and use method Download PDFInfo
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Abstract
The invention relates to a microfluidic device for exosome separation and surface protein detection and a using method thereof. The invention can realize the separation and detection of exosomes on the same chip system, provides an integration platform and a method for the separation and detection of exosomes, which have the advantages of simple operation, low cost and small sample amount, and has good application prospect.
Description
Technical Field
The invention belongs to the field of exosome detection, and particularly relates to a microfluidic device for exosome separation and surface protein detection and a using method thereof.
Background
Exosomes (exosomes) are one type of Extracellular Vesicles (EVs), which are microvesicles coated with a phospholipid bilayer, with a size between 30-150nm, actively secreted by cells into the extracellular environment. The exosome contains various biomolecular components including protein, DNA, RNA, etc. Exosome surfaces have multiple antigens such as CD9, CD63, EpCAM, etc. Exosomes, after secretion, can be transferred through the peripheral blood circulation, targeting parental cellular information and regulatory elements to the target region. This exosome is closely related to the occurrence, development and metastasis of cancer. Given the important role played by exosomes in cancer development and metastasis, we can discriminate analysis by isolating and detecting exosomes, detect the presence of early stage cancer and monitor cancer development.
As a marker for liquid biopsy, Circulating Tumor Cells (CTCs) are only a fewIn 1ml of human peripheral blood, compared with 10 exosomes9And has obvious advantages. The difficulty of exosome separation detection mainly lies in that the exosome separation detection is small in size (30-150nm), and the size of the particle size of a nanometer level makes traditional size sorting difficult to realize. If the separation and enrichment of exosomes can be realized, the number and molecular characteristics of exosomes can be analyzed, and a powerful basis is provided for cancer diagnosis analysis and targeted drug therapy.
The classical separation methods of exosomes today are ultracentrifugation and gradient centrifugation, which require separation at very high centrifugation rates (100,000 × g), and have the problems of high sample consumption, high impurity content, low exosome purity, etc. And other methods, e.g. kit methods (e.g. ExoQuick)TMKit) whose separation components are kept secret by the merchant, the specific separation product is actually unknown, the academic community is currently in doubt, the membrane filtration method is inefficient, and it is not possible to separate exosomes from other impurities of similar size, such as other hetero-proteins.
The micro-fluidic chip technology is a new technology developed in recent years, is rapidly developed to a plurality of fields such as biology, chemical medicine and the like, and shows wide application prospect. The technology has the advantages of low consumption, high sensitivity, wide applicability and the like, and has proved to have great development potential in the fields. The micro-channel of the micro-fluidic chip is generally in the micron level, can be freely designed and combined to realize different requirements, and can make up the defects of the current exosome separation method. The current microfluidic technology is increasingly widely applied in the field of biological detection, and researches on exosomes are gradually developed, but the current microfluidic chip researches on exosomes have some defects, such as low detection sensitivity, complex detection instruments and materials, difficulty in realizing separation and detection integration and the like.
Disclosure of Invention
The invention aims to solve the technical problem of providing a micro-fluidic device for exosome separation and surface protein detection and a using method thereof, and overcoming the defects of complex operation, single function, low specificity, complex process, high cost and the like in the traditional exosome separation and detection method.
The invention provides a microfluidic control device for exosome separation and surface protein detection, which is sequentially connected with a sample injection pump, a microinjector, a microfluidic chip provided with a magnet and a waste liquid collector, and is characterized in that: the microfluidic chip comprises an exosome separation area and a detection area; wherein, the separation area comprises a sample inlet, a serpentine channel and a circular incubation cavity; the detection area comprises a micro-column array and a sample outlet; the separation region and the detection region are connected by a channel.
The microfluidic chip is formed by bonding a cover glass substrate and a PDMS chip.
The sample inlet is connected with the serpentine channel through a gradually reduced transition region.
Preferably, the transition region body is 900 μm wide.
The micro-column array is an array formed by long-strip micro-columns with cylindrical sections and used for capturing and uniformly distributing immune complexes. The capture sites are the interval between every two micro-columns, wherein the interval between the micro-columns is slightly smaller than the size of the immune complex, so that the immune complex can be intercepted to avoid loss.
The last row of the micro-column array is provided with a blocking column so as to avoid the omission of immune complexes under the condition of excess.
The magnet is arranged above and below the circular incubation cavity and used for generating a magnetic field in the vertical direction in the incubation micro-cavity so as to capture and retain the immunomagnetic beads.
The size of the snake-shaped channel, the size, the number and the arrangement mode of the circular incubation micro-cavity and the micro-column array in the micro-fluidic chip can be changed according to actual requirements. The size and spacing of the micropillar array are designed to better capture immune complexes, while avoiding situations where the immune complexes are missing from the gap or cannot be captured.
The heights of the micro-fluidic chips are uniform, and are preferably 20-30 μm and most preferably 20 μm.
Preferably, in the microfluidic chip, the cross-sectional length of the cylindrical micro-column in the micro-column array is 90 μm, the width is 30 μm, the radius is 15 μm, and the distance between every two micro-columns is 14 μm. The inlet and outlet ends of the micro-column array are distributed up and down to generate gradient liquid flow pressure from top to bottom and from left to right. The lower right part is a sample outlet area, is connected with the circular sample outlet through a linear micro-channel and is connected with a waste liquid collector.
Preferably, the width of the serpentine channel is 100 μm.
Preferably, the radius of the circular incubation cavity is 800 μm.
The serpentine channel of the invention functions as follows: the snake-shaped channel is used for stabilizing liquid flow, so that the magnetic microspheres can stably run in the microfluidic chip, the blockage and the escape are avoided, and the subsequent capture, retention and release of large-size immune complexes (d is 15 mu m) in the microfluidic chip are realized.
The function of the micro-column array of the invention is as follows: when the 'magnetic bead-exosome-quantum dot' immune complexes are released, the immune complexes enter the array and are uniformly and orderly arranged at the micro-column gap under the action of pressure difference generated by fluid dynamics to observe the immunofluorescence intensity of each independent immune complex, so that signal cross interference among the complex spheres is avoided.
The magnet of the invention has the following functions: the magnetic field strength was further increased by using a cover glass as a substrate to reduce the distance between the two magnets for placement above and below the circular incubation microcavity to generate a longitudinal magnetic field in the incubation experiment. During the detection process, the magnetic poles are removed to facilitate the immune complex to flow into and arrange in the detection area.
The invention adopts the micro-injector and the sample pump, and adopts the mode of combining positive pressure sample introduction and negative pressure extraction, thereby accurately controlling the sample introduction flow rate. Positive pressure sample introduction is used in the process of exosome separation labeling, and negative pressure extraction is adopted when immune complexes are arranged in the micro-column array.
The capture principle of the immune complex in the microfluidic chip is based on the fluid diffusion principle of transverse and longitudinal fluid pressure difference.
The invention also provides a use method of the exosome separation and surface protein detection microfluidic device, which comprises the following steps:
(1) sequentially and positively introducing the experimental sample groups into the microfluidic chip for controlled separation and incubation marking;
(2) after the experimental sample group is sequentially introduced into the microfluidic chip and is incubated and marked, introducing washing liquor to wash the microfluidic chip; then, negative pressure extraction is carried out;
(3) the arrangement condition of the immune complex and the intensity of quantum dots under a bright field and a fluorescent field are observed in real time under a microscope, the protein expression of different samples in an experiment is analyzed and distinguished, and the fluorescence intensity is measured through microscopic shooting and software analysis.
The experimental sample group in the step (1) comprises capture magnetic beads, a sample to be detected and a quantum dot detection probe.
The introduction flow rate in the step (1) is 1-2 μ l/min.
The negative pressure extraction flow rate in the step (2) is 10-30 ml/h.
In the using process, a buffer solution is introduced to wash the microfluidic chip, and then the immune complexes are sequentially and orderly arranged in the micro-column array gaps of the microfluidic chip under the action of a negative liquid flow to realize single detection, so that the fluorescence interference caused by the stacking of magnetic beads is avoided.
According to the invention, before sample introduction, a sample is subjected to conventional pretreatment, a conventional centrifuge is used for gradient centrifugation, the highest centrifugation speed is 10000 g, the sample is used for removing pollutants such as cell debris precipitation and the like in the sample, the adsorption is avoided to generate interference fluorescence, and the requirement can be met by using the centrifuge in a common laboratory. And the chip structure is small, 4 structure integrated chips are realized on one glass slide at present, and the simultaneous detection of a plurality of samples can be realized through a multi-channel sample injection pump according to actual requirements.
The invention separates and detects two modules separately, which can avoid cross interference of fluorescence and is convenient for replacing modules. If exosome is required to be separated and then is led out for other detection, only the front half part can be manufactured and used, and only the rear half part can be used in the same way. And the operation and detection do not need the advanced chip surface modification process, the separation and the marking of the exosome are finished on the chip, and the off-chip pre-incubation process is not needed.
Advantageous effects
(1) The invention simultaneously realizes the separation and detection of exosome without additional processing methods such as filtration, precipitation and the like,
(2) the invention greatly improves the micro-dosage sample (as low as 10000 times of 1 mul diluted sample (2.4 x 10)3Mu/l)) has the advantages of high efficiency, low detection limit, high specificity, convenient operation and the like.
(3) The chip is low in cost, and on one hand, PDMS can be repeatedly poured for utilization after the silicon chip mould is etched; on the other hand, the chip consumables are only the cover glass and the PDMS, and the magnet pair can be repeatedly used; meanwhile, the microchip system structure is a single layer, so that the manufacture of a double-layer microstructure is avoided, and an extra electrode field or an extra sound wave field is not required to be added, so that when a channel of the microchip system is etched on a die, the process can be finished at one time, the steps of alignment, alignment and the like are not required, the process is simple, and the complex procedures of later chip bonding alignment, electrode modification and the like are omitted.
Drawings
FIG. 1 is a schematic diagram of a microfluidic chip according to the present invention;
FIG. 2 is a schematic structural diagram of an exosome-separating region in a microfluidic chip of the present invention;
FIG. 3 is a schematic structural diagram of an exosome-detecting region in a microfluidic chip of the present invention;
FIG. 4 is a graph showing the actual distribution of immune complexes in the exosome-detecting region in the microfluidic chip of the present invention;
FIG. 5 is a graph showing the results of the test performed by the microfluidic chip of the present invention when the samples injected in example 3 are positive circulating tumor cells H446, negative control human endothelial cells HUVEC, and a culture medium blank control group, respectively;
FIG. 6 is a statistical chart of the intensities detected by the microfluidic chip of the present invention when the sample injected in example 3 is positive circulating tumor cell H446, negative control human endothelial cell HUVEC and culture medium blank control group, respectively.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Example 1
Preparation of microfluidic chip
(1) As shown in fig. 1, the required structural graph is drawn by using AutoCAD drawing software, and the structural dimension is as described above. And manufacturing a mask plate, coating glue on a four-inch monocrystalline silicon wafer serving as a substrate, photoetching, deep reactive ion etching (deep RIE) to etch the height of a chip channel, removing the glue, and cleaning to obtain the silicon wafer mold with the microstructure.
(2) Placing a silicon chip mould and an open centrifugal tube filled with 10 mul of fluorosilane in a vacuum pan, vacuumizing to negative pressure to vaporize and spread fluorosilane in the vacuum pan, and standing the mould in the fluorosilane steam atmosphere for 12 hours. In a ventilation kitchen, the vacuum pan is opened, and after ventilation for 1 hour, the silicon wafer is taken out. The purpose of this step is to deposit a thin layer of fluorosilane on the surface of the silicon wafer, thereby avoiding the adhesion of the PDMS chip to the silicon wafer.
(3) Respectively weighing PDMS prepolymer and curing agent according to a ratio of 10:1(w/w), mixing and stirring uniformly, placing in a vacuum pan for vacuumizing, and standing for 30min under the condition of keeping negative pressure. And after the PDMS has no bubbles, pouring the PDMS on a silicon wafer mould, standing for 30min, and then putting the PDMS in an oven at 95 ℃ for heating for 1 h. And finally, stripping the cured PDMS layer from the mold, punching according to the sample inlet and outlet on the pattern, and cutting off the excessive part outside the structure. And finally, putting the structural surface of the PDMS chip upwards and the cover glass substrate into a plasma cleaning machine for cleaning for 1min, and quickly attaching the structural surface and the cover glass substrate together after being taken out, thereby completing the packaging of the microfluidic chip.
Example 2
Functional modification of polystyrene microsphere
(1) Mu.l of stock solution of carboxyl microspheres was mixed in 90. mu.l of PBS phosphate buffer solution and resuspended by pipetting. After this time, the mixture was centrifuged at 6000r/min for 5min, the supernatant was removed, the pellet was retained, and the procedure was repeated twice to wash the microspheres, and finally resuspended in 80. mu.l of PBS.
(2) EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) powders were mixed in acidic (pH 5) PBS at a concentration of 10 mg/ml. Incubate at room temperature with shaking for 30min, then repeat the procedure
(1) And resuspended in 100. mu.l of PBS buffer.
(3) Mu.g of anti-CD9 capture antibody was added to the microsphere suspension in step (2), vortexed to mix well, and then incubated for 4 hours at 37 ℃ using a mixer comfort incubator. After the incubation, the centrifugation in step (1) was repeated and finally resuspended in 100. mu.l of PBS buffer.
(4) The modified microspheres were resuspended in 1% BSA (bovine serum albumin) in PBS to block non-specific binding and then kept at 4 ℃ until use.
Example 3
Separation and detection of exosomes of samples using microfluidic chip prepared in example 1
(1) The sample group to be tested (anti-CD 9 labeled magnetic beads prepared in example 2, sample to be tested, quantum dots with antibody attached, and the dosage of the three in turn 5. mu.l, 1. mu.l, and 10. mu.l) was aspirated into a micro syringe for standby. The chip packaged in example 1 is placed in a vacuum pan and vacuumized for 30min, a pipette is used for sucking 100 μ l of PBS solution, then the pipette head is inserted into the chip and stands for 10min, and the PBS solution spontaneously enters and fills the whole microstructure chip by means of negative pressure in the microfluidic channel.
(2) Through the method of microscope alignment, place the magnet in circular incubation microcavity about the top down to catch the magnetic bead in incubating the chamber, pay attention to the position and align, avoid the magnetic pole deviation to cause the reduction of catching efficiency.
(3) A microsyringe filled with magnetic beads labeled with immune antibodies (CD9) in advance is connected to a sample injection pump, the microsyringe is connected with the chip through a thin tube and is introduced into the round incubation microcavity cavity, and the microsyringe flows into the round incubation microcavity cavity and is retained in the round incubation microcavity cavity, wherein the preferred flow rate is 2 mul/min.
(4) Replacing the micro-injector, introducing the experimental sample to be tested into the microchip system through the sample inlet, stopping sample introduction through the sample inlet, and incubating at room temperature in situ, wherein the preferable parameter is the flow rate of 1 mul/min, and the incubation time of 1min is 1min after circulation.
(5) Replacing the micro-injector, introducing the quantum dot solution marked with the antibody into the microchip system through the injection port, stopping injecting the sample through the injection port, and performing in-situ room temperature incubation at the same time, wherein the preferable parameter is the flow rate of 1 mu l/min, the flow is 10min, and the incubation time is 20 min. The sampling process of the flow rate quantum dot detection probe solution needs light-proof treatment, and the influence on efficiency caused by light excitation of quantum dots is avoided.
(6) After incubation, the magnet was removed and the injection port was changed to a tip connection with 100 μ l pbs added to flush the channel and prevent air from entering through the injection port. The sample outlet is connected with the upper needle cylinder, the sample is extracted outwards in a negative pressure mode, and fluid pressure gradient in the microfluidic system is formed through negative pressure of air in the tube, so that the magnetic bead-exosome-quantum dot immune complexes are guided to be uniformly and orderly arranged in gaps of the cylindrical microcolumn array; the preferred negative pressure extraction flow rate is 10ml/h to 30 ml/h.
(7) Under the condition of keeping out of the sun, an inverted microscope equipped with a mercury lamp is used for observing the complex arrangement and immunofluorescence in a bright field and a fluorescence field, and image pro plus is used for shooting and carrying out intensity analysis statistics.
(8) When the sample samples are positive circulating tumor cells H446, negative control human endothelial cells HUVEC and a culture medium blank control group respectively, the experimental result detected by the microfluidic chip is shown in figure 5, and the positive samples and the negative samples show obvious difference visible to naked eyes.
(9) The experimental procedure of this example was completed within 76min, with high efficiency compared to ultracentrifugation (sample separation required more than 2 hours, not including testing).
(10) As shown in fig. 6, the positive sample showed a macroscopic difference in fluorescence intensity from the negative sample, and the fluorescence intensity analysis software showed that the intensity of the positive sample was 10 times or more that of the negative sample (control group 4.15, experimental group 41.06).
Claims (6)
1. A method of using a microfluidic device for exosome separation and surface protein detection, comprising:
(1) sequentially and positively introducing the experimental sample groups into the microfluidic chip for controlled separation and incubation marking; the micro-fluidic device is sequentially connected with a sample injection pump, a micro-injector, a micro-fluidic chip provided with a magnet and a waste liquid collector, and the micro-fluidic chip comprises an exosome separation area and a detection area; the separation area comprises a sample inlet, a serpentine channel and a circular incubation cavity; the detection area comprises a micro-column array and a sample outlet; the separation area and the detection area are connected through a channel; the magnets are arranged above and below the circular incubation cavity; the experimental sample group comprises a capture magnetic bead, a sample to be detected and a quantum dot detection probe;
(2) after the experimental sample group is sequentially introduced into the microfluidic chip and is incubated and marked, introducing washing liquor to wash the microfluidic chip; then, negative pressure extraction is carried out;
(3) the arrangement condition of the immune complex and the intensity of quantum dots under a bright field and a fluorescent field are observed in real time under a microscope, the protein expression of different samples in an experiment is analyzed and distinguished, and the fluorescence intensity is measured through microscopic shooting and software analysis.
2. Use according to claim 1, characterized in that: the microfluidic chip in the step (1) is formed by bonding a cover glass substrate and a PDMS chip.
3. Use according to claim 1, characterized in that: and (2) the sample inlet in the step (1) is connected with the serpentine channel through a gradually reduced transition area.
4. Use according to claim 1, characterized in that: and (2) arranging interception columns in the last row of the micro-column array in the step (1).
5. Use according to claim 1, characterized in that: the introduction flow rate in the step (1) is 1-2 μ l/min.
6. Use according to claim 1, characterized in that: the negative pressure extraction flow rate in the step (2) is 10-30 ml/h.
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| CN111208297A (en) * | 2020-01-07 | 2020-05-29 | 何凤屏 | Method for detecting exosome GPC1 protein by using microfluidic chip and application of exosome GPC1 protein in early diagnosis of pancreatic cancer |
| CN112269023B (en) * | 2020-08-07 | 2023-11-21 | 宁波大学 | Microfluidic Raman chip and method for detecting exosomes in blood based on microfluidic Raman chip |
| CN113481096A (en) * | 2021-06-04 | 2021-10-08 | 生物岛实验室 | Detection chip and use method thereof |
| CN113654953A (en) * | 2021-07-29 | 2021-11-16 | 山东大学深圳研究院 | Method for detecting environmental behaviors and biological effects of nano-particle pollutants |
| CN117085751B (en) * | 2023-07-26 | 2024-04-02 | 湖南瑞生科生物科技有限公司 | Microfluidic chip and exosome separation and detection method based on microfluidic chip |
| CN117511879B (en) * | 2024-01-04 | 2024-05-03 | 北京理工大学 | Method for integrally realizing exosome enrichment and micromolecule extraction based on microfluidic chip |
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