WO2021115047A1

WO2021115047A1 - Microfluidic chip and whole blood separation method based on microfluidic chip

Info

Publication number: WO2021115047A1
Application number: PCT/CN2020/129487
Authority: WO
Inventors: 陈思卉; 陈希; 杨慧; 张翊
Original assignee: 深圳先进技术研究院
Priority date: 2019-12-13
Filing date: 2020-11-17
Publication date: 2021-06-17
Also published as: CN111001451A

Abstract

The present invention provides a microfluidic chip, comprising a first micropillar array, and a whole blood microchannel and a plasma microchannel, which are separated by the first micropillar array. When whole blood enters the whole blood microchannel, the pores between the micropillar arrays filter the larger blood cells, and also provide a capillary force to separate the plasma, and the separated plasma enters the plasma microchannel, realizing the separation of blood cells and plasma. The microfluidic chip provided by the present invention filters the blood cells by means of the tiny pores and also provides a sufficient capillary force for blood so as to achieve whole blood separation without control from an external power; in addition, the continuous pore structure can solve the problem of the stagnation of whole blood separation caused by the clogging of blood cells, thereby improving the efficiency of whole blood separation.

Description

Microfluidic chip and whole blood separation method based on microfluidic chip

Technical field

The invention relates to the technical field of microfluidic technology, in particular to a microfluidic chip and a whole blood separation method based on the microfluidic chip.

Background technique

As the most popular sample in clinical diagnosis, blood contains a large proportion of disease markers in the human body. It is suitable for various tests such as immunodiagnosis, clinical biochemistry, and molecular diagnosis. It is currently the most commonly used sample object. Most of the tests performed on blood require the removal of blood cells from the blood for plasma or serum extraction during the sample pretreatment stage, or the need to enrich the cells in the blood for the next step of the relevant test. Different from traditional methods of whole blood separation such as centrifugation, filtration, and salting out, in the emerging field of in vitro diagnostics such as microfluidic technology and lab-on-a-chip, corresponding new miniaturized, highly integrated whole blood separation methods are required.

The current whole blood separation methods based on microfluidic technology can be divided into active and passive types according to whether external physical fields provide power or not. Active methods are not easy to integrate with other microfluidic functional devices because they involve complex control structures such as magnetism, electricity, and ultrasound. The passive method mainly relies on fluid dynamics and microchannel geometry to achieve different behavioral control of blood. The preparation process is relatively simple and easy to integrate. It shows advantages in the development of miniaturized point-of-care instruments. According to different separation principles, passive microfluidic whole blood separation methods can be divided into three types: filtration, sedimentation, and directional cell migration. Different from the other two methods, the directional cell migration method achieves effective plasma extraction by directional control of the blood cell trajectory, which has advantages in the development of easy-to-manipulate high-purity whole blood separation devices.

At present, the directional cell migration method is mainly used for the separation of whole blood in the following two realization schemes: 1. Deterministic lateral displacement method, the blood passes through a series of regular and orderly microstructures in the laminar flow process, and the cells will be of different sizes according to their own Deterministic lateral displacement occurs, thereby realizing the separation of plasma and blood cells; 2. The hydrodynamic method uses the inertial force, viscous force, Dean vortex and other methods of the vertical or curved tube wall on the cell to achieve the orientation of the cell Migrate laterally to achieve the purpose of separation from plasma.

Due to the high viscosity of the blood itself, the plasma will further increase after separation. Therefore, the current whole blood separation based on the above two methods requires a negative pressure pump or a fluid pump to provide power control to the blood, which is not easy for the microscopic separation of the overall separation device. miniaturization.

technical problem

In view of this, it is necessary to provide a microfluidic chip with high whole blood separation efficiency and a whole blood separation method based on the microfluidic chip in view of the defects of the prior art.

Technical solutions

In order to achieve the above objectives, the present invention adopts the following technical solutions:

In one aspect, the present invention provides a microfluidic chip including a first micropillar array and a whole blood microchannel and a plasma microchannel separated by the first micropillar array. When the whole blood is composed of the whole blood microchannel When entering, the pores between the microcolumn arrays filter the blood cells with a larger size, and at the same time provide capillary force to separate the plasma, and the separated plasma enters the plasma microchannel to realize the separation of blood cells and plasma.

In some preferred embodiments, the first micropillar array is spirally distributed.

In some preferred embodiments, the micropillars of the first micropillar array are cylindrical.

In some preferred embodiments, the diameter of the cylinder is 10-1000 microns, and the distance between adjacent cylinders is 0.5-2.5 microns.

In some preferred embodiments, a capillary pump structure is provided at the bottom of the whole blood microchannel and the plasma microchannel.

In some preferred embodiments, the capillary pump structure includes a second micropillar array structure.

In some preferred embodiments, the second micropillar array structure is arranged regularly.

In some preferred embodiments, the microfluidic chip has a single-layer or multi-layer structure, and the microchannel depth of the whole blood microchannel and the plasma microchannel is 10-2000 micrometers and the width is 10-5000 micrometers.

In addition, the present invention also provides a whole blood separation method of the microfluidic chip, which includes the following steps:

The movement of the blood to be separated spreads to the nearest micro-column of the first micro-column and stagnates at the gap between the micro-columns. The blood to be separated is driven by the capillary force along the whole blood micro-channel, and at the same time in the process After passing through the micro-column gap in turn and stagnating, the pores between the micro-column arrays filter larger blood cells while providing capillary force to separate the plasma; the separated plasma will pass through the micro-column gap through the starting point and travel along the plasma micro-columns. The channel is self-driving forward and triggers the micro-column gap flowing through one by one, continuously filtering out plasma from the whole blood;

Blood and plasma enter the capillary pump structure at the bottom ends of the whole blood microchannel and the plasma microchannel respectively, and the capillary pump structure collects and measures blood and plasma.

Beneficial effect

The advantages of the present invention using the above technical solution are:

The microfluidic chip provided by the present invention includes a first micropillar array and a whole blood microchannel and a plasma microchannel separated by the first micropillar array. When whole blood enters through the whole blood microchannel, the microchannel The pores between the column arrays filter larger blood cells and provide capillary force to separate the plasma. The separated plasma enters the plasma microchannel to realize the separation of blood cells and plasma. The microfluidic chip provided by the present invention utilizes tiny pores. While filtering blood cells, it provides sufficient capillary force to achieve whole blood separation without external power control; in addition, the continuous pore structure can solve the problem of stagnant separation of whole blood caused by clogging of blood cells, thereby improving The efficiency of whole blood separation.

Description of the drawings

FIG. 1 is a schematic structural diagram of a microfluidic chip provided by an embodiment of the present invention;

Figure 2 is a schematic structural diagram of a capillary pump structure provided by an embodiment of the present invention;

Fig. 3 is a side view of a microfluidic chip provided by an embodiment of the present invention.

Fig. 4 is a process principle diagram of a microfluidic chip provided by an embodiment of the present invention.

The best mode of the present invention

The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

Example one

Please refer to FIG. 1, which is a schematic structural diagram of a microfluidic chip provided by Embodiment 1 of the present invention, including: a first micropillar array 110 and a whole blood microchannel 120 and a plasma microchannel separated by the first micropillar array 110 130.

Specifically, the first micropillar array 110 is spirally distributed. It can be understood that the micropillar array 110 may be distributed in an inner spiral or an outer spiral, and the orientation may be distributed from the outside to the inside or from the inside to the outside.

It can be understood that the distribution shape of the first micro-pillar array 110 is not limited to a spiral shape, and may be any unclosed revolving shape.

Specifically, the micropillars of the first micropillar array 110 are cylindrical. The diameter of the cylinder is 10-1000 microns, and the distance between adjacent cylinders is 0.5-2.5 microns. It can be understood that the cylindrical shape should correspond to the diameter of the inner recess of the red blood cell in the filtered blood with a smaller size.

It can be understood that the micro-pillars are not limited to cylindrical shapes, but can also have various shapes such as squares and diamonds. The size and adjacent spacing can be designed in different sizes under the condition of ensuring the feature size, so that the chip can handle different samples in different samples. Cells of different sizes, such as white blood cells with a diameter of 6-20 microns, platelets with a thickness of 0.5-1.5 microns, etc.

In the above-mentioned microfluidic chip, when whole blood enters through the whole blood microchannel, the pores between the micropillar arrays filter the blood cells with a larger size, and at the same time provide capillary force to separate the plasma, and the separated plasma enters the plasma microchannel , In order to achieve the separation of blood cells and plasma.

Referring to FIG. 2, the bottoms of the whole blood microchannel 120 and the plasma microchannel 130 are both provided with a capillary pump structure 140, and the capillary pump structure 140 can collect and measure blood and plasma.

Specifically, the capillary pump structure 140 includes a second micropillar array structure 141, and the second micropillar array structure 141 is arranged in an orderly and regular manner.

It can be understood that the shape, size, and size of the micro-pillars of the second micro-pillar array structure 141 can be designed with various parameters under the condition of ensuring the characteristic size. In addition, the capillary pump structure 141 includes, but is not limited to, structures such as a Christmas tree shape, a serpentine channel shape, and the like.

Please refer to FIG. 3, which is a side view of a microfluidic chip provided by an embodiment of the present invention.

In this embodiment, the microfluidic chip has a single-layer structure, that is, the channel depth of the whole blood microchannel 120 and the plasma microchannel 130 is equal to the depth of the first micropillar array 110.

Specifically, the channel depth of the whole blood microchannel 120 and the plasma microchannel 130 is 10 to 2000 microns, and the width is 10 to 5000 microns.

It should be noted that the capillary force of blood in the microchannel will increase as the channel size decreases, with a higher flow rate, so that the microfluidic chip will achieve higher separation efficiency of whole blood; while the microfluidic control The overall flux of the chip decreases as the channel size decreases. Therefore, the width and depth of the microchannel can be designed with different sizes according to the different focus requirements of different occasions, so that the final chip can achieve different application requirements.

The microfluidic chip provided by the above-mentioned embodiments of the present invention uses tiny pores to filter blood cells, while providing sufficient capillary force to the blood to achieve whole blood separation without external power control; in addition, the continuous pore structure can solve the problem of blood cells. The problem of stagnation of whole blood separation caused by blockage, thereby improving the efficiency of whole blood separation.

Example two

In this embodiment, the microfluidic chip provided by the present invention may be a material that can be manufactured by a micro-nano processing method, such as silicon-based materials such as monocrystalline silicon, silicon oxide, and silicon nitride, and specifically may also be glass materials such as quartz. , Or can be polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA) and other polymer materials. For different materials and different sizes of channels in the chip, preparation methods include, but are not limited to, laser etching, 3D printing, photolithography, plasma etching and other micro-nano processing methods.

Please refer to FIG. 4, which is a process schematic diagram of a microfluidic chip provided by an embodiment of the present invention. The specific steps are as follows:

S1: A layer of photoresist is suspended on the substrate, a corresponding microchannel mask is used to form a microchannel etching window through a photolithography process, and the anode film sacrificial layer is removed by an etching process.

In this embodiment, the substrate is glass or silicon. It can be understood that the substrate includes, but is not limited to, glass materials such as quartz and silicon-based materials such as silicon oxide and silicon nitride.

In this embodiment, the positive film sacrificial layer includes but is not limited to photoresist (including positive photoresist, negative photoresist and other photoresists), silicon oxide, silicon nitride, silicon carbide, or chromium, Metal materials such as aluminum.

It can be understood that the above-mentioned microfluidic chip preparation process is not limited to the photoresist plasma etching method in the embodiment, and the microfluidic channel can be etched on glass or PMMA material by laser etching, and the microfluidic channel can be etched by photolithography. The method is to prepare a micropillar array on a glass or silicon substrate, and then bond the two; or use a laser direct writing method to form patterns such as fluid channels on the surface of glass or organic materials.

S2: Using the cation film layer on the substrate as a mask, etch the base material of the part where the microchannel is located to form the final microchannel, and remove the remaining cation film layer.

In this embodiment, etching includes but is not limited to plasma etching, deep silicon etching, and wet etching. The removal process includes, but is not limited to, cleaning with concentrated sulfuric acid and hydrogen peroxide, and cleaning with other organic solvents. Craft.

S3: Perform surface chemical treatment on the microfluidic chip with a microchannel structure to realize the hydrophilic treatment of the surface of the microfluidic chip and improve the stability and uniformity of the physical and chemical properties of the chip surface.

The surface chemical treatment method for the microfluidic chip with the microchannel structure is not limited to chemical methods such as immersion, fumigation, spraying, etc., and electrochemical, thermal processing, vapor deposition and other methods can also be used.

In this embodiment, the chemical treatment reagents used include, but are not limited to, polyethylene glycol (PEG) and 3-aminopropyl triethoxysilane ((3-aminopropyl) triethoxysilane, APTES), etc. The treatment method used Including but not limited to fumigation, soaking, spraying, etc.

It can be understood that the following functions of the microfluidic chip can be achieved through surface chemical treatment: (1) Hydrophilization, which is beneficial to improve the capillary force driving ability of the whole blood sample on the surface; (2) Improve the uniformity and stability of the physical and chemical properties of the chip surface It can prevent the substrate from non-specific adsorption of cells and proteins in the blood, so that the whole blood sample is not prone to adhesion and stagnation when passing through.

The microfluidic chip prepared by the above steps includes a first micropillar array and a whole blood microchannel and a plasma microchannel separated by the first micropillar array. When whole blood enters through the whole blood microchannel, the The pores between the microcolumn arrays filter the blood cells of larger size, and provide capillary force to separate the plasma, and the separated plasma enters the plasma microchannel to realize the separation of blood cells and plasma.

The microfluidic chip provided by the above-mentioned embodiments of the present invention uses tiny pores to filter blood cells, while providing sufficient capillary force to the blood to achieve whole blood separation without external power control; in addition, the continuous pore structure can solve the problem of blood cells. The problem of stagnant whole blood separation caused by blockage, thereby improving the efficiency of whole blood separation

Example three

The present invention also provides a whole blood separation method based on the microfluidic chip, which includes the following steps:

Step S110: the movement of the blood to be separated spreads to the nearest micro-pillar of the first micro-pillar, and stops at the gap between the micro-pillars, and the blood to be separated is self-driving along the whole blood microchannel under the action of capillary force, At the same time, the process passes through the subsequent micro-column gaps and stagnates. The pores between the micro-column arrays filter larger blood cells and provide capillary force to separate the plasma; the separated plasma will pass through the micro-column gap through the starting point and travel along the path. The plasma microchannel is self-driving forward and triggers the microcolumn gaps flowing through one by one to continuously filter the plasma from the whole blood.

Specifically, the blood sample to be separated is added to the injection port of the microfluidic chip (A in Figure 3 is the injection port), and the blood to be separated has the injection port movement and spreads to the nearest first microcolumn的微柱处。 At the micro-column.

It can be understood that the inlet of the microfluidic chip is not limited to one sample inlet, and can be designed as multiple inlets. Each inlet can be connected to a single or multiple first micropillar arrays, and each first micropillar array can also be Access single or multiple entrances.

Step S110: blood and plasma enter the capillary pump structure at the bottom ends of the whole blood microchannel and the plasma microchannel respectively, and the capillary pump structure collects and measures blood and plasma.

In addition, the microfluidic chip provided by the present invention adopts a capillary force self-driving manner to realize whole blood separation, does not require an external power control device, and has more potential for the development of integration and miniaturization.

The microfluidic chip provided by the present invention is not limited to whole blood/plasma separation, but can also be used to separate and enrich different levels of biological objects in a solution system, such as circulating tumor cells at the cell level, such as extracellular vesicles at the subcellular level. Bubbles, liposomes, etc.

The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, all possible combinations of the various technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, All should be considered as the scope of this specification.

Of course, the positive electrode material of the microfluidic chip of the present invention can also have various transformations and modifications, and is not limited to the specific structure of the above-mentioned embodiment. In short, the protection scope of the present invention should include those alterations or substitutions and modifications that are obvious to those of ordinary skill in the art.

Claims

A microfluidic chip, which is characterized by comprising a first micropillar array and a whole blood microchannel and a plasma microchannel separated by the first micropillar array. When whole blood enters from the whole blood microchannel, The pores between the microcolumn arrays filter blood cells with a larger size and provide capillary force to separate the plasma, and the separated plasma enters the plasma microchannel to realize the separation of blood cells and plasma.
8. The microfluidic chip of claim 1, wherein the first micropillar array is spirally distributed.
The microfluidic chip of claim 1, wherein the micropillars of the first micropillar array are cylindrical.
The microfluidic chip of claim 3, wherein the diameter of the cylindrical shape is 10 to 1000 microns, and the distance between adjacent cylinders is 0.5 to 2.5 microns.
The microfluidic chip of claim 1, wherein the bottoms of the whole blood microchannel and the plasma microchannel are both provided with a capillary pump structure.
8. The microfluidic chip of claim 5, wherein the capillary pump structure comprises a second micropillar array structure.
7. The microfluidic chip of claim 6, wherein the second micropillar array structure is arranged regularly.
The microfluidic chip of claim 1, wherein the microfluidic chip has a single-layer or multi-layer structure, and the microchannel depth of the whole blood microchannel and the plasma microchannel is 10 to 2000 microns, The width is 10~5000 microns.
A whole blood separation method based on the microfluidic chip according to any one of claims 1 to 8, characterized in that it comprises the following steps:

The movement of the blood to be separated spreads to the nearest micro-column of the first micro-column and stagnates at the gap between the micro-columns. The blood to be separated is driven by the capillary force along the whole blood micro-channel, and at the same time in the process After passing through the micro-column gap in turn and stagnating, the pores between the micro-column arrays filter larger blood cells while providing capillary force to separate the plasma; the separated plasma will pass through the micro-column gap through the starting point and travel along the plasma micro-columns. The channel is self-driving forward and triggers the micro-column gap flowing through one by one, continuously filtering out plasma from the whole blood;

Blood and plasma enter the capillary pump structure at the bottom ends of the whole blood microchannel and the plasma microchannel respectively, and the capillary pump structure collects and measures blood and plasma.