CN111250182A - High-flux microfluidic electrophoresis screening chip and preparation method and application method thereof - Google Patents
High-flux microfluidic electrophoresis screening chip and preparation method and application method thereof Download PDFInfo
- Publication number
- CN111250182A CN111250182A CN202010086231.4A CN202010086231A CN111250182A CN 111250182 A CN111250182 A CN 111250182A CN 202010086231 A CN202010086231 A CN 202010086231A CN 111250182 A CN111250182 A CN 111250182A
- Authority
- CN
- China
- Prior art keywords
- separation
- glass layer
- chip
- electrophoresis
- glass
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
Abstract
The invention provides a high-throughput microfluidic electrophoresis screening chip which comprises a glass layer and a polydimethylsiloxane PDMS cover plate, wherein a separation concentration area is arranged in the middle of the glass layer, the polydimethylsiloxane PDMS cover plate is in pressure bonding with the separation concentration area of the glass layer, a mask pattern is etched on the glass layer of the separation concentration area, the bonded polydimethylsiloxane PDMS cover plate is collapsed at the position corresponding to the mask pattern, and a microchannel is formed between the periphery of the collapsed position and adjacent glass and is used for electrophoresis screening. According to the high-throughput microfluidic electrophoresis screening chip disclosed by the invention, an unordered gel mesh structure is replaced by the ordered two-dimensional mesh screening structure, so that the surface modification of a microchannel is facilitated, and the reduction of non-specific adsorption is facilitated.
Description
Technical Field
The invention belongs to the field of microfluidic chips, and particularly relates to a high-flux microfluidic electrophoresis screening chip and a manufacturing and using method thereof.
Background
Micro-fluidic chip electrophoresis (microfluidic chip electrophoresis) utilizes glass, quartz or various polymer materials to process micron-sized channels, and takes a high-voltage direct-current electric field as a driving force to sample, separate and detect a sample. Since 1990, the method has been widely used due to its characteristics of small size, low cost, high integration level, high portability, high analysis speed, high automation degree and the like.
In recent years, with the intensive development of proteomics research, how to realize the rapid and efficient separation of a plurality of protein mixtures becomes one of the key problems to be solved urgently. The microfluidic capillary gel electrophoresis developed based on the microfluidic chip electrophoresis can realize the separation of proteins with different molecular weights and shapes on the microfluidic chip. However, this method has disadvantages such as low separation efficiency and difficulty in recovering a sample, and cannot well separate a complex mixture for downstream analysis.
Therefore, in recent years, research work of replacing disordered porous gel media with ordered molecular sieve structures draws wide attention of researchers, the combination of the ordered sieve structures and the electrophoresis method can improve the analysis speed and resolution of multi-component samples, overcome the inherent batch processing mode of the gel electrophoresis method, realize real-time sample adding and separation of objects to be detected, and greatly improve the flux of a separation system.
However, the fabrication of the molecular sieve chip based on the nano-microstructure is similar to the fabrication and packaging process of the integrated circuit, and has a high technical threshold and a high fabrication cost. In addition, due to the limitation of the manufacturing process, the width of the micro-channel of the conventional micro-fluidic chip is usually not less than 10 μm, and the protein size sieving level is difficult to achieve. And the controllable elastic collapse phenomenon (the PDMS top plate of the microchannel or the cavity formed by bonding starts to be sunken from the center under the action of external pressure until the PDMS top plate is contacted with the lower glass substrate and is permanently sealed) in the irreversible bonding of Polydimethylsiloxane (PDMS) and the glass surface is utilized, so that the submicron (100nm-1 mu m) level microchannel can be manufactured, and has different steric hindrance on protein molecules with different sizes and shapes.
The two-dimensional electrophoresis screening chip constructed by utilizing the characteristics of the PDMS material can realize the high-flux and high-selectivity separation of protein molecules with different molecular weights and shapes.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a high-flux microfluidic electrophoresis screening chip based on PDMS elastic collapse phenomenon, a preparation method and a corresponding use method thereof, and real-time and high-flux separation is realized according to the difference of the molecular weight and the shape of protein.
The invention provides the following technical scheme:
the utility model provides a high flux micro-fluidic electrophoresis screening chip, includes glass layer, polydimethylsiloxane PDMS cover plate glass layer both ends are equipped with the liquid storage tank, are equipped with the electrode respectively in the liquid storage tank, are equipped with the concentrated region of separation in the centre of glass layer, polydimethylsiloxane PDMS cover plate and the concentrated region pressure bonding of separation of glass layer, the glass layer sculpture in the concentrated region of separation has the mask pattern, and the polydimethylsiloxane PDMS cover plate sinks in the position that corresponds the mask pattern after the bonding, forms the microchannel between the periphery of the department of collapsing and adjacent glass for the electrophoresis screening, caves in respectively at the concentrated region both ends of separation and forms introduction port, goes out the appearance mouth.
Furthermore, the separation concentration area comprises a separation area, a plurality of raised patterns arranged in a matrix are formed after the glass of the separation area is etched, each pattern is respectively composed of two long microcolumns and two short microcolumns which are not connected with each other, a thick channel is formed between the bonded polydimethylsiloxane PDMS cover sheet and the connected short microcolumns after the bonded polydimethylsiloxane PDMS cover sheet is collapsed, and a thin channel is formed between the bonded polydimethylsiloxane PDMS cover sheet and the connected long microcolumns.
Furthermore, the included angle between the long side of the long microcolumn and the positive direction of the x axis is-20 degrees to-35 degrees, and the included angle between the long side of the short microcolumn and the positive direction of the x axis is 85 degrees to 70 degrees, and the included angle between the adjacent long microcolumn and the adjacent short microcolumn is 90 degrees to 60 degrees.
Further, the height of the long and short micro-columns is 0.5-1 μm, the size of the short micro-column is 200-50 μm ﹡ 50-20 μm, the size of the long micro-column is 200 μm ﹡ 50-20 μm, the width of the fine channel is 60-100nm, and the width of the coarse channel is 250-300 nm.
Furthermore, a post-concentration area is arranged at the downstream of the separation area of the separation and concentration area, and a plurality of rows of micro-columns arranged in a splayed shape are arranged in the post-concentration area.
A preparation method of a high-throughput microfluidic electrophoresis screening chip comprises the following steps:
preparing a glass layer, designing a mask pattern by using drawing software, printing a PET negative film to obtain a photoetching mask, etching glass by adopting a photoetching-wet method by using a glass substrate deposited with a chromium nitride-chromium oxynitride film and coated with a photoresist, transferring the mask pattern to the glass substrate, and etching to a depth of 0.5-1 mu m;
preparing a polydimethylsiloxane PDMS cover plate, namely performing silanization treatment on a monocrystalline silicon plate, mixing and degassing polydimethylsiloxane PDMS and a curing agent, casting the mixture on the silicon plate, heating and curing the mixture, separating the mixture from the silicon plate, and cutting the mixture into pieces with the same size as a separation and concentration area of the glass layer;
and step three, chip bonding, namely after the glass layer and the polydimethylsiloxane PDMS cover plate are subjected to plasma treatment, aligning the cover plate to the separation and concentration area, pressing and bonding, wherein the pressure intensity is 50kPa, under the action of external pressure, the polydimethylsiloxane corresponding to the concave part of the glass layer collapses until the polydimethylsiloxane contacts with the lower glass substrate and is permanently sealed, and channels with different widths are formed according to different relative positions.
A method for separating protein samples by a high-throughput microfluidic electrophoresis screening chip comprises the following steps:
step a, respectively injecting a protein sample and an electrophoresis buffer solution into liquid storage tanks at two ends of a chip, and completely covering the liquid storage tanks at the two ends with a parafilm before electrophoresis begins;
and b, applying an x-axis positive electric field to platinum electrodes in the liquid storage tanks at two ends, enabling the protein sample to enter a separation and concentration area from a sample inlet, enabling protein molecules with different sizes or shapes to have different deflection angles in the separation and concentration area, and enabling the protein molecules to enter different microchannels according to the difference of traveling routes generated by the deflection angles, so that the separation of the protein sample is realized.
Further, a step a0 is included before the step a, the protein sample to be separated is fluorescently labeled, and a step c is included after the step b, the fluorescence intensity of the sample separating fluid is analyzed by using the fluorescence microscope imaging of the CCD camera.
Further, step a1 is included after step a0, the surface of the chip microchannel is treated with a surface modification solution, and then the microchannel and the two-end reservoirs are filled with an electrophoresis buffer solution, and excess air bubbles are removed by pre-electrophoresis.
By adopting the technical scheme, the invention has the following beneficial effects:
1. the high-throughput microfluidic electrophoresis screening chip overcomes the inherent batch processing mode of microfluidic chip gel electrophoresis, can realize continuous sample introduction and long-time stable operation, and has the characteristics of small volume, high resolution and high throughput.
2. According to the high-throughput microfluidic electrophoresis screening chip disclosed by the invention, an unordered gel mesh structure is replaced by the ordered two-dimensional mesh screening structure, so that the surface modification of a microchannel is facilitated, and the reduction of non-specific adsorption is facilitated.
3. The high-throughput microfluidic electrophoresis screening chip disclosed by the invention utilizes the elastic collapse phenomenon of PDMS (polydimethylsiloxane) to manufacture a submicron screening structure, belongs to a non-silicon micromachining technology, and avoids high cost brought by a high-precision semiconductor integrated circuit process.
4. The high-throughput microfluidic electrophoresis screening chip disclosed by the invention not only can be used for carrying out real-time imaging on a separation result, but also can be used for recycling purified protein for downstream analysis.
Drawings
FIG. 1 is a schematic diagram of the structure of a high throughput microfluidic electrophoresis sieving chip according to the present invention;
FIG. 2 is a front view of a high throughput microfluidic electrophoresis sieving chip according to an embodiment of the present invention;
FIG. 3 is a schematic diagram showing the structure of the separation and concentration section in the embodiment of the present invention.
Wherein: 1-glass layer, 2-liquid storage tank, 3-electrode, 4-separation zone, 5-post concentration zone, 6-PDMS cover sheet, 7-long microcolumn, 8-short microcolumn, 9-PDMS elastic collapse zone, 10-fine channel, 11-coarse channel, 12-splayed microcolumn
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the block diagrams and specific examples are set forth only for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Example 1
As shown in fig. 1-3, a high-throughput microfluidic electrophoresis screening chip comprises a glass layer 1 and polydimethylsiloxane PDMS cover plates 6, wherein liquid storage tanks 2 are arranged at two ends of the glass layer, electrodes 3 are respectively arranged in the liquid storage tanks, a separation concentration region is arranged in the middle of the glass layer, the polydimethylsiloxane PDMS cover plates are in pressure bonding with the separation concentration region of the glass layer, a mask pattern is etched on the glass layer of the separation concentration region, the bonded polydimethylsiloxane PDMS cover plates collapse 9 at positions corresponding to the mask pattern, a microchannel is formed between the periphery of the collapse position and adjacent glass and used for electrophoresis screening, and a sample inlet and a sample outlet are respectively formed at two ends of the separation concentration region in a sunken manner. The sample inlet and the sample outlet are also grooves formed by the depressions on the glass, are exposed and are not covered by the PDMS cover plate.
The separation concentration area comprises a separation area 4, a plurality of raised patterns arranged in a matrix are formed after glass of the separation area is etched, each pattern is composed of two long microcolumns 7 and two short microcolumns 8 which are not connected with each other, a thick channel 11 is formed between the bonded polydimethylsiloxane PDMS cover sheet and the connected short microcolumns after the bonded polydimethylsiloxane PDMS cover sheet is collapsed, and a thin channel 10 is formed between the bonded polydimethylsiloxane PDMS cover sheet and the connected long microcolumns.
The included angle between the long side of the long microcolumn and the positive direction of the x axis is (-20 degrees) - (-35 degrees), and the included angle between the long side of the short microcolumn and the positive direction of the x axis is 85 degrees to 70 degrees, and the included angle between the adjacent long microcolumn and the short microcolumn is 90 degrees to 60 degrees. The angle formed between the thick and thin channels in fig. 3 is preferably a right angle. The deflection angle of the microcolumn can be adjusted according to the screening requirement.
The height of the long and short micro-columns is 0.5-1 μm, the size of the short micro-column is 200-50 μm ﹡ 50-20 μm, the size of the long micro-column is 500-200 μm ﹡ 50-20 μm, the width of the fine channel is 60-100nm, and the width of the coarse channel is 250-300 nm.
The downstream of the separation area of the separation and concentration area is provided with a post-concentration area 5, and the post-concentration area is provided with a plurality of rows of micro-columns 12 arranged in a splay shape.
The two channels are vertically arranged to form a two-dimensional mesh screening structure, and protein molecules with different sizes or shapes follow different tracks in the two-dimensional mesh screening structure, so that separation can be realized according to the difference of the molecular weight and the shape of the protein; the rear concentration area is provided with 5-16 rows of splayed supporting microcolumns, liquid is more and more concentrated along the enrichment structure of a plurality of microcolumns, an enrichment effect is achieved, the liquid is used for contraction of separated sample flow, and the peak type in subsequent analysis can be sharper. The method has the advantages of high separation efficiency, high sample recovery rate, high-throughput continuous flow screening and the like; the micro-channel is convenient for surface modification and is beneficial to reducing nonspecific adsorption. These advantages, combined with the ease of sample recovery, make the method useful for the isolation and purification of complex multicomponent biological samples, which is of great significance for proteomic studies and biomarker discovery.
Forming a submicron two-dimensional mesh screening structure after elastic collapse of PDMS in the separation area, wherein the submicron two-dimensional mesh screening structure comprises a small-angle deflection fine channel forming (-20 degrees) - (-35 degrees) with the positive direction of an x axis and a large-angle deflection coarse channel forming 85 degrees to 70 degrees with the positive direction of the x axis, and under the action of an electric field in the positive direction of the x axis, small-molecular-weight or long-linear protein molecules are easy to quickly pass through the separation area along the small-angle deflection fine channel due to small steric hindrance; due to larger steric hindrance, large molecular weight or globular protein is difficult to or cannot pass through a thinner channel but can only deflect a thicker channel through a large angle, different protein molecules are subjected to continuous screening, the deflection angles generated when the different protein molecules pass through a chip separation area are different, and different molecular flows and advancing routes are formed according to the sizes of the protein molecules, so that separation is realized.
Example 2
The invention provides a preparation method of a high-flux microfluidic electrophoresis screening chip, which comprises the following steps:
preparing a glass layer, designing a mask pattern by using drawing software, printing a PET negative film to obtain a photoetching mask, etching glass by adopting a photoetching-wet method by using a glass substrate deposited with a chromium nitride-chromium oxynitride film and coated with a photoresist, transferring the mask pattern to the glass substrate, and etching to a depth of 0.5-1 mu m;
preparing a polydimethylsiloxane PDMS cover plate, namely performing silanization treatment on a monocrystalline silicon plate, mixing and degassing polydimethylsiloxane PDMS and a curing agent, casting the mixture on the silicon plate, heating and curing the mixture, separating the mixture from the silicon plate, and cutting the mixture into pieces with the same size as a separation and concentration area of a glass layer;
and step three, chip bonding, namely after the glass layer and the polydimethylsiloxane PDMS cover plate are subjected to plasma treatment, aligning the cover plate to the separation and concentration area, pressing and bonding, wherein the pressure intensity is 50kPa, under the action of external pressure, the dimethyl siloxane is sunken until the dimethyl siloxane is contacted with the lower glass substrate and sealed, and channels with different widths are formed according to different relative positions.
Because of the relatively low elastic modulus of the PDMS material, the PDMS top plate of the micro-chamber formed by bonding collapses corresponding to the polydimethylsiloxane at the concave part of the glass layer under the action of external pressure until contacting and sealing with the lower glass substrate, and two kinds of fine micro-channels with different widths are formed according to different relative positions.
In addition, the preparation of the glass layer, the mask pattern is designed by using AutoCAD software, the required photoetching mask is obtained by printing on a PET negative film by using a high-resolution laser phototypesetter, and then the mask pattern is transferred to a quartz glass substrate by adopting a standard photoetching-wet etching quartz glass manufacturing process and is etched to a certain depth.
The electrode is manufactured by taking a platinum sheet as an electrode and implanting strip-shaped platinum electrodes into the liquid storage tanks at two ends to provide a stable electric field.
The invention is manufactured by adopting a standard photoetching-wet etching process, a quartz glass substrate which is deposited with a chromium nitride-chromium oxynitride film and coated with a photoresist is used, a mask pattern is transferred to the glass substrate, and the depth of 0.5-1 mu m is etched.
Example 3
The invention provides a method for separating a protein sample by a high-throughput microfluidic electrophoresis screening chip, which comprises the following steps:
step a, respectively injecting a protein sample and an electrophoresis buffer solution into liquid storage tanks at two ends of a chip, and completely covering the liquid storage tanks at the two ends with a parafilm before electrophoresis begins;
and b, applying an x-axis positive electric field to platinum electrodes in the liquid storage tanks at two ends, enabling the protein sample to enter a separation and concentration area from a sample inlet, enabling protein molecules with different sizes or shapes to have different deflection angles in the separation and concentration area, and enabling the protein molecules to enter different microchannels according to the difference of traveling routes generated by the deflection angles, so that the separation of the protein sample is realized.
Wherein, step a0 is also included before step a, the protein sample to be separated is fluorescently labeled, and step c is also included after step b, the fluorescence intensity of the sample separating fluid is analyzed by utilizing the fluorescence microscope imaging of the CCD camera.
Step a1 is further included after step a0, the surface of the chip microchannel is treated with a surface modification solution, and then the microchannel and the two-end reservoirs are filled with an electrophoresis buffer solution, and excess air bubbles are removed by pre-electrophoresis.
Wherein, the fluorescent dye for labeling the protein to be separated can be selected from one of the following: FITC, Alexa Fluor488, Cy2, etc., and the labeled solution was transferred to an ultrafiltration tube and centrifuged to remove excess fluorescent dye.
The electrophoresis buffer solution is 10 XTris-boric acid (TBE) buffer solution, and the surface modification solution for processing the surface of the chip microchannel comprises 0.2% of polyvinylpyrrolidone or 0.1% of POP-6 polymer separation gel dissolved in the electrophoresis buffer solution. The surface treatment solution was introduced into the chip microchannel by electroosmotic flow after application of an electric field and treated for 2 hours, followed by washing the microchannel for 1 hour with an electrophoresis buffer.
The loading process can be performed continuously by a capillary connected to a computer controlled syringe pump to achieve high throughput electrophoretic screening of proteins.
The protein to be separated can be selected from two or more of bovine serum albumin (66kDa, pl ═ 4.7), ovalbumin (44.5kDa, pl ═ 4.5), human growth hormone (22.1kDa, pl ═ 4.9), α -lactalbumin (14.3kDa, pl ═ 4.8).
In the process of pre-electrophoresis and electrophoresis, the electrode at the sample inlet end is grounded, the voltage applied to the electrode at the sample outlet end is 100-150V, and after 30-60min, the sample flow in the concentration area behind the chip reaches a stable state, so that fluorescence imaging can be performed.
After continuous screening, different protein molecules generate different deflection angles when passing through the separation area of the chip, different molecular flows and advancing routes are formed according to the sizes of the protein molecules, and finally the protein molecules are enriched at different positions of the chip, and separation liquid is sucked at different positions by using an injection pump to achieve the separation effect.
The above-mentioned embodiments only express the embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (9)
1. The utility model provides a high flux micro-fluidic electrophoresis screening chip, includes glass layer, polydimethylsiloxane PDMS cover plate, its characterized in that glass layer both ends are equipped with the liquid storage tank, are equipped with the electrode respectively in the liquid storage tank, are equipped with the separation concentrated region in the centre of glass layer, polydimethylsiloxane PDMS cover plate and the separation concentrated region pressure bonding of glass layer, the glass layer sculpture in separation concentrated region has the mask pattern, and the polydimethylsiloxane PDMS cover plate sinks in the position that corresponds the mask pattern after the bonding, forms the microchannel between the periphery of the department of collapsing and the adjacent glass for electrophoresis screening, caves in respectively at separation concentrated region both ends and forms introduction port, goes out the sample mouth.
2. The high-throughput microfluidic electrophoresis screening chip of claim 1, wherein the separation concentration region comprises a separation region, a plurality of raised patterns arranged in a matrix are formed after the glass of the separation region is etched, each pattern is composed of two long micro-columns and two short micro-columns which are not connected with each other, a thick channel is formed between the bonded polydimethylsiloxane PDMS cover sheet and the connected short micro-columns after the bonded polydimethylsiloxane PDMS cover sheet is collapsed, and a thin channel is formed between the bonded polydimethylsiloxane PDMS cover sheet and the connected long micro-columns.
3. The high-throughput microfluidic electrophoretic screening chip according to claim 2, wherein the included angle between the long side of the long microcolumn and the positive direction of the x-axis is-20 ° -35 °, and the included angle between the long side of the short microcolumn and the positive direction of the x-axis is 85 ° -70 °, and the included angle between the adjacent long microcolumn and short microcolumn is 90 ° -60 °.
4. The high throughput microfluidic electrophoresis sieving chip of claim 3, wherein the height of the long and short microcolumns is 0.5-1 μm, the size of the short microcolumns is 200-50 μm ﹡ 50-20 μm, the size of the long microcolumns is 500-200 μm ﹡ 50-20 μm, the width of the fine channel is 60-100nm, and the width of the coarse channel is 250-300 nm.
5. The high throughput microfluidic electrophoretic screening chip of claim 3, wherein a post-concentration region is disposed downstream of the separation region of the separation-concentration region, and the post-concentration region is provided with a plurality of rows of splayed micro-pillars.
6. The method for preparing the high-throughput microfluidic electrophoretic screening chip according to claim 1, comprising the following steps:
preparing a glass layer, designing a mask pattern by using drawing software, printing a PET negative film to obtain a photoetching mask, etching glass by adopting a photoetching-wet method by using a glass substrate deposited with a chromium nitride-chromium oxynitride film and coated with a photoresist, transferring the mask pattern to the glass substrate, and etching to a depth of 0.5-1 mu m;
preparing a polydimethylsiloxane PDMS cover plate, namely performing silanization treatment on a monocrystalline silicon plate, mixing and degassing polydimethylsiloxane PDMS and a curing agent, casting the mixture on the silicon plate, heating and curing the mixture, separating the mixture from the silicon plate, and cutting the mixture into pieces with the same size as a separation and concentration area of the glass layer;
and step three, chip bonding, namely after the glass layer and the polydimethylsiloxane PDMS cover plate are subjected to plasma treatment, aligning the cover plate to the separation and concentration area, pressing and bonding, wherein the pressure intensity is 50kPa, under the action of external pressure, the polydimethylsiloxane corresponding to the concave part of the glass layer collapses until the polydimethylsiloxane contacts with the lower glass substrate and is permanently sealed, and channels with different widths are formed according to different relative positions.
7. A method for separating protein samples according to the high throughput microfluidic electrophoretic screening chip of claim 1, comprising the steps of:
step a, respectively injecting a protein sample and an electrophoresis buffer solution into liquid storage tanks at two ends of a chip, and completely covering the liquid storage tanks at the two ends with a parafilm before electrophoresis begins;
and b, applying an x-axis positive electric field to platinum electrodes in the liquid storage tanks at two ends, enabling the protein sample to enter a separation and concentration area from a sample inlet, enabling protein molecules with different sizes or shapes to have different deflection angles in the separation and concentration area, and enabling the protein molecules to enter different microchannels according to the difference of traveling routes generated by the deflection angles, so that the separation of the protein sample is realized.
8. The method of isolating a protein sample according to claim 7,
step a0 is also included before step a, the protein sample to be separated is fluorescently labeled, and step c is also included after step b, the fluorescence intensity of the sample separating fluid is analyzed by the fluorescence microscope imaging of a CCD camera.
9. The method for separating protein samples according to claim 8, further comprising a step a1 after the step a0, wherein the surface of the microchannel of the chip is treated with a surface modification solution, and then the microchannel and the two-end reservoirs are filled with an electrophoresis buffer solution, and excess air bubbles are removed by pre-electrophoresis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010086231.4A CN111250182B (en) | 2020-02-11 | 2020-02-11 | High-flux microfluidic electrophoresis screening chip and preparation method and application method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010086231.4A CN111250182B (en) | 2020-02-11 | 2020-02-11 | High-flux microfluidic electrophoresis screening chip and preparation method and application method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111250182A true CN111250182A (en) | 2020-06-09 |
| CN111250182B CN111250182B (en) | 2021-03-19 |
Family
ID=70949250
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010086231.4A Active CN111250182B (en) | 2020-02-11 | 2020-02-11 | High-flux microfluidic electrophoresis screening chip and preparation method and application method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111250182B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112433278A (en) * | 2020-11-27 | 2021-03-02 | 宁波东旭成新材料科技有限公司 | Preparation method of novel light diffusion film |
Citations (26)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030020915A1 (en) * | 1998-03-23 | 2003-01-30 | Schueller Olivier J. A. | Optical modulator/detector based on reconfigurable diffraction grating |
| US20030106799A1 (en) * | 2001-12-06 | 2003-06-12 | Nanostream, Inc | Adhesiveless microfluidic device fabrication |
| US20040179972A1 (en) * | 2003-03-14 | 2004-09-16 | Nanostream, Inc. | Systems and methods for detecting manufacturing defects in microfluidic devices |
| US7182371B1 (en) * | 2003-01-24 | 2007-02-27 | Sandia National Laboratories | Edge compression manifold apparatus |
| CN101153281A (en) * | 2006-09-29 | 2008-04-02 | 中国科学院大连化学物理研究所 | DNA on-line separating microcurrent control chip and analytical method thereof |
| CN101274469A (en) * | 2007-12-29 | 2008-10-01 | 重庆大学 | Construction method of micro point array in microchannel |
| EP2049262A2 (en) * | 2006-07-19 | 2009-04-22 | Bionanomatrix, Inc. | Nanonozzle device arrays: their preparation and use for macromolecular analysis |
| CN101657717A (en) * | 2007-04-17 | 2010-02-24 | Nxp股份有限公司 | A fluid separation structure and a method of manufacturing a fluid separation structure |
| US20120196376A1 (en) * | 2009-08-21 | 2012-08-02 | Cornell University | Nanofilter devices using elastomeric micro to nanochannel interfaces and methods based thereon |
| WO2012170560A2 (en) * | 2011-06-06 | 2012-12-13 | Cornell University | Microfluidic device for extracting, isolating, and analyzing dna from cells |
| US8394324B2 (en) * | 2007-06-11 | 2013-03-12 | Wako Pure Chemical Industries, Ltd. | Microchip large-volume PCR with integrated real-time CE detection |
| US20140038854A1 (en) * | 2011-02-03 | 2014-02-06 | Albert-Ludwigs-Universitaet Freiburg | Device and method for the generation of molecular microarrays |
| CN104722342A (en) * | 2009-03-24 | 2015-06-24 | 芝加哥大学 | Slip chip device and method |
| CN105170208A (en) * | 2015-10-15 | 2015-12-23 | 华中科技大学 | Preparation method of microarray chip and product thereof |
| CN106423313A (en) * | 2016-08-31 | 2017-02-22 | 中国药科大学 | Method for detecting lactic acid by visual electrochemical luminescence sensor based on bipolar electrode array-micro-fluidic chip |
| CN107123625A (en) * | 2017-05-18 | 2017-09-01 | 华南理工大学 | A kind of through-type electrohydraulic dynamic Micropump |
| CN107876112A (en) * | 2017-10-20 | 2018-04-06 | 河南工业大学 | A kind of method of glass Direct Bonding artistic glass base microfluidic channel sealing-in |
| CN108151949A (en) * | 2017-12-20 | 2018-06-12 | 深圳先进技术研究院 | A kind of flexible electronic pressure sensor device and preparation method thereof |
| CN108753596A (en) * | 2018-04-09 | 2018-11-06 | 暨南大学 | A kind of microorganism growth image detection microchamber microfluidic system |
| CN109456879A (en) * | 2018-12-18 | 2019-03-12 | 北京化工大学 | For cell sorting and the dielectrophoresis micro-fluidic chip of focusing and its exempt to be directed at micro-processing method |
| CN109453827A (en) * | 2018-12-19 | 2019-03-12 | 清华大学天津高端装备研究院 | The micro-fluidic chip of flow control is realized based on the microarray of lyophily and/or lyophoby |
| CN109847818A (en) * | 2019-03-08 | 2019-06-07 | 北京理工大学 | A kind of high throughput microarray detection chip and preparation method thereof, application method |
| CN109975265A (en) * | 2019-04-22 | 2019-07-05 | 中国矿业大学 | A kind of three-dimensional reducing and expansion micro-fluidic device and method of multidirectional induction Dean stream |
| CN110007072A (en) * | 2019-05-07 | 2019-07-12 | 北京理工大学 | A kind of construction method and its application method of microbiological sensor |
| CN110327994A (en) * | 2019-07-11 | 2019-10-15 | 北京理工大学 | A kind of multidimensional micro-fluidic electrophoresis chip and detection device, detection method |
| CN110628568A (en) * | 2019-09-30 | 2019-12-31 | 北京化工大学 | Slide rail type dielectrophoresis electrode structure for high-throughput continuous flow cell separation |
-
2020
- 2020-02-11 CN CN202010086231.4A patent/CN111250182B/en active Active
Patent Citations (26)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030020915A1 (en) * | 1998-03-23 | 2003-01-30 | Schueller Olivier J. A. | Optical modulator/detector based on reconfigurable diffraction grating |
| US20030106799A1 (en) * | 2001-12-06 | 2003-06-12 | Nanostream, Inc | Adhesiveless microfluidic device fabrication |
| US7182371B1 (en) * | 2003-01-24 | 2007-02-27 | Sandia National Laboratories | Edge compression manifold apparatus |
| US20040179972A1 (en) * | 2003-03-14 | 2004-09-16 | Nanostream, Inc. | Systems and methods for detecting manufacturing defects in microfluidic devices |
| EP2049262A2 (en) * | 2006-07-19 | 2009-04-22 | Bionanomatrix, Inc. | Nanonozzle device arrays: their preparation and use for macromolecular analysis |
| CN101153281A (en) * | 2006-09-29 | 2008-04-02 | 中国科学院大连化学物理研究所 | DNA on-line separating microcurrent control chip and analytical method thereof |
| CN101657717A (en) * | 2007-04-17 | 2010-02-24 | Nxp股份有限公司 | A fluid separation structure and a method of manufacturing a fluid separation structure |
| US8394324B2 (en) * | 2007-06-11 | 2013-03-12 | Wako Pure Chemical Industries, Ltd. | Microchip large-volume PCR with integrated real-time CE detection |
| CN101274469A (en) * | 2007-12-29 | 2008-10-01 | 重庆大学 | Construction method of micro point array in microchannel |
| CN104722342A (en) * | 2009-03-24 | 2015-06-24 | 芝加哥大学 | Slip chip device and method |
| US20120196376A1 (en) * | 2009-08-21 | 2012-08-02 | Cornell University | Nanofilter devices using elastomeric micro to nanochannel interfaces and methods based thereon |
| US20140038854A1 (en) * | 2011-02-03 | 2014-02-06 | Albert-Ludwigs-Universitaet Freiburg | Device and method for the generation of molecular microarrays |
| WO2012170560A2 (en) * | 2011-06-06 | 2012-12-13 | Cornell University | Microfluidic device for extracting, isolating, and analyzing dna from cells |
| CN105170208A (en) * | 2015-10-15 | 2015-12-23 | 华中科技大学 | Preparation method of microarray chip and product thereof |
| CN106423313A (en) * | 2016-08-31 | 2017-02-22 | 中国药科大学 | Method for detecting lactic acid by visual electrochemical luminescence sensor based on bipolar electrode array-micro-fluidic chip |
| CN107123625A (en) * | 2017-05-18 | 2017-09-01 | 华南理工大学 | A kind of through-type electrohydraulic dynamic Micropump |
| CN107876112A (en) * | 2017-10-20 | 2018-04-06 | 河南工业大学 | A kind of method of glass Direct Bonding artistic glass base microfluidic channel sealing-in |
| CN108151949A (en) * | 2017-12-20 | 2018-06-12 | 深圳先进技术研究院 | A kind of flexible electronic pressure sensor device and preparation method thereof |
| CN108753596A (en) * | 2018-04-09 | 2018-11-06 | 暨南大学 | A kind of microorganism growth image detection microchamber microfluidic system |
| CN109456879A (en) * | 2018-12-18 | 2019-03-12 | 北京化工大学 | For cell sorting and the dielectrophoresis micro-fluidic chip of focusing and its exempt to be directed at micro-processing method |
| CN109453827A (en) * | 2018-12-19 | 2019-03-12 | 清华大学天津高端装备研究院 | The micro-fluidic chip of flow control is realized based on the microarray of lyophily and/or lyophoby |
| CN109847818A (en) * | 2019-03-08 | 2019-06-07 | 北京理工大学 | A kind of high throughput microarray detection chip and preparation method thereof, application method |
| CN109975265A (en) * | 2019-04-22 | 2019-07-05 | 中国矿业大学 | A kind of three-dimensional reducing and expansion micro-fluidic device and method of multidirectional induction Dean stream |
| CN110007072A (en) * | 2019-05-07 | 2019-07-12 | 北京理工大学 | A kind of construction method and its application method of microbiological sensor |
| CN110327994A (en) * | 2019-07-11 | 2019-10-15 | 北京理工大学 | A kind of multidimensional micro-fluidic electrophoresis chip and detection device, detection method |
| CN110628568A (en) * | 2019-09-30 | 2019-12-31 | 北京化工大学 | Slide rail type dielectrophoresis electrode structure for high-throughput continuous flow cell separation |
Non-Patent Citations (6)
| Title |
|---|
| J.HAN: "Separation of Long DNA Molecules in a Microfabricated Entropic Trap Array", 《SCIENCE》 * |
| LIN FENGMING: "A novel microfluidic chip electrophoresis strategy for simultaneous, label-free, multi-protein detection based on a graphene energy transfer biosensor", 《ANALYST》 * |
| PARK, SEUNG-MIN: "A method for nanofluidic device prototyping using elastomeric collapse", 《PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA》 * |
| SAHORE, VISHAL: "Automated microfluidic devices integrating solid-phase extraction, fluorescent labeling, and microchip electrophoresis for preterm birth biomarker analysis", 《ANALYTICAL AND BIOANALYTICAL CHEMISTRY》 * |
| 吕雪飞等: "基于微流控芯片的核酸检测技术", 《生命科学仪器》 * |
| 汪骅等: "聚二甲基硅氧烷微流控芯片电泳的临床应用研究", 《国际检验医学杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112433278A (en) * | 2020-11-27 | 2021-03-02 | 宁波东旭成新材料科技有限公司 | Preparation method of novel light diffusion film |
| CN112433278B (en) * | 2020-11-27 | 2022-07-29 | 宁波东旭成新材料科技有限公司 | Preparation method of light diffusion film |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111250182B (en) | 2021-03-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Slentz et al. | Nanoliter capillary electrochromatography columns based on collocated monolithic support structures molded in poly (dimethyl siloxane) | |
| Zhang et al. | High-speed free-flow electrophoresis on chip | |
| Turgeon et al. | Micro free-flow electrophoresis: theory and applications | |
| Kwak et al. | Continuous-flow biomolecule and cell concentrator by ion concentration polarization | |
| Novo et al. | Current advances and challenges in microfluidic free-flow electrophoresis—A critical review | |
| US7727363B2 (en) | Microfluidic device and methods for focusing fluid streams using electroosmotically induced pressures | |
| Cabodi et al. | Continuous separation of biomolecules by the laterally asymmetric diffusion array with out‐of‐plane sample injection | |
| US20060065528A1 (en) | Nanostructured devices for separation and analysis | |
| US20100108519A1 (en) | Polymeric Nanopillars and Nanotubes, Their Manufacture and Uses | |
| JP2004045357A (en) | Separating apparatus and its manufacturing method | |
| Jezierski et al. | Multistep liquid-phase lithography for fast prototyping of microfluidic free-flow-electrophoresis chips | |
| Sassi et al. | Rapid, parallel separations of D1S80 alleles in a plastic microchannel chip | |
| CN111250182B (en) | High-flux microfluidic electrophoresis screening chip and preparation method and application method thereof | |
| JP4735119B2 (en) | Reactor and production method thereof | |
| US20090250347A1 (en) | Microfluidic devices & processes for electrokinetic transport | |
| Chen et al. | Applications and theory of electrokinetic enrichment in micro-nanofluidic chips | |
| WO2009005476A1 (en) | Capillary sample separation apparatus | |
| Mohamadi et al. | Nanotechnology for genomics & proteomics | |
| WO2004008132A1 (en) | Bio-molecule separation cell, manufacturing method thereof, and dna fragmentation apparatus | |
| Tsai et al. | Development of a microchip for 2-dimensional capillary electrophoresis | |
| Loughran et al. | Separation of DNA in a versatile microchip | |
| US20090250345A1 (en) | Microfluidic electroelution devices & processes | |
| JP2012002801A (en) | Substrate for fixing gel, reaction instrument for electrophoresis, method for manufacturing reaction instrument for electrophoresis, and kit for electrophoresis | |
| Li et al. | Microfluidic lab-on-a-chip | |
| Ugaz et al. | Electrophoresis in microfluidic systems |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |