CN113522384A

CN113522384A - Microfluidic chip and preparation and application thereof

Info

Publication number: CN113522384A
Application number: CN202110761828.9A
Authority: CN
Inventors: 朱庆夫; 娄豆豆; 刘飞
Original assignee: Eye Hospital of Wenzhou Medical University
Current assignee: Eye Hospital of Wenzhou Medical University
Priority date: 2021-07-06
Filing date: 2021-07-06
Publication date: 2021-10-22

Abstract

The invention provides a microfluidic chip which comprises a channel layer and a substrate layer, wherein the channel layer comprises a sample injection area, a mixing area and a detection area which are sequentially communicated, the mixing area comprises a plurality of mixing units, each mixing unit comprises at least 1 wide channel and at least 1 narrow channel, the width of each wide channel is 100-300 mu m, the width of each narrow channel is 1/5-1/2 of the width of each wide channel, a target sample can be efficiently mixed, functional areas including mixing and separation detection are simultaneously integrated on the chip, requirements such as reaction, separation and detection can be met, and the production or research efficiency can be effectively improved. The obtained micro-fluidic chip can also be applied to the field of separation of plasma lipoprotein and exosome, and is beneficial to promoting the application of the lipoprotein and the exosome in the research and diagnosis of disease mechanisms.

Description

Microfluidic chip and preparation and application thereof

Technical Field

The invention relates to the technical field of microfluidics, in particular to a microfluidic chip.

Background

With the development of semiconductor processing technology and microfluidic technology, chemical and biological chips based on the microfluidic technology are emerging continuously. The micro-fluidic technology is based on micro-processing technology, and functional units such as micro-channels and micro-reactors are processed on matrix materials such as glass and high molecular polymers, so that basic operation units related to fields such as biology and chemistry, such as pretreatment, sample adding, reaction, separation, analysis, cell culture and the like are integrated or basically integrated on a chip with a square size, and a technical platform with various functions in a conventional chemical or biological laboratory is replaced. Specifically, the microfluidic chip significantly improves the mixing efficiency of the sample by reducing the channel size to the micrometer level, but at the same time, the throughput of the sample per unit time is limited. Therefore, how to continuously optimize the structure of the micro-channel and improve the mixing efficiency of the sample has important significance.

The existing cardioid mixing channel designed by corning corporation, in which the reaction materials flow rapidly, can perform effective heterogeneous mixing. However, the structure is complex, the manufacturing cost is high, and the device is mainly used for mixing gas-liquid or liquid-liquid two-phase samples, and the samples containing solid phase may stay or even block the fine structure of the heart-shaped mixing channel, which has adverse effects on the mixing efficiency of the samples and the service life of the channel. In addition, the current microfluidic process usually adopts a soft lithography technology, for example, SU-8 photoresist is used to make a microchannel male mold, and Polydimethylsiloxane (PDMS) is cured, molded and inverted, and then bonded with a glass or organic polymer substrate to form a chip, but this method can only prepare highly uniform microstructures.

Further specifically to the field of cell biology, microfluidic chips also have potential for exosome isolation. The exosome is a vesicle structure with 30-150nm secreted by cells and can be used as a potential biomarker for tumor diagnosis. However, due to the high overlap of density and size with lipoproteins, and the lack of an effective separation method, plasma exosomes obtained by using the conventional exosome extraction and detection technology often contain a large amount of lipoproteins, and the requirement of extracting and detecting biological samples required for liquid biopsy with high sensitivity is far from being met. The existing three-dimensional nano-structure microfluidic chip designed by the professor of Liu Pen Feng of Huazhong university of science and technology, wherein a micro-column array is functionalized by using crossed multi-walled carbon nanotubes through chemical deposition, and exosomes can be efficiently captured through a specific recognition molecule (CD63) and a unique topological nano-material, but the method is high in cost, is still in a laboratory research stage, and is difficult to popularize and apply in a short period.

Disclosure of Invention

Aiming at the problems in the prior art, the invention designs the micro-fluidic chip which is provided with the wide and narrow mixing channels, can efficiently mix a target sample, meets the requirements such as reaction, separation, detection and the like, can be prepared by a 3D printing technology, can be applied to the field of plasma lipoprotein and exosome separation, and is beneficial to promoting the application of the lipoprotein and exosome in disease mechanism research and diagnosis.

The technical scheme adopted by the invention is as follows:

a microfluidic chip comprises a channel layer and a substrate layer, wherein the channel layer is positioned on the substrate layer;

the channel layer comprises a sample introduction area, a mixing area and a detection area which are sequentially communicated;

the sample injection region comprises a sample injection port and a sample injection channel, and a sample is injected from the sample injection port and enters the mixing region through the sample injection channel;

the mixing region comprises a plurality of mixing units, each mixing unit comprises at least 1 wide channel and at least 1 narrow channel, the width of the wide channel is 100-300 μm, the width of the narrow channel is 1/5-1/2 of the width of the wide channel, and the sample enters the detection region through the mixing region;

the detection area comprises at least 1 chamber, and the height of the chamber is not less than 0.5 mm.

In other optimized technical solutions, the mixing area includes not less than 100 mixing units, each mixing unit includes 1 wide channel and 1 narrow channel; the length of the wide channel is 200-500 mu m, and the length of the narrow channel is not more than the length of the wide channel.

In other optimized technical solutions, the detection region includes a first chamber and a second chamber which are communicated in sequence; the mixing region is in communication with the first chamber, which is in communication with the second chamber.

In other optimized technical schemes, the height of the first cavity is 0.5-1.5 mm, and the volume is 5-10 muL; the microfluidic chip further comprises a magnet, and the magnet is arranged below the first chamber.

In other optimized technical schemes, the height of the second chamber is 5-10 mm, and the volume of the second chamber is 140-300 mu L.

In other optimized technical schemes, the channel layer is made of any one of PDMS, PMMA, PET and PBT; and/or the substrate layer is a glass plate.

In other optimized technical schemes, the surface of the glass plate is modified with distearoyl phosphatidyl ethanolamine-polyethylene glycol.

In other optimized technical schemes, the preparation method of the microfluidic chip comprises the following steps:

respectively preparing the channel layer and the substrate layer, and assembling the channel layer and the substrate layer to obtain the microfluidic chip; the preparation method of the channel layer comprises the following steps:

s1: printing the microstructure of the channel layer by using a 3D printing technology;

s2: and copying the microstructure by using a reverse mode method to obtain the channel layer.

In another preferred embodiment, the assembling method of the channel layer and the substrate layer includes: bonding the channel layer on the base layer by surface plasma treatment.

In other optimized technical schemes, the microfluidic chip is used for separating plasma lipoprotein and exosome, and/or detecting the lipoprotein and the exosome respectively, wherein the microfluidic chip is prepared by any one of the preparation methods.

The invention has the beneficial effects that:

the micro-fluidic chip provided by the invention adopts the mixing units with alternate widths, the mixing efficiency of a sample can be effectively improved, and functional areas including mixing and separation detection are integrated on the chip, so that the production or research efficiency can be effectively improved.

Further optimization, the 3D printing technology is adopted to prepare the micro-fluidic chip, so that a higher cavity can be prepared on one hand, and channels with different heights can be prepared on the other hand, and the method is simple and easy to implement and is convenient for industrial popularization and application. Compared with the traditional technology, the micro-fluidic chip designed by the invention has higher efficiency when being used for separating plasma lipoprotein and exosome.

Drawings

In order to more clearly illustrate the technical solution of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without any creative effort.

FIG. 1 is a schematic view of a channel layer of a microfluidic chip in example 1;

FIG. 2 is a simulation of the mixing effect of a conventional rectangular microchannel (a) and a mixing channel (b) of example 1;

FIG. 3 is a channel layer microstructure mold printed by 3D printing technique as in example 1;

FIG. 4 is a PDMS chip obtained by back-molding PDMS in example 1 and an enlarged view thereof;

FIG. 5 is a schematic view of a channel layer of the microfluidic chip in example 2;

FIG. 6 shows the results of staining the lipoprotein targeting quantum dot Dye (Qdot 605-antitAPOE) and the cell membrane green fluorescent Dye (DIO Dye) in the lipoprotein detection chamber and the exosome detection chamber, respectively, in example 2;

fig. 7 is a diagram illustrating two-dimensional code addresses for storing the color original drawings of fig. 2 and fig. 5 to 6.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that, unless otherwise specifically limited, operations such as "assembling", "copying", "bonding", etc. in the description of the present invention are to be construed broadly and operations that can achieve the objects of the present invention are within the scope of the present invention. All directional indicators (such as up, down, left, right, front, and rear … …) in the embodiments of the present invention are only used to explain the relative positional relationship between the components, the movement, and the like in a specific state (as shown in the drawings), and if the specific state changes, the directional indicator changes accordingly. In the description of the present invention, the measurement direction of "length" is the flow direction of the sample, the measurement direction of "width" is the direction perpendicular to the flow direction of the sample and parallel to the base layer, and the measurement direction of "height" is the direction perpendicular to the base layer. In the present invention, the terms "connected," "communicating," and the like are to be construed broadly unless otherwise explicitly specified or limited.

Example 1:

the embodiment provides a microfluidic chip, which comprises a channel layer and a substrate layer, wherein the channel layer is bonded on the substrate layer through surface plasma treatment.

The channel layer has further structural characteristics as shown in fig. 1, and comprises a sample injection region 1, a mixing region 2 and a detection region 3 which are sequentially communicated. In this embodiment, the sample injection region 1 includes two sample injection ports, and the sample injection channel is in a "Y" shape, and both the width and the height of the "Y" shaped channel are in the micrometer scale, specifically, in this embodiment, the width of the "Y" shaped channel is designed to be 200 μm, and the height is designed to be 100 μm. The sample A and the sample B are respectively input from the two sample inlets and then enter the mixing area 2 through the sample inlet channels.

In other embodiments, the sample introduction channel may also be designed to be "T" shaped, "S" shaped, and the like, and the height and width of the channel may also be adjusted, and in a specific application scenario, as long as the shape and size of the channel that can meet the corresponding sample introduction requirement are within the protection scope of the present invention.

With continued reference to fig. 1, the mixing area 2 includes a plurality of mixing units, the specific number of the mixing units can be determined according to the mixing system in the specific application, the microfluidic chip of the embodiment includes not less than 100 mixing units, and each mixing unit includes 1 wide channel and 1 narrow channel. The width of the wide channel can be adjusted between 100-300 μm, such as 200 μm, the width of the narrow channel can be 1/5-1/2 of the width of the wide channel, such as 50 μm, the length of the wide channel is 400 μm, the height is 100 μm, the length of the narrow channel is 200 μm, and the height is 100 μm. Compared with a microchannel with a pure rectangular structure, the mixing units with different widths can effectively improve the mixing efficiency, and reference can be made to the simulation result shown in fig. 2.

Wherein, the attached figure 2-a is a common rectangular micro-channel with a width of 200 μm, the attached figure 2-B is a mixing channel with a wide-narrow alternate structure in the embodiment, and the mixing area of the two is a vertical channel in the figure, the sample injection area is a horizontal channel in the figure, wherein, the length of the vertical channel is 5.2mm, the length of the horizontal channel is 4.2mm (from the meeting time of the two samples), the sample injection speed of the sample A and the sample B is 5mm/s, the sample temperature is 20 ℃, the viscosity is 1 mPa.s, and the diffusion coefficient is 1e^-9m²The simulation software is COMSOL Multiphysics 5.6, and the results are shown in FIG. 2: in the sample introduction area, the flow state of the fluid is laminar flow, and the sample A and the sample B are still in a layered state and are not effectively mixed. After entering the mixing region, the flow state of the fluid in fig. 2-a is still laminar flow, and the mixing is started gradually in the second half of the mixing channel, but at this time, it can still be seen that the two have a relatively obvious concentration gradient, and the mixing effect is poor; after entering the wide-narrow mixing unit, the fluid in figure 2-b forms a 'turbulent-like flow', and passes through the wide-narrow mixing unitAfter 4 wide and narrow mixing units, better mixing is realized, and at the moment, the fluid in the channel is uniformly mixed, and a concentration gradient hardly exists, so that the mixing efficiency of the wide and narrow interphase mixing structure in the embodiment is high.

With continued reference to FIG. 1, the sample passes through the mixing region 2 and into the detection region 3. In this embodiment, the detection region 3 includes a first chamber 31 and a second chamber 32, and the mixing region 2 is communicated with the first chamber 31, and the first chamber 31 is communicated with the second chamber 32.

The height of the first chamber can be adjusted between 0.5mm and 1.5mm, mainly for the convenience of accommodating samples transiting from the mixing area, in the embodiment, the height of the first chamber is designed to be 1mm, and the volume is designed to be 5-10 muL; the height of the second chamber can be adjusted between 5mm and 10mm, and in the embodiment, the volume of the second chamber is designed to be larger and can reach 140 mu L to 300 mu L, so that a corresponding sample can be fully contained. However, the above design of the size and capacity of the chamber is determined according to the application requirements, and does not limit the scope of the technical solution to be protected by the present invention.

Further, the microfluidic chip can be prepared by the following method:

firstly, respectively preparing a channel layer and a substrate layer, and assembling the channel layer and the substrate layer to obtain a target microfluidic chip; the preparation method of the channel layer comprises the following steps:

s1: printing the microstructure on a piece of resin by using a 3D printing technology, and determining the size of the cavity by controlling the height and the width of the microstructure to obtain the microstructure of the channel layer; as shown in fig. 3, the channel layer microstructure mold obtained by printing through the 3D printing technique in this embodiment is shown;

s2: the microstructure is replicated using a reverse-mode method. Specifically, the channel layer can be obtained by copying the microstructure onto Polydimethylsiloxane (PDMS) in the form of a PDMS reverse mold. FIG. 4 shows a PDMS chip obtained by back-molding PDMS in this embodiment and an enlarged view thereof;

and finally, bonding the PDMS chip on a glass plate through surface plasma treatment so as to finish the manufacture of the target microfluidic chip. The preparation method has the following remarkable advantages: the method can be used for preparing a cavity with a higher height (such as a cavity with a height of 1-10 mm), and channels or cavities with different heights are more efficient and convenient to use through 3D printing, so that the method is very suitable for manufacturing a micro-channel structure mold to be protected by the invention.

Example 2:

the present embodiment provides a microfluidic chip, which includes a channel layer and a substrate layer, wherein the channel layer is bonded to the substrate layer by surface plasma treatment, and the specific preparation method is the same as that in embodiment 1.

Further, the structure of the channel layer in this embodiment is the same as that in embodiment 1. Further, the channel layer in this embodiment may be made of PDMS, but PMMA, a vinyl polymer, or the like may be used instead.

The substrate layer can be made of a glass plate or a polyethylene plate, the glass plate is used as a material for making the substrate layer in the embodiment, and distearoyl phosphatidyl ethanolamine-polyethylene glycol (DSPE-PEG) is modified on the surface of the glass plate.

Further, the microfluidic chip of this embodiment further includes a magnet, as shown in fig. 5, disposed below the first chamber.

Next, plasma lipoproteins and exosomes were separated and detected separately using the microfluidic chip in this example.

As shown in the attached figure 5, a pretreated plasma sample and a CB (Ciba Bayland) magnetic bead sample are respectively injected from two injection ports at the same flow rate (1-10 muL/min), and enter a mixing area 2 through an injection area 1, and are fully mixed in the mixing area 2, so that the CB magnetic bead can better grab lipoproteins, and then the plasma sample and the lipoproteins grabbed by the magnetic bead enter a first chamber 31 along with the magnetic bead, in the first chamber, the magnetic bead is placed on a magnetic paste under a microfluidic chip for fixing, while the plasma continuously flows and enters a second chamber 32 at the back, the sample is accumulated in the second chamber 32, wherein exosome in the plasma sample is grabbed by distearoylethanolamine-polyethylene glycol (DSPE-PEG) modified on the surface of a substrate layer, and the microfluidic chip is used for separating and detecting the lipoproteins and exosome in the plasma, an amount of about 100. mu.L of plasma can be treated at one time.

Further, in this embodiment, most of the plasma abundant proteins are removed from the plasma by ultrafiltration (100K) before the plasma enters the microfluidic chip, and the raw materials for preparing CB magnetic beads include amino magnetic beads (1 μm in diameter, 10mg dispersed in PBS), Cibacron Blue 3GA (0.02 g dispersed in deionized water), and sodium chloride (0.20 g dispersed in deionized water), and the three raw materials are mixed and reacted at 40 ℃ for 2 hours.

Finally, a sample in the first chamber 31 is taken and is marked as a sample 1, a sample in the second chamber 32 is taken and is marked as a sample 2, and lipoproteins and exosomes in the sample 1 and the sample 2 are stained by using a cell membrane green fluorescent Dye (DIO Dye) and a lipoprotein-targeting quantum dot Dye (Qdot605-anti apoe), and quantitative analysis is performed, and the result is shown in fig. 6.

Wherein, fig. 6-a is a representation image of a sample 1 after Qdot605-anti APOE staining, fig. 6-b is a representation image of a sample 2 after Qdot605-anti APOE staining, and fig. 6-c is a Qdot signal intensity histogram of the sample 1 and the sample 2 after Qdot605-anti APOE staining; fig. 6-d is a characterization image of the sample 1 after DIO Dye staining, fig. 6-e is a characterization image of the sample 2 after DIO Dye staining, and fig. 6-f is a DIO signal intensity histogram of the sample 1 and the sample 2 after DIO Dye staining.

As can be seen from FIG. 6, the Qdot605 fluorescence signal on sample 1 is strong and the Qdot605 fluorescence signal on sample 2 is weak, demonstrating that a large amount of lipoproteins are captured on the CB beads, while almost no signals of lipoproteins are found on the DSPE. The DIO Dye staining results prove that vesicles with membrane structures are found on the magnetic beads in the first cavity and the DSPE in the second cavity, the lipoprotein mainly exists on the magnetic beads, and the exosome is mainly captured by the DSPE, so that the purpose of efficiently and conveniently separating plasma lipoprotein and exosome is achieved, the exosome and the lipoprotein can be respectively detected, and the application research of the exosome and the exosome in disease mechanism and diagnosis can be promoted.

To more clearly illustrate the technical solutions and the technical effects achieved by the embodiments of the present invention, the two-dimensional code viewing addresses of the color original drawings shown in fig. 2 and fig. 5 to 6 are attached, as shown in fig. 7.

The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent structural changes made by using the contents of the specification and drawings of the present invention or other related technical fields directly/indirectly using the technical idea of the present invention shall fall within the scope defined by the claims of the present invention without departing from the spirit of the present invention.

Claims

1. A microfluidic chip, characterized in that:

the device comprises a channel layer and a substrate layer, wherein the channel layer is positioned on the substrate layer;

2. The microfluidic chip of claim 1, wherein:

the mixing region comprises not less than 100 mixing units, each mixing unit comprises 1 wide channel and 1 narrow channel; the length of the wide channel is 200-500 mu m, and the length of the narrow channel is not more than the length of the wide channel.

3. The microfluidic chip of claim 1, wherein:

the detection area comprises a first chamber and a second chamber which are communicated in sequence; the mixing region is in communication with the first chamber, which is in communication with the second chamber.

4. A microfluidic chip as claimed in claim 3, wherein:

the height of the first chamber is 0.5-1.5 mm, and the volume of the first chamber is 5-10 mu L; the microfluidic chip further comprises a magnet, and the magnet is arranged below the first chamber.

5. A microfluidic chip as claimed in claim 3, wherein:

the height of the second chamber is 5-10 mm, and the volume of the second chamber is 140-300 mu L.

6. The microfluidic chip of claim 1, wherein:

the channel layer is made of any one of PDMS, PMMA, PET and PBT; and/or the substrate layer is a glass plate.

7. The microfluidic chip of claim 6, wherein:

the surface of the glass plate is modified with distearoyl phosphatidyl ethanolamine-polyethylene glycol.

8. The method for preparing a microfluidic chip according to any one of claims 1 to 7, wherein:

9. The method for preparing a microfluidic chip according to claim 8, wherein:

the assembling method of the channel layer and the substrate layer comprises the following steps: bonding the channel layer on the base layer by surface plasma treatment.

10. Use of a microfluidic chip according to any of claims 1 to 7, wherein:

the microfluidic chip is used for separating plasma lipoprotein and exosome, and/or detecting the lipoprotein and the exosome respectively, wherein the microfluidic chip is prepared by the preparation method of claim 8 or 9.