Microfluidic plasma separation system and method, system for detecting biomarkers in blood

Abstract

The application provides a micro-fluidic plasma separation system, which comprises a deposition micro-fluidic module and a separation micro-fluidic module. The deposition micro-fluidic module comprises a first polymer sheet and a second polymer sheet which are bonded to each other, and a first chamber is arranged between the first polymer sheet and the second polymer sheet. A first end of the first chamber is arranged as an inlet of blood to be separated. The separation micro-fluidic module comprises a silicon-based substrate and a third polymer sheet which are bonded to each other, a second chamber is arranged between the silicon-based substrate and the third polymer sheet, and a third chamber and a fourth chamber are arranged on two sides of the second chamber respectively. A second end of the second chamber is connected to a second end of the first chamber through a connecting device, the connecting device converts longitudinally layered blood cells and plasma into transversely layered blood cells and plasma, and makes the blood keep transversely layered in the second chamber. The second end of the second chamber is connected to a first end of the third chamber and the fourth chamber through a channel respectively. The plasma separation system of the application 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 using plasma or serum because blood cells and their constituent substances can falsify measurement results. Plasma separation reduces cellular interference 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 typically utilizes centrifugation and membrane filtration. These methods leverage 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 operational efficiency. For example, centrifuges are generally labor-intensive, time-consuming, and bulky, and even require operators to be well-trained, possessing specialized knowledge, and possess certain skills. Furthermore, centrifugation methods typically require manual pipetting from centrifuge tubes, which inevitably leads to human error and incorrect results. Membrane filtration also faces significant clogging issues.

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

[0005] Microfluidic systems typically 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 an external force field (such as acoustic or magnetic force) to operate, while passive separation technology relies solely 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 technologies typically utilize fluid dynamics, such as the Zweifacher-von effect, deterministic lateral displacement, and inertial microfluidics, to design and manufacture various microfluidic chips for plasma separation. However, these methods can damage cells, cause channel clogging by blood cells, or require high dilution of the blood, resulting in ineffective plasma separation. Furthermore, these methods cannot achieve satisfactory plasma quality, leading to ineffective testing. Summary of the Invention

[0007] The present invention aims to address the problems existing in passive separation in existing microfluidic systems and to provide a microfluidic plasma separation system and method, which utilizes gravity as a natural field to achieve plasma separation in blood.

[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 includes 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 for 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 provided between the silicon-based substrate and the third polymer sheet, and a third chamber and a fourth chamber are respectively provided on both sides of the second chamber;

[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 layers to transverse layers and flows them 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, 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 cavity for supporting the first cavity; the width of the ribs is 0.8 mm to 1.2 mm, and the length is 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 connected to the first end of the second channel and the first end of the third channel respectively, 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, thereby forming a connection between the second chamber, the third chamber and the fourth chamber.

[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, a method for separating plasma from blood is provided, wherein the separation is performed using the aforementioned microfluidic plasma separation system, comprising the following steps:

[0021] 1x PBS was injected into the microfluidic plasma separation system to clean and moisten the entire separation system. Subsequently, diluted whole blood was injected from the first end of the first chamber. After the blood entered the first chamber, the blood cells aggregated and precipitated due to gravity and settled to the bottom of the first chamber, forming a longitudinal layer of blood cells and plasma in the blood.

[0022] The longitudinally layered blood is converted into transversely layered blood cells and blood plasma by the connecting device, and enters the second chamber of the separation microfluidic module to make the blood in a laminar flow state, and the blood cells and blood plasma in the blood continue to be transversely layered; 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 blood plasma in the second chamber enters the fourth chamber through the channel and then enters the blood plasma collection device.

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

[0024] According to a third aspect of the object of the present application, a detection system of a biomarker in blood is provided, comprising the aforementioned microfluidic plasma separation system, and

[0025] The detection system is arranged according to a target biomarker, and is used for detecting the target biomarker in the blood plasma separated by the plasma separation system.

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

[0027] As can be seen from the technical solutions of the present application, the microfluidic plasma separation system provided by the present application separates the blood cells and blood plasma in the blood in a longitudinal direction by using the gravity effect to make the blood cells aggregate and deposit, and then converts the longitudinally separated blood cells and blood plasma into transverse separation by the connecting device, and enters the second chamber of the separation microfluidic module to form a laminar flow state, so that the cells flow along their own flow lines without disturbing the blood plasma, thereby flowing into the chambers on the corresponding side, and the blood cells and blood plasma are separated to achieve effective separation of the blood plasma.

[0028] The microfluidic plasma separation system of the present application can realize high-throughput plasma separation, does not need to use a complex external force field, and does not need to be operated by a professional, so that high-purity and high-yield blood plasma can be obtained from the diluted whole blood under limited conditions, and the plasma separation system of the present application is very stable and has a low failure rate. BRIEF DESCRIPTION OF DRAWINGS

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

[0030] Figure 2 is a structural schematic diagram of another exemplary microfluidic plasma separation system of the present application.

[0031] Figure 3 is a schematic diagram of the connecting device converting the longitudinally layered blood cells and blood plasma into transverse layering under the connecting mode of Figure 1 is a schematic diagram of the connecting device converting the longitudinally layered blood cells and blood plasma into transverse layering under the connecting mode of

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

[0033] Figure 5 Schematic diagram of the movement of whole blood in the deposition microfluidic module in an 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 Schematic 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 below with reference to the accompanying drawings.

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

[0039] Microfluidic plasma separation system

[0040] Combine Figures 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 arranged as an inlet for blood to be separated, and the second end 132 of the first chamber is connected to the separation micro-fluidic control module 2.

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

[0044] The first end of the second chamber is connected to the second end of the first chamber through the connecting device 3, the connecting device 3 converts the longitudinal layering of blood cells and plasma into horizontal layering, and the blood is kept in horizontal layering in the second chamber 23; 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 through channels respectively.

[0045] The first end of the second chamber is connected to the second end of the first chamber through the connecting device 3, the connecting device 3 converts the longitudinal layering of blood cells and plasma into horizontal layering, and the blood is kept in horizontal layering in the second chamber 23; 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 through channels respectively.

[0046] It can be understood that the deposition micro-fluidic control module 1 and the separation micro-fluidic control module 2 can be at the same level or have a height difference, and 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 size of the hose and the steel needle is matched with the connecting hole diameter.

[0048] The purpose of the connecting device is to introduce the blood in the first chamber into the second chamber, and at the same time, convert the longitudinal layering of blood cells at the bottom and plasma at the top in the first chamber into horizontal layering of blood cells on the left and plasma on the right, so that the blood in the second chamber presents horizontal layering of blood cells on the left and plasma on the right, which is convenient for subsequent separation, therefore, the setting of the connecting device only needs to meet the conversion of the layering mode of blood cells and plasma; for example, when the deposition micro-fluidic control module 1 and the separation micro-fluidic control module 2 are at the same level, the connecting device 3 can also be directly an L-shaped steel needle with two ends; when the deposition micro-fluidic control module 1 and the separation micro-fluidic control 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 micro-fluidic control module 1 and separation micro-fluidic control module 2, the steel needle, hose and steel needle connected in sequence are used here to illustrate how to connect to achieve the purpose of converting the longitudinal layering of blood cells and plasma into horizontal layering.

[0050] As shown in Figure 1 When the deposition microfluidic module 1 and the separation microfluidic module 2 are at the same level, a first L-shaped steel needle is inserted into the second end of the first chamber from above the deposition microfluidic module 1, 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] As shown in Figure 3 In this connection mode, the longitudinally layered blood cells (red in the figure) and plasma (yellow in the figure) flow along the first L-shaped steel needle and the hose, and when the blood flows onto the second L-shaped steel needle, the layering mode changes, and the blood cells are on the left and the plasma is on the right, which is a horizontal layering, and the blood cells and the plasma enter the second chamber in this layering state, and the blood cells and the plasma remain horizontally layered in the second chamber (i.e., left-right layering as shown in Figure 3

[0052] As shown in Figure 2 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 below the deposition microfluidic module 1, 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.

[0053] As shown in Figure 4 In this connection mode, the longitudinally layered blood cells (red in the figure) and plasma (yellow in the figure) flow along the first L-shaped steel needle and the hose, and when the blood flows onto the second L-shaped steel needle, the layering mode changes, and the blood cells are on the left and the plasma is on the right, which is a horizontal layering, and the blood cells and the plasma enter the second chamber in this layering state, and the blood cells and the plasma remain horizontally layered in the second chamber (i.e., left-right layering as shown in Figure 4

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

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

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

[0057] As an optional example, the size of the first chamber 13 is: length 3cm-5cm, width 1.5cm-3cm, height 500μm-1000μm; this size setting shortens the absolute height required for red blood cells to drop, so that red blood cells can settle to the bottom of the first chamber in a shorter time to realize the longitudinal separation of the cell layer-plasma layer; on the other hand, the channel height also cannot be too low, such height can maintain the continuous flow of blood in the deposition channel on the basis of shortening the red blood cell sedimentation time, avoiding the channel being blocked.

[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 inclined angle with the horizontal plane, and the angle is 1°-15°, and is particularly preferably 5°; using this inclined arrangement can make it more difficult for cells to flow into the separation port, improving the layering effect.

[0059] As an optional example, at least two rib beams 133 are arranged in the first chamber 13 for supporting the first chamber, and the positions of the rib beams 133 are as evenly as possible to divide the space of the first chamber, for example, in the case of two rib beams, it is appropriate to divide the space of the first chamber into three equal parts, and it can be understood that the number and position of the rib beams include but are not limited to this, as long as they can support the first chamber without collapsing.

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

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

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

[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 as needed.

[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 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 first polymer sheet 11 , the second polymer sheet 12 and the third polymer sheet 22 are all made of 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 comes into contact with 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), thereby achieving chemical bonding.

[0073] Method of making a 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 the 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 cutter to cut the acrylic sheet into the designed shape and size, and fasten it to a smooth, flat, millimeter-scale acrylic sheet as a mold for the upper layer of the deposition microfluidic module; take another smooth, flat acrylic sheet as a mold for the lower layer of the deposition microfluidic module.

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

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

[0080] Steps 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] Steps 1-5: Place the two PDMS sheets together in a plasma cleaning machine and clean them at 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 spread the surface of the silicon wafer on a spreader, 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 to a glass cup, stir thoroughly to prepare a PDMS solution, then pour it on the exposed SU-8 mold and place it in a vacuum tank to remove bubbles.

[0086] Step 2-3: 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.

[0087] Step 2-4: After the PDMS solidifies, use a knife to cut off the portion with the separation channel pattern, and use a punch 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 700V for 20s. After cleaning, 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 of plasma separation in 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 single red blood cells to settle due to gravity, forming a plasma layer and a blood cell layer. However, due to the small difference in mass density between red blood cells and plasma, the sedimentation rate of a single red blood cell is very slow, only about 0.13μm / 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] As Figure 5 As shown, after the blood enters the first chamber 13, the blood cells begin to aggregate and precipitate due to the effect of gravity and settle at the bottom of the first chamber 13, which will form a two-phase flow of blood cells-plasma, and the flow rate of the blood cells will be lower than that of the plasma, so the plasma will reach the second end 132 of the first chamber faster.

[0097] In the preferred example, the first end 131 of the first chamber is lower than the second end 132 of the first chamber, and due to the presence of an inclined angle at the bottom of the first chamber, it makes it more difficult for the cells to flow into the separation port due to the effect of gravity, and the combined polymeric material (e.g., PDMS) has a certain adsorption capacity for cells, a large number of red blood cells will stay in the deposition channel, so that a clear color stratification (more or less red blood cells) can be seen, and when the blood reaches the second end 132 of the first chamber, the blood has formed a longitudinal stratification, i.e., the upper part is cell-free plasma, and the lower part is blood cells.

[0098] After that, the blood with longitudinal stratification is converted into horizontal stratification of blood cells and plasma by the connecting device 3 from the first chamber 13, and enters the second chamber 23 of the separation microfluidic module to make the blood in a laminar flow state, and the blood cells and plasma in the blood continue to maintain horizontal stratification.

[0099] Finally, the blood cells and plasma in the second chamber 23 flow into the corresponding chambers to form separation and flow into the corresponding collection devices for collection.

[0100] As an optional example, the rate of injecting whole blood is 10 μL / min, which ensures that the blood in the first chamber has sufficient time for sedimentation.

[0101] In another optional example, the diluted whole blood is rapidly injected at a speed of 200 μL / min, and then stands for 10 min to wait for the blood cells to precipitate.

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

[0103] System for detecting biomarkers in blood

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

[0105] A detection system is arranged according to the target biomarker, for detecting the target biomarker in the plasma separated by the plasma separation system, for example, antigen-antibody (carbohydrate antigen CA19-9), aptamer, etc.

[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 capable of capturing the carbohydrate antigen CA19-9 can be selected, and then the plasma separated by the aforementioned microfluidic chip-based plasma separation system is added dropwise to the channel of the carbon nanotube field effect transistor, so as to be fully combined with the biological probe, and then the uncombined target is washed away, and 0.1xPBS is added dropwise as a gate for applying voltage. When the concentration of biomolecules in the solution environment to be detected increases or decreases, the carrier hole concentration in the semiconductor material carbon nanotube in the channel region will increase or decrease, thereby affecting the current size of the sensor, so as to detect.

[0107] The blood to be separated in the present application is blood subjected to anticoagulation treatment, and therefore the separated product is plasma.

[0108] In order to facilitate better understanding, the present application will be further described below in combination with several specific examples, but the preparation process is not limited thereto, and the content of the present application is not limited thereto.

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

[0110] Example 1

[0111] [Preparation of microfluidic plasma separation system]

[0112] {Deposition of microfluidic module}

[0113] 1. A 0.8mm acrylic plate is cut according to the designed shape and size using a laser cutting machine, and is tightly adhered to a smooth and flat 5mm acrylic plate as a mold for making the upper layer of the microfluidic chip deposition channel. Another smooth and flat 5mm acrylic plate is used as the lower layer of the deposition channel mold.

[0114] 2. According to the mass ratio of 10:1, PDMS prepolymer and curing agent (Dow Corning SYLGARD 184 silicone rubber) are added to a glass cup, stirred until dense bubbles are generated, and a PDMS solution is prepared, which is then poured into the acrylic mold with a pouring height of about 5mm, and is placed in a vacuum tank for vacuumizing to 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, about 2 cm wide, the deposition channel height is about 800 μm, and the inclination angle is 5°) and the lower smooth surface (about 5 cm long and about 4 cm wide), and use a punch to punch holes at the liquid inlet and outlet.

[0117] 5. Place the two pieces of PDMS together in a plasma cleaner 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 spread the surface of the silicon wafer on a spreader, 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 SYLGARD 184 silicone rubber) to a glass in a mass ratio of 10:1 and stir until dense bubbles are generated to prepare a PDMS solution. Then pour it onto the exposed SU-8 mold (the pouring height is about 3mm) and place 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 punch to punch holes at the liquid inlet and two liquid outlets.

[0123] 5. Place the glass sheet and the patterned PDMS in a plasma cleaner and clean it at 700V for 20s. After cleaning, 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, one L-shaped steel needle is inserted into the second end of the first chamber, another L-shaped steel needle is inserted into the first end of the second chamber, and the two L-shaped steel needles are connected by a hose; the liquid inlet of the deposition microfluidic module is connected with a syringe through the L-shaped steel needle hose, and the liquid outlet of the separation microfluidic module is connected with a collection device through the L-shaped steel needle and a short tube; wherein the inner and outer diameters of the steel needle are 0.4*0.6 mm, and the inner diameter of the hose is 0.5 mm.

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

[0128] Example 2

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

[0130] 2 mL of 1x PBS was introduced into the plasma separation system to clean and wet the channel, and then 1 mL of five-fold diluted whole blood was introduced at a flow rate of 10 μL / min, and the blood formed a vertical stratification of red blood cells at the bottom and plasma at the top in the deposition microfluidic module.

[0131] The blood flowed from the deposition microfluidic module through the steel needle-hose-steel needle to form a horizontal stratification and flowed into the separation microfluidic module, and the horizontally stratified whole blood passed through the separation microfluidic module, and the blood cells and plasma were separated and flowed into the blood cell collection device and the plasma collection device, respectively.

[0132] Example 3

[0133] [Observation of the number of red blood cells in the separated plasma under a microscope and comparison with the original input blood cell content]

[0134] The separated plasma was gently dispersed, 5 μL of the separated plasma was taken with a pipette, dropped onto a glass slide, and then covered with a cover glass in the middle of the plasma, and then the number of cells was observed under a microscope, the cover glass was divided into nine grids, and the number of red blood cells was counted randomly in nine different areas. The results are shown in Figure 6 .

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

[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] While the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Persons skilled in the art will readily appreciate that various modifications and variations can be made without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by 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 longitudinal layers of blood cells and plasma in blood to be separated; The first end (131) of the first chamber is configured as an inlet for 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) comprises 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, and 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 cavity for supporting the first cavity; the width of the ribs (133) is 0.8 mm to 1.2 mm, and the length is 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), thereby forming a connection between the second chamber (23), the third chamber (24) and the fourth chamber (25).

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 was injected into the microfluidic plasma separation system to clean and moisten the entire separation system. Subsequently, diluted whole blood was injected from the first end of the first chamber. After the blood entered the first chamber, the blood cells aggregated and precipitated due to gravity and settled to the bottom of the first chamber, forming a longitudinal layer of blood cells and plasma in the blood. The blood that has formed longitudinal stratification is converted into transverse stratification of blood cells and plasma through the connecting device, and enters the second chamber 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; 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 was 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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