CN107185612B - Microfluid chip applied to exosome capture and preparation method thereof - Google Patents
Microfluid chip applied to exosome capture and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a microfluid biological microfluid chip for exosome capture and a preparation method thereof. The micro-fluid chip combines a micro-array column and specific affinity recognition to realize the synergistic high-efficiency and high-purity capture of exosomes in cell culture supernatant and human body fluid. The microfluid chip is packaged by an upper cover plate and a lower substrate through irreversible bonding. The cover plate is provided with a liquid feeding hole and a liquid outlet hole. The lower substrate is provided with a buffer chamber and a separation chamber, and the separation chamber is modified with affinity recognition biological specificity molecules and microarray columns with geometric arrangement. The exosomes were captured by microarray column and antibody affinity purification. The biological microfluid chip can efficiently realize the enrichment and extraction of target exosomes, and has the advantages of simple structure, convenient operation, no pollution, low energy consumption and the like.
Description
Technical Field
The invention belongs to biotechnology and microfluidics technology, and particularly relates to design and preparation of an exosome-capturing microfluidics chip.
Background
Exosomes (exosomes) are vesicles with a bilayer plasma membrane structure, about 30-150nm in diameter, released by cells into the extracellular space or into biological fluids by exocytosis; in recent years, exosomes are receiving more and more attention and become new research hotspots, wherein the exosomes contain various biomolecules such as mRNAs, microRNAs and proteins specific to source cells and play an important role in signal transduction and an immune system, and researches show that the exosomes are closely related to tumor metastasis, nervous system diseases and the like, and the content of the exosomes is also a marker molecule for clinical detection and diagnosis of various diseases.
The existing exosome separation technology comprises an ultracentrifugation method, a filtration centrifugation method, a density gradient ultracentrifugation method, an immunomagnetic bead combined ultracentrifugation method, a chromatography method and the like; these techniques are highly demanding for instrumentation and time consuming and therefore require efficient exosome capture techniques to solve these problems.
Based on the above, the invention intends to combine the microfluidic technology, the microarray technology and the specific affinity capture technology to prepare a micro-column array fluid chip based on biospecific molecules and apply the micro-column array fluid chip to capture exosomes.
Disclosure of Invention
The invention aims to provide a micro-column array fluid chip with specific molecular modification, which is used for separating and capturing exosomes.
The technical scheme of the invention is as follows: the device comprises an upper layer cover plate and a lower layer substrate, wherein the upper layer cover plate is provided with a sample inlet hole and a sample outlet hole;
a separation chamber is arranged on the lower substrate, buffer chambers connected with the separation chamber are arranged at the two ends of the separation chamber,
the separation chamber is a serpentine channel, a micro-column array which is arranged in a crossed way is arranged in the serpentine channel,
the surface of the micro-column array is modified with specific recognition molecules.
The size of the microfluidic chip is 40mm x 30mm x 2mm,
the buffer chamber is square, 10-20um wide, 1-10mm long and 5-20um high;
the width of the separation chamber is 10-20um, and the height is 5-20 um.
The top of the micro-column array is one of a circle, a triangle, an ellipse, a drop shape and a polygon;
the diameter of the micro-column array is 0.5-5um, the height is 4-9um, and the distance is 0.5-2 um.
The specific recognition molecule comprises one of an aptamer and an antibody, wherein the aptamer specifically recognizes the exosome surface marker.
The upper cover plate is a glass plate with the size of 40mm multiplied by 30mm, and the positions of the sample inlet hole and the sample outlet hole on the upper cover plate correspond to the positions of the buffer chamber on the lower substrate.
The present invention relates to a method for affinity capture of exosomes in a sample by modifying specific biomolecules on a microarray in a microfluidic chip.
The method is characterized in that the method comprises the steps of preparing a microfluid chip and modifying the surface of a microarray column, the contact area of a sample and the microfluid chip of the microfluid chip is increased mainly through the arrangement of the microarray column, and specific biomolecules on the surface of the column have affinity with exosomes, so that the exosomes are separated and captured.
The preparation method comprises the following steps:
preparing a microfluid chip: the manufacture of the microstructure is realized by utilizing a standard photoetching process; the whole microfluidic chip is about 40mm × 30mm × 2mm, and the preparation process is as follows:
1) preparing materials: preparing a silicon wafer and an upper cover plate with the same size,
2) and silicon wafer treatment: cleaning a silicon wafer, drying, spin coating (SU-8 photoresist), spin coating, pre-drying, exposing, thick drying, developing and the like to manufacture a mold;
uniformly mixing PDMS and a curing agent in a mass ratio of 10: 1, removing bubbles in the mixture, pouring the mixture on a mold, heating and curing at 90 ℃ for 1 hour, and carefully stripping the mold and the PDMS cured material to obtain a lower substrate of the microfluidic chip;
plasma processing the underlying substrate of the microfluidic chip,
carrying out surface silanization modification on a lower substrate of the microfluid chip by GPTMS absolute ethyl alcohol solution;
the cover and base of the microfluidic chip are irreversibly bonded,
punching sample inlet and outlet holes on the upper cover plate by using punching needles; and obtaining the prepared microfluid chip.
(II) specific biomolecule modification: after the microfluid chip of the microfluid chip is made, carry on the modification biological specificity molecule to the chip surface of microfluid, the preparation process is as follows:
introducing a mixed solution of 0.1mol/l,2- (N-morpholine) ethanesulfonic acid (MES), 2mol/l N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide (EDC) and 5mol/l N-hydroxysuccinimide (NHS) into a microfluidic chip for reaction for 30min, washing the microfluidic chip by 0.1mol/l Phosphate Buffer Solution (PBS), introducing 10ug/ml specific antibody working solution, washing a serpentine channel of the microfluidic chip by sterilized deionized water after reacting for 4 hours at room temperature, blocking the serpentine channel by BSA solution, slowly washing by 0.01mol/l PBS, and storing for later use at 4 ℃.
When the chip is used, a sample is introduced into the microfluidic chip through the sample inlet, the flow rate of the sample is adjusted to be 0-10mL/h, the sample flows through the buffer chamber and the separation chamber of the serpentine channel, and the specificity of exosome in the sample is acted and combined with the modified specific molecule on the microarray column, so that exosome is captured.
The invention has the advantages that: the micro-array column structure is added in the micro-fluid chip, so that the area/volume ratio of the fluid environment is obviously increased, and the biomolecule specifically combined with the exosome is modified on the micro-column array, so that the reaction efficiency is greatly improved, the consumption of samples and reagents is reduced, and the cost is reduced.
Drawings
FIG. 1 is a schematic diagram of the overall structure of a microfluidic array microfluidic chip according to the present invention;
FIG. 2 is a schematic diagram of the structure of an upper cover sheet in a microfluidic array chip according to the present invention;
FIG. 3 is a schematic diagram of the structure of the lower substrate of the microfluidic array chip of the present invention;
FIG. 4 is a schematic diagram of a microarray column on a serpentine channel of a microfluidic array microfluidic chip according to the present invention,
FIG. 5 is a schematic view of surface modification of a microarray column according to the present invention;
in the figure, 1 is the upper cover sheet, 2 is the lower substrate, 3 is the sample inlet, 4 is the sample outlet, 5 is the separation chamber, 6 is the buffer chamber.
Detailed Description
The present invention is further illustrated by the following detailed description taken in conjunction with the accompanying drawings, which are included to provide a further understanding of the invention, and are not intended to limit the scope of the invention, which is defined by the appended claims, as modified by those skilled in the art, after reading this disclosure.
The present invention will be described in further detail below for the understanding of the present invention.
As shown in the figure, comprises an upper cover sheet 1 and a lower substrate sheet 2, wherein the upper cover sheet 1 is preferably a glass sheet, and can also be PDMS,
the upper cover plate 1 is provided with a sample inlet hole 3 and a sample outlet hole 4;
a separation chamber 5 is arranged on the lower substrate 2, buffer chambers 6 connected with the separation chamber 5 are arranged at two ends of the separation chamber 5,
the separation chamber 5 is a serpentine channel, a micro-column array which is arranged in a crossed way is arranged in the serpentine channel,
the surface of the micro-column array is modified with specific recognition molecules.
The size of the microfluidic chip is 40mm x 30mm x 2mm,
the buffer chamber 6 is square, 10-20um wide, 1-10mm long and 5-20um high;
the separating chamber 5 is 10-20um wide and 5-20um high.
The top of the micro-column array is one of a circle, a triangle, an ellipse, a drop shape and a polygon;
the diameter of the micro-column array is 0.5-5um, the height is 4-9um, and the distance is 0.5-2 um.
The specific recognition molecule comprises one of an aptamer and an antibody, wherein the aptamer specifically recognizes the exosome surface marker.
Example 1
Preparing a microfluid chip: a schematic of the microstructure in a microfluidic chip is shown in fig. 1; the manufacture of the microstructure is realized by utilizing a standard photoetching process; the volume size of whole microfluid chip is about 40mm x 30mm x 2mm, and the microcolumn is cylindrical, diameter 0.5um, and the separation chamber is wide 10um, high 5um, microcolumn array diameter 0.5um, high 4um, interval 0.5 um.
The preparation process comprises the following steps:
1) preparing materials: preparing a silicon wafer and an upper cover plate 1 with the same size in advance, wherein the silicon wafer is used as a base material,
2) and silicon wafer treatment: cleaning a silicon wafer, drying, spin coating (SU-8 photoresist), spin coating, pre-drying, exposing, thick drying, developing and the like to manufacture a mold;
uniformly mixing PDMS and a curing agent in a mass ratio of 10: 1, removing bubbles in the mixture, pouring the mixture on a mold, heating and curing at 90 ℃ for 1 hour, and carefully stripping the mold and the PDMS cured material to obtain a lower substrate 2 of the microfluidic chip;
the plasma treats the underlying substrate 2 of the microfluidic chip,
carrying out surface silanization modification on a lower substrate 2 of the microfluid chip by GPTMS absolute ethyl alcohol solution;
the upper cover sheet 1 and the lower substrate 2 of the microfluidic chip are irreversibly bonded,
punching a sample inlet hole 3 and a sample outlet hole 4 on the upper-layer cover plate 1 by using a punching needle; and obtaining the prepared microfluid chip.
Specific biomolecule modification: after the manufacture of the microfluid chip is completed, the surface of the microfluid chip is modified with biospecific molecules, and the preparation process is as follows:
introducing a mixed solution of 0.1mol/l,2- (N-morpholine) ethanesulfonic acid (MES), 2mol/l N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide (EDC) and 5mol/l N-hydroxysuccinimide (NHS) into the microfluidic chip for reaction for 30min, washing the microfluidic chip with 0.1mol/l Phosphate Buffer Solution (PBS), introducing 10ug/ml specific antibody working solution, reacting for 4 hours at room temperature, washing the serpentine channel of the microfluidic chip with sterilized deionized water, blocking the serpentine channel with BSA solution, slowly washing with 0.01mol/l PBS, and storing at 4 ℃ for later use.
When the chip is used, a sample is introduced into the microfluidic chip through the sample inlet hole 3, the flow rate of the sample is adjusted to be 0-10mL/h, the sample flows through the buffer chamber 6 and the separation chamber 5 of the serpentine channel, and the specificity of exosome in the sample is acted and combined with the modified specific molecule on the microarray column, so that exosome is captured.
Example 2
Preparing a microfluid chip: a schematic of the microstructure in a microfluidic chip is shown in fig. 1; the manufacture of the microstructure is realized by utilizing a standard photoetching process; the whole microfluid chip size is about 40mm x 30mm x 2mm, and the microcolumn is cylindrical, diameter 2.5um, and the separation chamber is wide 15um, high 13um, and microcolumn array diameter 2.5um, high 6.5um, interval 1.5 um.
The preparation process comprises the following steps:
1) preparing materials: preparing a silicon wafer and an upper cover plate 1 with the same size in advance, wherein the silicon wafer is used as a base material,
2) and silicon wafer treatment: cleaning a silicon wafer, drying, spin coating (SU-8 photoresist), spin coating, pre-drying, exposing, thick drying, developing and the like to manufacture a mold;
uniformly mixing PDMS and a curing agent in a mass ratio of 10: 1, removing bubbles in the mixture, pouring the mixture on a mold, heating and curing at 90 ℃ for 1 hour, and carefully stripping the mold and the PDMS cured material to obtain a lower substrate 2 of the microfluidic chip;
the plasma treats the underlying substrate 2 of the microfluidic chip,
carrying out surface silanization modification on a lower substrate 2 of the microfluid chip by GPTMS absolute ethyl alcohol solution;
the upper cover sheet 1 and the lower substrate 2 of the microfluidic chip are irreversibly bonded,
punching a sample inlet hole 3 and a sample outlet hole 4 on the upper-layer cover plate 1 by using a punching needle; and obtaining the prepared microfluid chip.
Specific biomolecule modification: after the manufacture of the microfluid chip is completed, the surface of the microfluid chip is modified with biospecific molecules, and the preparation process is as follows:
introducing a mixed solution of 0.1mol/l,2- (N-morpholine) ethanesulfonic acid (MES), 2mol/l N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide (EDC) and 5mol/l N-hydroxysuccinimide (NHS) into the microfluidic chip for reaction for 30min, washing the microfluidic chip with 0.1mol/l Phosphate Buffer Solution (PBS), introducing 10ug/ml specific antibody working solution, reacting for 4 hours at room temperature, washing the serpentine channel of the microfluidic chip with sterilized deionized water, blocking the serpentine channel with BSA solution, slowly washing with 0.01mol/l PBS, and storing at 4 ℃ for later use.
When the chip is used, a sample is introduced into the microfluidic chip through the sample inlet hole 3, the flow rate of the sample is adjusted to be 0-10mL/h, the sample flows through the buffer chamber 6 and the separation chamber 5 of the serpentine channel, and the specificity of exosome in the sample is acted and combined with the modified specific molecule on the microarray column, so that exosome is captured.
Example 3
Preparing a microfluid chip: a schematic of the microstructure in a microfluidic chip is shown in fig. 1; the manufacture of the microstructure is realized by utilizing a standard photoetching process; the whole microfluid chip size is about 40mm x 30mm x 2mm, and the microcolumn is equilateral triangle, length of side 5um, separation chamber are wide 20um, high 10um, microcolumn array diameter 5um, high 9um, interval 2 um.
The preparation process comprises the following steps:
1) preparing materials: preparing a silicon wafer and an upper cover plate 1 with the same size in advance, wherein the silicon wafer is used as a base material,
2) and silicon wafer treatment: cleaning a silicon wafer, drying, spin coating (SU-8 photoresist), spin coating, pre-drying, exposing, thick drying, developing and the like to manufacture a mold;
uniformly mixing PDMS and a curing agent in a mass ratio of 10: 1, removing bubbles in the mixture, pouring the mixture on a mold, heating and curing at 90 ℃ for 1 hour, and carefully stripping the mold and the PDMS cured material to obtain a lower substrate 2 of the microfluidic chip;
the plasma treats the underlying substrate 2 of the microfluidic chip,
carrying out surface silanization modification on a lower substrate of the microfluid chip by GPTMS absolute ethyl alcohol solution;
the upper cover sheet 1 and the lower substrate 2 of the microfluidic chip are irreversibly bonded,
punching a sample inlet hole 3 and a sample outlet hole 4 on the upper-layer cover plate 1 by using a punching needle; and obtaining the prepared microfluid chip.
Specific biomolecule modification: after the manufacture of the microfluid chip is completed, the surface of the microfluid chip is modified with biospecific molecules, and the preparation process is as follows:
introducing a mixed solution of 0.1mol/l,2- (N-morpholine) ethanesulfonic acid (MES), 2mol/l N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide (EDC) and 5mol/l N-hydroxysuccinimide (NHS) into the microfluidic chip for reaction for 30min, washing the microfluidic chip with 0.1mol/l Phosphate Buffer Solution (PBS), introducing 10ug/ml specific antibody working solution, reacting for 4 hours at room temperature, washing the serpentine channel of the microfluidic chip with sterilized deionized water, blocking the serpentine channel with BSA solution, slowly washing with 0.01mol/l PBS, and storing at 4 ℃ for later use.
When the chip is used, a sample is introduced into the microfluidic chip through the sample inlet hole 3, the flow rate of the sample is adjusted to be 0-10mL/h, the sample flows through the buffer chamber 6 and the separation chamber 5 of the serpentine channel, and the specificity of exosome in the sample is acted and combined with the modified specific molecule on the microarray column, so that exosome is captured.
Claims (3)
1. A preparation method of a microfluid chip applied to exosome capture is characterized in that the microfluid chip applied to exosome capture comprises an upper layer cover plate and a lower layer substrate, wherein the upper layer cover plate is provided with a sample inlet hole and a sample outlet hole;
a separation chamber is arranged on the lower substrate, buffer chambers connected with the separation chamber are arranged at the two ends of the separation chamber,
the separation chamber is a serpentine channel, a micro-column array which is arranged in a crossed way is arranged in the serpentine channel,
the surface of the micro-column array is modified with specific recognition molecules;
the size of the microfluidic chip is 40mm x 30mm x 2mm,
the buffer chamber is square, 10-20um wide, 1-10mm long and 5-20um high,
the width of the separation chamber is 10-20um, and the height is 5-20 um;
the top of the micro-column array is one of a circle, a triangle, an ellipse, a drop shape and a polygon;
the diameter of the micro-column array is 0.5-5um, the height is 4-9um, and the distance is 0.5-2 um;
the specific recognition molecule comprises one of an aptamer and an antibody, wherein the aptamer specifically recognizes the exosome surface marker;
the upper cover plate is a glass plate with the size of 40mm multiplied by 30mm, and the positions of the sample inlet hole and the sample outlet hole on the upper cover plate correspond to the positions of the buffer chamber on the lower substrate;
the preparation method comprises the following steps:
preparing a microfluid chip: the manufacture of the microstructure is realized by utilizing a standard photoetching process; the size of the whole microfluid chip is about 40mm multiplied by 30mm multiplied by 2mm, the microcolumn is cylindrical, the diameter is 0.5um, the separation chamber is 10um wide, 5um high, the diameter of the microcolumn array is 0.5um, 4um high, the interval is 0.5 um;
the preparation process comprises the following steps:
1) preparing materials: preparing a silicon wafer and an upper cover plate with the same size in advance, wherein the silicon wafer is used as a base material,
2) and silicon wafer treatment: cleaning a silicon wafer, drying, spin coating, pre-drying, exposing, thick drying and developing to manufacture a die;
uniformly mixing PDMS and a curing agent in a mass ratio of 10: 1, removing bubbles in the mixture, pouring the mixture on a mold, heating and curing at 90 ℃ for 1 hour, and carefully stripping the mold and the PDMS cured material to obtain a lower substrate of the microfluidic chip;
plasma processing the underlying substrate of the microfluidic chip,
carrying out surface silanization modification on a lower substrate of the microfluid chip by GPTMS absolute ethyl alcohol solution;
the upper cover sheet and the lower substrate of the microfluid chip are bonded irreversibly,
punching sample inlet and outlet holes on the upper cover plate by using punching needles; obtaining a prepared microfluid chip;
(II) specific biomolecule modification: after the manufacture of the microfluid chip is completed, the surface of the microfluid chip is modified with biospecific molecules, and the preparation process is as follows:
introducing a mixed solution of 0.1mol/l,2- (N-morpholine) ethanesulfonic acid (MES), 2mol/l N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide (EDC) and 5mol/l N-hydroxysuccinimide (NHS) into the microfluidic chip for reaction for 30min, washing the microfluidic chip with 0.1mol/l Phosphate Buffer Solution (PBS), introducing 10ug/ml specific antibody working solution, reacting for 4 hours at room temperature, washing the serpentine channel of the microfluidic chip with sterilized deionized water, blocking the serpentine channel with BSA solution, slowly washing with 0.01mol/l PBS, and storing at 4 ℃ for later use;
when the chip is used, a sample is introduced into the microfluidic chip through the sample inlet, the flow rate of the sample is adjusted to be 0-10mL/h, the sample flows through the buffer chamber and the separation chamber of the serpentine channel, and the specificity of exosome in the sample is acted and combined with the modified specific molecule on the microarray column, so that exosome is captured.
2. A method for preparing a microfluidic chip for exosome capture according to claim 1, comprising the following steps:
the manufacture of the microstructure is realized by utilizing a standard photoetching process; the size of the whole microfluid chip is about 40mm multiplied by 30mm multiplied by 2mm, the microcolumn is cylindrical, the diameter is 2.5um, the width of the separation chamber is 15um, the height is 13um, the diameter of the microcolumn array is 2.5um, the height is 6.5um, and the distance is 1.5 um;
the preparation process comprises the following steps:
1) preparing materials: preparing a silicon wafer and an upper cover plate 1 with the same size in advance, wherein the silicon wafer is used as a base material,
2) and silicon wafer treatment: cleaning a silicon wafer, drying, spin coating, pre-drying, exposing, thick drying and developing to manufacture a die;
uniformly mixing PDMS and a curing agent in a mass ratio of 10: 1, removing bubbles in the mixture, pouring the mixture on a mold, heating and curing at 90 ℃ for 1 hour, and carefully stripping the mold and the PDMS cured material to obtain a lower substrate of the microfluidic chip;
plasma processing the underlying substrate of the microfluidic chip,
carrying out surface silanization modification on a lower substrate of the microfluid chip by GPTMS absolute ethyl alcohol solution;
the upper cover sheet and the lower substrate of the microfluid chip are bonded irreversibly,
punching sample inlet and outlet holes on the upper cover plate by using punching needles; obtaining a prepared microfluid chip;
(II) specific biomolecule modification: after the manufacture of the microfluid chip is completed, the surface of the microfluid chip is modified with biospecific molecules, and the preparation process is as follows:
introducing a mixed solution of 0.1mol/l,2- (N-morpholine) ethanesulfonic acid (MES), 2mol/l N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide (EDC) and 5mol/l N-hydroxysuccinimide (NHS) into the microfluidic chip for reaction for 30min, washing the microfluidic chip with 0.1mol/l Phosphate Buffer Solution (PBS), introducing 10ug/ml specific antibody working solution, reacting for 4 hours at room temperature, washing the serpentine channel of the microfluidic chip with sterilized deionized water, blocking the serpentine channel with BSA solution, slowly washing with 0.01mol/l PBS, and storing at 4 ℃ for later use;
when the chip is used, a sample is introduced into the microfluidic chip through the sample inlet, the flow rate of the sample is adjusted to be 0-10mL/h, the sample flows through the buffer chamber and the separation chamber of the serpentine channel, and the specificity of exosome in the sample is acted and combined with the modified specific molecule on the microarray column, so that exosome is captured.
3. A method for preparing a microfluidic chip for exosome capture according to claim 1, comprising the following steps:
the manufacture of the microstructure is realized by utilizing a standard photoetching process; the size of the whole microfluid chip is about 40mm multiplied by 30mm multiplied by 2mm, the microcolumn is an equilateral triangle, the side length is 5um, the separation chamber is 20um wide and 10um high, the diameter of the microcolumn array is 5um, the height is 9um, and the distance is 2 um;
the preparation process comprises the following steps:
1) preparing materials: preparing a silicon wafer and an upper cover plate 1 with the same size in advance, wherein the silicon wafer is used as a base material,
2) and silicon wafer treatment: cleaning a silicon wafer, drying, spin coating, pre-drying, exposing, thick drying and developing to manufacture a die;
uniformly mixing PDMS and a curing agent in a mass ratio of 10: 1, removing bubbles in the mixture, pouring the mixture on a mold, heating and curing at 90 ℃ for 1 hour, and carefully stripping the mold and the PDMS cured material to obtain a lower substrate of the microfluidic chip;
plasma processing the underlying substrate of the microfluidic chip,
carrying out surface silanization modification on a lower substrate of the microfluid chip by GPTMS absolute ethyl alcohol solution;
the upper cover sheet and the lower substrate of the microfluid chip are bonded irreversibly,
punching sample inlet and outlet holes on the upper cover plate by using punching needles; obtaining a prepared microfluid chip;
(II) specific biomolecule modification: after the manufacture of the microfluid chip is completed, the surface of the microfluid chip is modified with biospecific molecules, and the preparation process is as follows:
introducing a mixed solution of 0.1mol/l,2- (N-morpholine) ethanesulfonic acid (MES), 2mol/l N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide (EDC) and 5mol/l N-hydroxysuccinimide (NHS) into the microfluidic chip for reaction for 30min, washing the microfluidic chip with 0.1mol/l Phosphate Buffer Solution (PBS), introducing 10ug/ml specific antibody working solution, reacting for 4 hours at room temperature, washing the serpentine channel of the microfluidic chip with sterilized deionized water, blocking the serpentine channel with BSA solution, slowly washing with 0.01mol/l PBS, and storing at 4 ℃ for later use;
when the chip is used, a sample is introduced into the microfluidic chip through the sample inlet, the flow rate of the sample is adjusted to be 0-10mL/h, the sample flows through the buffer chamber and the separation chamber of the serpentine channel, and the specificity of exosome in the sample is acted and combined with the modified specific molecule on the microarray column, so that exosome is captured.
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| CN100494960C (en) * | 2005-04-20 | 2009-06-03 | 中国科学院上海微系统与信息技术研究所 | Minitype gathering chip for biological sample, and preparation method |
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