Microfluidic plasma separation system and method and detection system for biomarkers in blood

Abstract

The invention provides a microfluidic plasma separation system, which comprises a deposition microfluidic module and a separation microfluidic module, the deposition microfluidic module comprises a first polymer sheet and a second polymer sheet which are bonded with each other, and a first chamber is arranged between the first polymer sheet and the second polymer sheet; the first end of the first chamber is arranged to be an inlet of blood to be separated; the separation micro-fluidic module comprises a silicon-based substrate and a third polymerization sheet which are bonded with each other, a second chamber is arranged between the silicon-based substrate and the third polymerization sheet, and a third chamber and a fourth chamber which are respectively arranged at two sides of the second chamber; the first end of the second chamber is connected with the second end of the first chamber through a connecting device, and the connecting device converts longitudinally-layered blood cells and blood plasma into transversely-layered blood cells and blood plasma and enables the blood to be transversely layered in the second chamber; the second end of the second cavity is connected with the first end of the third cavity and the first end of the fourth cavity through channels. The plasma separation system realizes effective separation of plasma in whole blood.

Description

Technical Field

[0001] The present invention relates to the field of microfluidic technology, and in particular to a microfluidic plasma separation system and method, and a detection system for biomarkers in blood. Background Art

[0002] Blood tests can provide valuable information for clinical diagnosis and drug development. Most blood biochemistry tests are performed with plasma or serum because blood cells and their constituent substances can falsify measurement results. Plasma separation reduces the interference of cells when detecting and analyzing different analytes, improving the accuracy and reliability of the process. Therefore, separating plasma from whole blood is the first step in performing biochemical analysis.

[0003] Laboratory plasma separation is usually performed by centrifugation and membrane filtration, which utilize physical properties such as cell density and size to provide effective plasma separation, but they are still largely limited by factors such as component purity, clogging, processing time and operating efficiency. For example, centrifuges are generally labor-intensive, time-consuming, large in size, and even require operators to be well-trained, have professional knowledge and skills to a certain extent. Centrifugation methods generally require manual pipetting from centrifuge tubes, which inevitably leads to human error and incorrect results, and membrane filtration also faces serious clogging problems.

[0004] In recent years, due to the development of microfluidics technology, the popularity of traditional centrifuges and membrane filtration methods has decreased over time. Microfluidics chip technology can use less blood sample volume and shorten the time to obtain plasma. Microfluidics chips integrate the entire blood testing process on a single chip, preventing human errors.

[0005] Microfluidic systems usually use the differences in the intrinsic properties of different cell populations (i.e., size, deformability, surface charge, and density) to achieve separation, and can be roughly divided into active separation technology and passive separation technology. Active separation technology relies on external force fields (such as acoustics or magnetism) to operate, while passive separation technology only relies on the geometry of the channel and inherent fluid dynamics to achieve its function. However, active separation technology requires more complex design and manufacturing because it requires the application of an external force field, while passive separation technology only needs to design the geometry of the channel to achieve the ultimate goal. Therefore, passive separation technology is more popular than active separation technology.

[0006] Passive separation technology is generally designed by using fluid dynamics, such as the Zweifacher-von effect, deterministic lateral displacement and inertial microfluidic effect, to design and manufacture different microfluidic chips for plasma separation. However, these methods may cause cell damage, or the channels may be blocked by blood cells, or the blood may need to be highly diluted, which makes it impossible to effectively separate plasma, and it is impossible to effectively detect the plasma because of the inability to obtain satisfactory plasma quality. Summary of the invention

[0007] The purpose of the present invention is to provide a microfluidic plasma separation system and method to solve the problems existing in passive separation in existing microfluidic systems, and to achieve plasma separation in blood by utilizing gravity as a natural field.

[0008] According to a first aspect of the present invention, a microfluidic plasma separation system and method are provided, comprising:

[0009] A deposition microfluidic module comprises a first polymer sheet and a second polymer sheet bonded to each other, wherein a first chamber is provided between the first polymer sheet and the second polymer sheet for forming a longitudinal layer of blood cells and plasma in the blood to be separated;

[0010] The first end of the first chamber is configured as an inlet of the blood to be separated, and the second end of the first chamber is connected to the separation microfluidic module;

[0011] A separation microfluidic module comprises a silicon-based substrate and a third polymer sheet bonded to each other, wherein a second chamber is disposed between the silicon-based substrate and the third polymer sheet, and a third chamber and a fourth chamber are disposed on both sides of the second chamber respectively;

[0012] The first end of the second chamber is connected to the second end of the first chamber via a connecting device, and the connecting device converts blood cells and plasma from longitudinal stratification to transverse stratification and flows into the second chamber; the second end of the second chamber is connected to the first end of the third chamber and the first end of the fourth chamber via channels respectively;

[0013] The second end of the third chamber is connected to the first collecting device, and the second end of the fourth chamber is connected to the second collecting device;

[0014] The side close to the blood cell layer in the second chamber is defined as the third chamber, and the side close to the plasma layer in the second chamber is defined as the fourth chamber; the blood cells in the second chamber enter the third chamber through the channel and then enter the first collecting device for collection, and the plasma in the second chamber enters the fourth chamber through the channel and then enters the second collecting device for collection.

[0015] As an optional embodiment, the first end of the first chamber is lower than the second end of the first chamber, so that the first end of the first chamber forms an inclination angle with the horizontal plane, and the angle is 1° to 15°; the dimensions of the first chamber are: length 3cm to 5cm, width 1.5cm to 3cm, height 500μm to 1000μm.

[0016] As an optional embodiment, at least two ribs are provided in the first chamber for supporting the first chamber; the ribs have a width of 0.8 mm to 1.2 mm and a length of 22 mm to 27 mm.

[0017] As an optional embodiment, the second chamber is a circle with a diameter of 1.0mm to 2.0mm and a height of 100μm to 500μm; the third chamber is a circle with a diameter of 1.0mm to 2.0mm and a height of 100μm to 500μm; the fourth chamber is a circle with a diameter of 1.0mm to 2.0mm and a height of 100μm to 500μm.

[0018] As an optional embodiment, the channel includes a first channel, a second channel and a third channel, the first end of the first channel is connected to the second end of the second chamber, the second end of the first channel is respectively connected to the first end of the second channel and the first end of the third channel, the second end of the second channel is connected to the first end of the third chamber, and the second end of the third channel is connected to the first section of the fourth chamber, so that the second chamber, the third chamber and the fourth chamber are connected.

[0019] As an optional embodiment, the width of the first channel is 50μm to 1000μm, and the height is 100μm to 500μm; the width of the second channel is 1 / 4 to 1 times the width of the first channel, and the height is 100μm to 500μm; the width of the third channel is the same as the width of the first channel, and the height is 100μm to 500μm; the lengths of the second channel and the third channel are equal.

[0020] According to a second aspect of the present invention, there is provided a method for separating plasma from blood, using the aforementioned microfluidic plasma separation system for separation, comprising the following steps:

[0021] 1x PBS is injected into the microfluidic plasma separation system to clean and moisten the entire separation system, and then diluted whole blood is injected from the first end of the first chamber. After the blood enters the first chamber, blood cells aggregate and precipitate due to gravity and settle to the bottom of the first chamber, so that blood cells and plasma in the blood form vertical stratification;

[0022] The blood that has formed longitudinal stratification is converted into transverse stratification of blood cells and plasma through a connecting device, and enters the second chamber of the separation microfluidic module to make the blood in a laminar state, and the blood cells and plasma in the blood continue to maintain transverse stratification; then, the blood cells in the second chamber enter the third chamber through the channel and then enter the blood cell collection device, and the plasma in the second chamber enters the fourth chamber through the channel and then enters the plasma collection device.

[0023] As an optional embodiment, the rate of infusing whole blood is 5-20 μL / min.

[0024] According to a third aspect of the present invention, a system for detecting biomarkers in blood is provided, comprising the aforementioned microfluidic plasma separation system, and

[0025] A detection system is set up according to the target biomarker and is used to detect the target biomarker in the plasma separated by the plasma separation system.

[0026] As an optional embodiment, the detection system includes a carbon-based field effect transistor biosensor.

[0027] It can be seen from the above technical solution of the present invention that the microfluidic plasma separation system proposed in the present invention, through the deposition microfluidic module, utilizes the action of gravity to aggregate and precipitate blood cells, thereby vertically separating the blood cells and plasma in the blood, and then converts the longitudinally separated blood cells and plasma into horizontal separation through the connecting device and enters the second chamber of the separation microfluidic module to form a laminar flow state, so that the cells move along their own streamlines without being disturbed by the plasma, and thus flow into the chambers on their respective sides, and the blood cells and plasma are diverted out, thereby realizing effective separation of plasma.

[0028] The microfluidic plasma separation system of the present invention can achieve high-throughput plasma separation, does not require the use of complex external force fields, and does not require professional personnel to operate. It can quickly obtain high-purity, high-yield plasma from diluted whole blood under limited conditions, and the plasma separation system of the present invention is very stable and has a low failure rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic structural diagram of an exemplary microfluidic plasma separation system of the present invention.

[0030] Figure 2 It is a schematic diagram of the structure of a microfluidic plasma separation system in another example of the present invention.

[0031] Figure 3 is Figure 1 Schematic diagram of the connection device converting the longitudinally layered blood cells and plasma into transverse layers under the connection mode.

[0032] Figure 4 is Figure 2 Schematic diagram of the connection device converting the longitudinally layered blood cells and plasma into transverse layers under the connection mode.

[0033] Figure 5 Schematic diagram of the movement of whole blood in the deposition microfluidic module in the example of the present invention.

[0034] Figure 6 Schematic diagram of the micrograph of the plasma after separation in the example of the present invention.

[0035] Figure 7 FIG. 1 is a microscopic diagram of blood before separation in an example of the present invention.

[0036] Explanation of the accompanying drawings: 1. Deposition microfluidic module; 11. First polymer sheet; 12. Second polymer sheet; 13. First chamber; 131. First end of the first chamber; 132. Second end of the first chamber; 133. Rib; 2. Separation microfluidic module; 21. Silicon-based substrate; 22. Third polymer sheet; 23. Second chamber; 24. Third chamber; 25. Fourth chamber; 26. First channel; 27. Second channel; 28. Third channel; 3. Connecting device; 31. First L-shaped steel needle; 32. Hose; 33. Second L-shaped steel needle. DETAILED DESCRIPTION

[0037] In order to better understand the technical content of the present invention, specific embodiments are given and described as follows in conjunction with the accompanying drawings.

[0038] Various aspects of the present invention are described in this disclosure with reference to the accompanying drawings, in which many illustrative embodiments are shown. The embodiments of the present disclosure are not necessarily intended to include all aspects of the present invention. It should be understood that the various concepts and embodiments introduced above, as well as those described in more detail below, can be implemented in any of many ways.

[0039] Microfluidic plasma separation system

[0040] Combination Figure 1-4 As shown, in an exemplary embodiment of the present invention, a microfluidic plasma separation system is provided, comprising a deposition microfluidic module 1 and a separation microfluidic module 2 .

[0041] like Figure 1 , 2 As shown, the deposition microfluidic module 1 includes a first polymer sheet 11 and a second polymer sheet 12 bonded to each other, and a first chamber 13 is provided between the first polymer sheet 11 and the second polymer sheet 12 for forming vertical layers of blood cells and plasma in the blood to be separated.

[0042] The first end 131 of the first chamber is configured as an inlet of the blood to be separated, and the second end 132 of the first chamber is connected to the separation microfluidic control module 2 .

[0043] The separation microfluidic module 2 comprises a silicon-based substrate 21 and a third polymer sheet 22 bonded to each other, a second chamber 23 is provided between the silicon-based substrate 21 and the third polymer sheet 22, and a third chamber 24 and a fourth chamber 25 are respectively provided on both sides of the second chamber.

[0044] The first end of the second chamber is connected to the second end of the first chamber via a connecting device 3, which converts the longitudinally stratified blood cells and plasma into transverse stratification and keeps the blood in the second chamber 23 in transverse stratification; the second end of the second chamber is connected to the first end of the third chamber and the first end of the fourth chamber respectively through channels.

[0045] The first end of the second chamber is connected to the second end of the first chamber via a connecting device 3, which converts blood cells and plasma from longitudinal stratification to transverse stratification and flows into the second chamber 23; the second end of the second chamber 23 is connected to the first end of the third chamber 24 and the first end of the fourth chamber 25 through channels.

[0046] It can be understood that the deposition microfluidic module 1 and the separation microfluidic module 2 can be in the same horizontal plane or there can be a height difference. Both modules can be placed on a solid platform for easy operation, for example, they can be placed on acrylic plates respectively.

[0047] As an optional example, the connecting device 3 is a first L-shaped steel needle 31, a hose 32 and a second L-shaped steel needle 33 connected in sequence, and the sizes of the hose and the steel needle can be adapted to the connecting hole diameter.

[0048] The purpose of the connecting device is to drain the blood in the first chamber into the second chamber, and at the same time transform the longitudinal stratification of blood cells at the bottom and plasma at the top in the first chamber into a transverse stratification of blood cells at the left and plasma at the right, so that the blood in the second chamber presents a transverse stratification of blood cells at the left and plasma at the right, which is convenient for subsequent separation. Therefore, the setting of the connecting device only needs to meet the conversion of the blood cell and plasma stratification modes; for example, when the deposition microfluidic module 1 and the separation microfluidic module 2 are in the same horizontal plane, the connecting device 3 can also be directly an L-shaped steel needle at both ends; when the deposition microfluidic module 1 and the separation microfluidic module 2 have a height difference, the connecting device 3 can also be directly a vertical steel needle.

[0049] In combination with the two positions of the aforementioned deposition microfluidic module 1 and the separation microfluidic module 2, a steel needle, a hose and a steel needle connected in sequence are used here to exemplify how to connect to achieve the purpose of converting the longitudinally layered blood cells and plasma into transverse layers.

[0050] like Figure 1 As shown, when the deposition microfluidic module 1 and the separation microfluidic module 2 are in the same horizontal plane, a first L-shaped steel needle is inserted into the second end of the first chamber from above the deposition microfluidic module 1, and a second L-shaped steel needle is inserted into the first end of the second chamber from above the separation microfluidic module 2, and the first L-shaped steel needle and the second L-shaped steel needle are connected by a hose.

[0051] like Figure 3 As shown, under this connection mode, the blood cells (red in the figure) and plasma (yellow in the figure) in the longitudinal stratification flow along the first L-shaped steel needle and the hose. When the blood flows to the second L-shaped steel needle, the stratification mode changes and is converted into a horizontal stratification with blood cells on the left and plasma on the right. The blood enters the second chamber in this stratified state and maintains the horizontal stratification in the second chamber (that is, as shown in FIG. Figure 3 left and right layers shown).

[0052] like Figure 2 As shown, when the deposition microfluidic module 1 and the separation microfluidic module 2 have a height difference, a first L-shaped steel needle is inserted into the second end of the first chamber from the bottom of the deposition microfluidic module 1, and a second L-shaped steel needle is inserted into the first end of the second chamber from the top of the separation microfluidic module 2, and the first L-shaped steel needle and the second L-shaped steel needle are connected by a hose.

[0053] like Figure 4 As shown, under this connection mode, the blood cells (red in the figure) and plasma (yellow in the figure) in the longitudinal stratification flow along the first L-shaped steel needle and the hose. When the blood flows to the second L-shaped steel needle, the stratification mode changes and is converted into a horizontal stratification with blood cells on the left and plasma on the right. The blood enters the second chamber in this stratified state and maintains the horizontal stratification in the second chamber (that is, as shown in FIG. Figure 4 left and right layers shown).

[0054] As an optional example, the channel includes a first channel 26, a second channel 27 and a third channel 28, the first end of the first channel 26 is connected to the second end of the second chamber, the second end of the first channel 26 is respectively connected to the first end of the second channel 27 and the first end of the third channel 28, the second end of the second channel 27 is connected to the first end of the third chamber 24, and the second end of the third channel 28 is connected to the first section of the fourth chamber 25, so that the second chamber 23, the third chamber 24 and the fourth chamber 25 are connected.

[0055] The second end of the third chamber 24 is connected to the first collecting device, and the second end of the fourth chamber 25 is connected to the second collecting device.

[0056] The side close to the blood cell layer in the second chamber is defined as the third chamber 24, and the side close to the plasma layer in the second chamber is defined as the fourth chamber 25; the blood cells in the second chamber enter the third chamber 24 through the first channel 26 and the second channel 27 and then enter the first collecting device for collection, and the plasma in the second chamber enters the fourth chamber 25 through the first channel 26 and the third channel 28 and then enters the second collecting device for collection.

[0057] As an optional example, the dimensions of the first chamber 13 are: length 3cm~5cm, width 1.5cm~3cm, height 500μm~1000μm; this dimension setting shortens the absolute height that the red blood cells need to descend, so that the red blood cells can settle to the bottom of the first chamber in a shorter time to achieve longitudinal separation of the cell layer-plasma layer; on the other hand, the channel height cannot be too low. Such a height can maintain the continuous fluidity of the blood in the sedimentation channel on the basis of shortening the red blood cell sedimentation time, thereby avoiding channel blockage.

[0058] As an optional example, the first end of the first chamber is lower than the second end of the first chamber, so that the first end of the first chamber forms an inclination angle with the horizontal plane, the angle is 1° to 15°, and 5° is particularly preferred; adopting this inclined setting can make it more difficult for cells to flow into the separation port, thereby improving the stratification effect.

[0059] As an optional example, at least two ribs 133 are provided in the first chamber 13 for supporting the first chamber. The positions of the ribs 133 are based on dividing the space of the first chamber equally as much as possible. For example, in the case of two ribs, it is advisable to divide the space of the first chamber into three equal parts. It can be understood that the number and position of the ribs include but are not limited to these, and it is only necessary to ensure that the first chamber can be supported and not collapse.

[0060] As an optional example, the rib beam 133 has a width of 0.8 mm to 1.2 mm and a length of 22 mm to 27 mm, and is particularly preferably 1 mm in width and 25 mm in length.

[0061] As an optional example, the second chamber 23 is a circle with a diameter of 1.0 mm to 2.0 mm and a height of 100 μm to 500 μm; the third chamber 24 is a circle with a diameter of 1.0 mm to 2.0 mm and a height of 100 μm to 500 μm; the fourth chamber 25 is a circle with a diameter of 1.0 mm to 2.0 mm and a height of 100 μm to 500 μm.

[0062] In a more preferred example, the second chamber 23 is a circle with a diameter of 1.5 mm and a height of 100 μm; the third chamber 24 is a circle with a diameter of 1.5 mm and a height of 100 μm; and the fourth chamber 25 is a circle with a diameter of 11.5 mm and a height of 100 μm.

[0063] It can be understood that the shapes and sizes of the second chamber 23, the third chamber 24 and the fourth chamber 25 include but are not limited to the above and can be selected according to actual conditions.

[0064] As an optional example, the width of the first channel 26 is 50μm to 1000μm, and the height is 100μm to 500μm; the width of the second channel 27 is 1 / 4 to 1 times the width of the first channel, and the height is 100μm to 500μm; the width of the third channel 28 is the same as the width of the first channel, and the height is 100μm to 500μm; the lengths of the second channel 27 and the third channel 28 are equal.

[0065] In a more preferred example, the first channel 26 has a width of 300 μm and a height of 100 μm; the second channel 27 has a width of 100 μm and a height of 100 μm; and the third channel 28 has a width of 300 μm and a height of 100 μm.

[0066] It can be understood that the parts that need to be connected in the present invention can be connected by steel needles and hoses, and its structure includes but is not limited to steel needles, hoses and steel needles connected in sequence, which can be arranged according to needs.

[0067] For example, the deposition microfluidic module and the separation microfluidic module are connected by inserting a steel needle into the second end of the first cavity and inserting another steel needle into the first end of the second cavity, and the two steel needles are connected by a hose.

[0068] For another example, an inlet for the blood to be separated is set at the first end of the first chamber, a steel needle is inserted into the inlet, the steel needle is connected to a hose, and then a syringe is used to connect the hose, so that blood is injected into the first chamber.

[0069] For another example, a hole is punched on the second end of the third cavity, a steel needle is inserted, a hose is connected to the steel needle, and the hose is extended into the first collecting device.

[0070] As an optional example, the materials of the first polymer sheet 11 , the second polymer sheet 12 and the third polymer sheet 22 are all PDMS.

[0071] As optional examples, the silicon-based substrate includes a glass wafer and a silicon wafer.

[0072] The bonding principle between the silicon-based substrate and the third polymer sheet is that when the plasma-treated PDMS surface contacts the silicon-based material, the hydroxyl groups on the surface can react with the silanol groups (Si-OH) on the surface of the silicon-based material to form silicon-oxygen bonds (Si-O-Si) to achieve chemical bonding.

[0073] Preparation method of microfluidic plasma separation system

[0074] [Preparation of deposition microfluidic module 1]

[0075] In a preferred embodiment of the present invention, the deposition microfluidic module 1 is prepared by using a laser cutting method to cut a thin acrylic plate into a designed shape and size, and fasten it to a smooth and flat thick acrylic plate as a mold for making the upper layer of the deposition channel of the microfluidic chip; and the lower layer of the deposition channel is directly cast using PDMS on a smooth and flat acrylic plate with a slight slope; after the upper and lower PDMS sheets are cast, they are heated and solidified, and finally the solidified PDMS is punched and placed in a plasma cleaning machine for bonding.

[0076] In one exemplary embodiment, the preparation process of the deposition microfluidic module 1 includes the following steps:

[0077] Step 1-1, use a laser cutting machine to cut the acrylic plate into the designed shape and size, and fasten it to a smooth and flat millimeter-level acrylic plate as a mold for making the upper layer of the deposition microfluidic module; take another smooth and flat acrylic plate as a mold for the lower layer of the deposition microfluidic module.

[0078] Step 1-2: Add PDMS prepolymer and curing agent into a glass cup, stir well to prepare a PDMS solution, then pour it on the upper and lower molds of the deposition microfluidic module respectively, and put it into a vacuum tank to extract bubbles.

[0079] Step 1-3: After all the bubbles are extracted, place the mold that has completed step 1-2 on a hot plate and bake it at 80°C for one hour until the PDMS solidifies.

[0080] Step 1-4: After the PDMS solidifies, use a knife to cut off the upper part with the deposition channel pattern and the lower smooth surface, and make the size of the lower smooth surface larger than the upper part with the deposition channel pattern, and use a puncher to punch holes at both ends of the upper layer of the part with the deposition channel pattern as the liquid inlet and outlet.

[0081] Step 1-5: Place the two pieces of PDMS together in a plasma cleaning machine and clean them at a power of 700V for 20s. After completion, bond the upper layer with the deposited channel pattern to the lower smooth surface.

[0082] [Preparation of separation microfluidic module 2]

[0083] In one exemplary embodiment, the preparation process of the separation microfluidic module 2 includes the following steps:

[0084] Step 2-1: Use SU-8 photoresist to coat the surface of the silicon wafer on a coater, use the photolithography process to expose the corresponding pattern, and then clean the excess SU-8 photoresist.

[0085] Step 2-2: Add PDMS prepolymer and curing agent into a glass cup, stir well to prepare a PDMS solution, then pour it on the exposed SU-8 mold and put it into a vacuum tank to extract bubbles.

[0086] Step 2-3: After all the bubbles are extracted, place it on a hot plate and bake it at 80°C for one hour until the PDMS solidifies.

[0087] Step 2-4: After the PDMS solidifies, use a knife to cut off the portion with the separation channel pattern, and use a puncher to punch holes at the liquid inlet and two liquid outlets.

[0088] Step 2-5: Place the glass sheet and the patterned PDMS into a plasma cleaning machine and clean them at 700 V for 20 seconds. After completion, bond the patterned PDMS to the glass sheet.

[0089] [Connection between deposition microfluidic module 1 and separation microfluidic module 2]

[0090] Insert the first L-shaped steel needle into the liquid outlet of the deposition microfluidic module, insert the second L-shaped steel needle into the liquid inlet of the separation microfluidic module, and use a hose to connect the first L-shaped steel needle and the second L-shaped steel needle.

[0091] In addition, the deposition microfluidic module 1 is also connected to the syringe through a steel needle and a hose, and the two liquid outlets of the separation microfluidic module 2 are respectively connected to the corresponding collection devices through a steel needle and a hose.

[0092] Method for separating plasma from blood

[0093] In another exemplary embodiment of the present invention, based on the aforementioned plasma separation system based on a microfluidic chip, a method for separating plasma from blood is provided, comprising the following steps:

[0094] 1x PBS is injected into the plasma separation system to clean and wet the entire separation system, and then diluted whole blood is injected from the first end 131 of the first chamber.

[0095] In the blood, the mass density of red blood cells is higher than that of plasma. The difference in mass density between red blood cells and plasma will cause a single red blood cell to settle due to gravity, forming a plasma layer and a blood cell layer. However, since the difference in mass density between red blood cells and plasma is very small, the sedimentation rate of a single red blood cell is very slow, only about 0.13 μm / min. When the blood is at a low shear rate (less than 50s -1 ) When flowing, red blood cells can reversibly aggregate. Because the sedimentation rate of particles depends on the square of the particle size, red blood cell aggregation greatly increases the sedimentation rate. Aggregation will increase the size of red blood cell aggregates, thereby accelerating the stratification of cells and plasma.

[0096] like Figure 5 As shown, after the blood enters the first chamber 13, the blood cells begin to aggregate and precipitate due to gravity and settle to the bottom of the first chamber 13, which will form a two-phase flow of blood cells and plasma. The flow rate of blood cells will be lower than the flow rate of plasma, so the plasma will reach the second end 132 of the first chamber faster.

[0097] In a preferred example, when the first end 131 of the first chamber is lower than the second end 132 of the first chamber, due to the inclination angle of the bottom of the first chamber, it is more difficult for cells to flow into the separation port due to gravity, and combined with the polymer sheet material (for example, PDMS) having a certain adsorption capacity for cells, a large number of red blood cells will remain in the sedimentation channel, so that obvious color stratification (more or less red blood cells) can be seen. When the blood reaches the second end 132 of the first chamber, the blood has formed a longitudinal stratification, that is, the upper layer is cell-free plasma, and the lower layer is blood cells.

[0098] Afterwards, the blood with longitudinal stratification is converted into transverse stratification of blood cells and plasma from the first chamber 13 through the connecting device 3, and enters the second chamber 23 of the separation microfluidic module to put the blood in a laminar state, and the blood cells and plasma in the blood continue to maintain transverse stratification.

[0099] Finally, the blood cells and plasma in the second chamber 23 flow into the corresponding chamber through the channel to be separated, and then flow into the corresponding collection device to be collected.

[0100] As an optional example, the rate of injecting whole blood is 10 μL / min, ensuring that the blood in the first chamber has enough time to settle.

[0101] In another optional example, diluted whole blood is rapidly introduced at a rate of 200 μL / min, and then the blood is allowed to stand for 10 minutes to allow blood cells to precipitate.

[0102] It can be understood that the blood to be separated is diluted with 1x PBS. The purpose of the dilution is to reduce the volume ratio of red blood cells in the blood to be separated, so as to achieve higher separation purity and better effect. The dilution multiple is preferably five times.

[0103] Detection system of biomarkers in blood

[0104] In other exemplary embodiments of the present invention, a system for detecting biomarkers in blood is also provided, comprising the aforementioned microfluidic plasma separation system, and

[0105] The detection system is set up according to the target biomarker and is used to detect the target biomarker in the plasma separated by the plasma separation system. For example, antigen-antibody (carbohydrate antigen CA19-9), aptamer, etc. can be detected.

[0106] As an optional example, the detection system includes a carbon-based field effect transistor biosensor. For example, when it is necessary to detect the carbohydrate antigen CA19-9 in the blood, a carbon nanotube field effect transistor biosensor that can capture the carbohydrate antigen CA19-9 can be selected. The plasma separated by the aforementioned microfluidic chip-based plasma separation system is then added to the channel of the carbon nanotube field effect transistor to allow it to fully combine with the biological probe, and then the unbound target is rinsed away, and 0.1×PBS is added as a gate for applying voltage. When the concentration of biological molecules in the solution environment to be tested increases or decreases, the concentration of carrier holes in the semiconductor material carbon nanotubes in the channel area will increase or decrease, thereby affecting the current size of the sensor for detection.

[0107] The blood to be separated in the present invention is blood that has been anticoagulated, so what is separated is plasma.

[0108] For better understanding, the present invention is further described below in conjunction with several specific examples, but the preparation process is not limited thereto, and the content of the present invention is not limited thereto.

[0109] Unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0110] Example 1

[0111] [Preparation of microfluidic plasma separation system]

[0112] {Deposition Microfluidics Module}

[0113] 1. Use a laser cutter to cut the 0.8 mm acrylic sheet into the designed shape and size, and fasten it to a smooth 5 mm acrylic sheet as a mold for the upper layer of the deposition channel of the microfluidic chip. Take another smooth 5 mm acrylic sheet as a mold for the lower layer of the deposition channel.

[0114] 2. Add PDMS prepolymer and curing agent (Dow Corning SYLGARD184 silicone rubber) into a glass cup in a ratio of 10:1, stir until dense bubbles are produced, prepare a PDMS solution, and then pour it on an acrylic mold with a pouring height of about 5 mm, and put it in a vacuum tank to evacuate and remove bubbles.

[0115] 3. After all the bubbles are removed, place it on a hot plate and bake it at 80°C for one hour until the PDMS solidifies.

[0116] 4. After the PDMS solidifies, use a knife to cut off the upper part with the deposition channel pattern (the deposition channel is about 4 cm long, 2 cm wide, 800 μm high, and has an inclination angle of 5°) and the lower smooth surface (about 5 cm long and 4 cm wide), and use a hole puncher to punch holes at the liquid inlet and outlet.

[0117] 5. Place the two pieces of PDMS into a plasma cleaner together and clean them at 700V for 20s. After completion, bond the PDMS to the PMDS.

[0118] {Separation Microfluidic Module}

[0119] 1. Use SU-8 photoresist to coat the surface of the silicon wafer on a coater, use the photolithography process to expose the corresponding pattern, and then clean the excess SU-8 photoresist.

[0120] 2. Add PDMS prepolymer and curing agent (Dow Corning SYLGARD184 silicone rubber) to a glass cup in a mass ratio of 10:1, stir until dense bubbles are generated, and prepare a PDMS solution. Then pour it on the exposed SU-8 mold (the pouring height is about 3mm, and put it in a vacuum tank to remove bubbles.

[0121] 3. After all the bubbles are removed, place it on a hot plate and bake it at 80°C for one hour until the PDMS solidifies.

[0122] 4. After the PDMS solidifies, use a knife to cut off the part with the separation channel pattern, and use a puncher to punch holes at the liquid inlet and two liquid outlets.

[0123] 5. Place the glass sheet and the patterned PDMS into a plasma cleaner and clean them at 700V for 20s. After completion, bond the PDMS to the glass sheet.

[0124] Specific dimensions of the separation microfluidic module: the second chamber is a circle with a diameter of 1.5 mm and a height of 100 μm; the third chamber is a circle with a diameter of 1.5 mm and a height of 100 μm; the fourth chamber is a circle with a diameter of 1.5 mm and a height of 100 μm; the first channel 27 has a width of 300 μm, a height of 100 μm and a length of 5 mm; the second channel 28 has a width of 100 μm, a height of 100 μm and a length of 5 mm; the third channel 29 has a width of 300 μm, a height of 100 μm and a length of 5 mm.

[0125] {Assemble to form plasma separation system}

[0126] The deposition microfluidic module and the separation microfluidic module are in the same horizontal plane. An L-shaped steel needle is inserted into the second end of the first chamber, and another L-shaped steel needle is inserted into the first end of the second chamber. The two L-shaped steel needles are connected by a hose. The liquid inlet of the deposition microfluidic module is connected to the syringe through the L-shaped steel needle hose, and the liquid outlet of the separation microfluidic module is connected to the collection device through the L-shaped steel needle and the short tube. The inner and outer diameters of the steel needle are 0.4*0.6mm, and the inner diameter of the hose is 0.5mm.

[0127] Formation: syringe-flexible steel needle-microfluidic deposition combination channel-steel needle flexible hose-steel needle-microfluidic separation channel-collecting device.

[0128] Example 2

[0129] [Separation of plasma using the microfluidic plasma separation system of Example 1]

[0130] 2 mL of 1x PBS was passed into the plasma separation system to clean and moisten the channel, and then 1 mL of five-fold diluted whole blood was passed at a flow rate of 10 μL / min. The blood formed a longitudinal layer with red blood cells at the bottom and plasma at the top in the sedimentation microfluidic module.

[0131] The blood is stratified horizontally from the deposition microfluidic module through the steel needle-hose-steel needle and flows into the separation microfluidic module. The horizontally stratified whole blood passes through the separation microfluidic module, and the blood cells and plasma are separated and flow into the blood cell collection device and the plasma collection device respectively.

[0132] Example 3

[0133] [The number of red blood cells in the separated plasma is observed under a microscope and compared with the blood cell content of the original transfused blood]

[0134] Gently break up the separated plasma, take 5 μL of separated plasma with a pipette, drop it onto a glass slide, and cover the middle of the plasma with a cover glass. Then observe the number of cells under a microscope, divide the cover glass into nine squares, and randomly observe and count the red blood cells in nine different areas. The results are as follows Figure 6 shown.

[0135] Since the red blood cell density of the 5x diluted blood is too high to be counted effectively using a microscope, we chose to dilute the 5x diluted blood 10 times again and then count the red blood cells of the original input blood according to the above method. The results are as follows Figure 7 shown.

[0136] As can be seen from the figure, the number of blood cells in the separated plasma is extremely low, while the number of blood cells in the original blood is dense, indicating that the plasma separation system based on the microfluidic chip of the present invention can effectively separate plasma and blood cells, and the separation rate can reach more than 99%, which is a high separation rate.

[0137] Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. A person with ordinary knowledge in the technical field to which the present invention belongs may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be determined by the definition of the claims.

Claims

1. A microfluidic plasma separation system, characterized in that: include: A deposition microfluidic module (1) comprises a first polymer sheet (11) and a second polymer sheet (12) bonded to each other, wherein a first chamber (13) is provided between the first polymer sheet (11) and the second polymer sheet (12) for forming a longitudinal layer of blood cells and plasma in blood to be separated; The first end (131) of the first chamber is configured as an inlet of the blood to be separated, and the second end (132) of the first chamber is connected to the separation microfluidic module; A separation microfluidic module (2) comprising a silicon-based substrate (21) and a third polymer sheet (22) bonded to each other, wherein a second chamber (23) is provided between the silicon-based substrate (21) and the third polymer sheet (22), and a third chamber (24) and a fourth chamber (25) are respectively provided on both sides of the second chamber; The first end of the second chamber (23) is connected to the second end of the first chamber (13) via a connecting device (3), and the connecting device (3) converts the blood cells and plasma from longitudinal stratification to transverse stratification and flows into the second chamber (23); the second end of the second chamber (23) is connected to the first end of the third chamber (24) and the first end of the fourth chamber (25) through channels respectively; The second end of the third chamber (24) is connected to the first collecting device, and the second end of the fourth chamber (25) is connected to the second collecting device; The side close to the blood cell layer in the second chamber is defined as the third chamber (24), and the side close to the plasma layer in the second chamber is defined as the fourth chamber (25); the blood cells in the second chamber (23) enter the third chamber (24) through the channel and then enter the first collecting device for collection, and the plasma in the second chamber (23) enters the fourth chamber (25) through the channel and then enters the second collecting device for collection.

2. The microfluidic plasma separation system according to claim 1, characterized in that: The first end (131) of the first chamber is lower than the second end (132) of the first chamber, so that the first end of the first chamber forms an inclined angle with the horizontal plane, and the angle is 1° to 15°; the dimensions of the first chamber (13) are: length 3cm to 5cm, width 1.5cm to 3cm, height 500μm to 1000μm.

3. The microfluidic plasma separation system according to claim 1, characterized in that: At least two ribs (133) are provided in the first chamber for supporting the first chamber; the ribs (133) have a width of 0.8 mm to 1.2 mm and a length of 22 mm to 27 mm.

4. The microfluidic plasma separation system according to claim 1, characterized in that: The second chamber (23) is a circle with a diameter of 1.0 mm to 2.0 mm and a height of 100 μm to 500 μm; the third chamber (24) is a circle with a diameter of 1.0 mm to 2.0 mm and a height of 100 μm to 500 μm; the fourth chamber (25) is a circle with a diameter of 1.0 mm to 2.0 mm and a height of 100 μm to 500 μm.

5. The microfluidic plasma separation system according to claim 1, characterized in that: The channel comprises a first channel (26), a second channel (27) and a third channel (28), wherein the first end of the first channel (27) is connected to the second end of the second chamber (23), the second end of the first channel (27) is connected to the first end of the second channel (27) and the first end of the third channel (28) respectively, the second end of the second channel (27) is connected to the first end of the third chamber (24), and the second end of the third channel (28) is connected to the first section of the fourth chamber (25), so that the second chamber (23), the third chamber (24) and the fourth chamber (25) are connected.

6. The microfluidic plasma separation system according to claim 5, characterized in that: The width of the first channel (26) is 50 μm to 1000 μm, and the height is 100 μm to 500 μm; the width of the second channel (27) is 1 / 4 to 1 times the width of the first channel (26), and the height is 100 μm to 500 μm; the width of the third channel (28) is the same as the width of the first channel (26), and the height is 100 μm to 500 μm; the lengths of the second channel (27) and the third channel (28) are equal.

7. A method for separating plasma from blood, characterized in that: The separation is performed using the microfluidic plasma separation system according to any one of claims 1 to 6, comprising the following steps: 1x PBS is injected into the microfluidic plasma separation system to clean and moisten the entire separation system, and then diluted whole blood is injected from the first end of the first chamber. After the blood enters the first chamber, blood cells aggregate and precipitate due to gravity and settle to the bottom of the first chamber, so that blood cells and plasma in the blood form vertical stratification; The blood that has formed longitudinal stratification is converted into transverse stratification of blood cells and plasma through a connecting device, and enters the second chamber of the separation microfluidic module to make the blood in a laminar state, and the blood cells and plasma in the blood continue to maintain transverse stratification; then, the blood cells in the second chamber enter the third chamber through the channel and then enter the blood cell collection device, and the plasma in the second chamber enters the fourth chamber through the channel and then enters the plasma collection device.

8. The method for separating plasma from blood according to claim 7, characterized in that: The rate of whole blood injection is 5-20 μL / min.

9. A system for detecting biomarkers in blood, characterized in that: The microfluidic system comprising any one of claims 1 to 6, and A detection system is set up according to the target biomarker and is used to detect the target biomarker in the plasma separated by the plasma separation system.

10. The blood biomarker detection system according to claim 9, characterized in that: The detection system includes a carbon-based field effect transistor biosensor.

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