A microfluidic chip for blood coagulation detection, a blood coagulation detection device and method

Abstract

The present invention discloses a microfluidic chip for blood coagulation detection, a blood coagulation detection device and method. The microfluidic chip is provided with a sample injection hole, an air extraction hole, a detection channel and a negative pressure constant pressure valve. The sample injection hole is connected to the inlet of the detection channel, the outlet of the detection channel is connected to the inlet of the negative pressure constant pressure valve, and the outlet of the negative pressure constant pressure valve is connected to the air extraction hole. The microfluidic chip has a simple structure, ingenious design and low manufacturing cost. The device and method are based on the above microfluidic chip. The device has a simple structure, is convenient to carry and has a low manufacturing cost. The method is simple to operate and can quickly complete the determination of prothrombin time.

Description

Technical Field

[0001] The present invention relates to the technical field of coagulation detection, and particularly relates to a microfluidic chip for blood coagulation detection, a blood coagulation detection device and a method. Background Art

[0002] Blood coagulation is a dynamic blood activity that requires the interaction of a large number of plasma proteins, cells and coagulation factors to form a cross-linked fibrin network to stop bleeding. Morphologically, blood changes from a liquid to a gel state during the coagulation process, in which soluble fibrinogen is converted into insoluble fibrin, which is the result of a series of coagulation factor enzyme chain reactions.

[0003] In recent years, thromboembolic diseases such as cardiovascular and cerebrovascular diseases have gradually become the biggest killers leading to the death of the Chinese population, and the increasing incidence rate has caused great harm to human health. With the continuous progress and development of society, medicine and science, medical experts and medical staff have a more comprehensive and in-depth understanding and research on thrombosis formation and hemostasis pathology. The improvement of clinical diagnosis, pathological treatment and prognosis evaluation of many diseases are closely related to the detection of coagulation function, and it is indispensable in the diagnosis of thrombosis, hemostasis and other related diseases. Therefore, it is very necessary to achieve rapid and convenient blood coagulation detection.

[0004] Microfluidic chip technology refers to integrating the basic operation processes of biological, chemical and other experiments onto a chip. There are at least micron- or even nanoscale structures in the internal structure of the chip, which can automatically complete the whole process of experiment and analysis. Due to the advantages of microfluidic chips such as integration, high throughput, rapid detection, convenient operation, small sample volume required, low energy consumption and low cost, they have shown broad application prospects in the fields of drug screening, environmental detection, forensic detection, clinical diagnosis and biomedical research in recent years.

[0005] At present, there are mainly three coagulation detection schemes applied to automatic coagulation analysis and detection equipment: namely, the optical method, the magnetic bead method and the current method. The optical method has become the most widely used method in existing fully automatic coagulation analysis and detection devices due to its high sensitivity. It includes two detection methods: transmission turbidimetry and scattering turbidimetry. By detecting the turbidity change during blood coagulation to judge the coagulation index, it has high accuracy and large throughput, but its machine itself is large in size and high in price, and it is not very suitable for small hospitals, point-of-care or personal use. Moreover, the anti-interference ability of most current instruments using the turbidimetry principle does not match their price. Summary of the Invention

[0006] To solve the problems existing in the above-mentioned prior art, the present invention provides a microfluidic chip for blood coagulation detection, a blood coagulation detection device and method. The microfluidic chip has a simple structure, ingenious design and low manufacturing cost. The device and method are based on the above-mentioned microfluidic chip. The device has a simple structure, is easy to carry and has a low manufacturing cost. The method is simple to operate and can quickly complete the determination of prothrombin time.

[0007] The technical solution adopted to achieve the above object of the present invention is as follows:

[0008] A microfluidic chip for blood coagulation detection, characterized in that: the microfluidic chip is provided with a sample inlet hole, an air extraction hole, a detection channel and a negative pressure constant pressure valve. The sample inlet hole is connected to the inlet of the detection channel, the outlet of the detection channel is connected to the inlet of the negative pressure constant pressure valve, and the outlet of the negative pressure constant pressure valve is connected to the air extraction hole.

[0009] The microfluidic chip includes a cover plate made of optical glass material and a substrate made of a breathable material. A detection channel is formed on one surface of the cover plate, and the detection channel is in a serpentine shape;

[0010] A first channel structure and a second channel structure are formed on one surface of the substrate. The first channel structure includes a gas distribution blind hole, a first main channel and a plurality of first branch channels distributed at equal intervals. The first branch channel array formed by the plurality of first branch channels and the gas distribution blind hole are respectively located on both sides of the first main channel. One end of each first branch channel is connected to the first main channel, and the gas distribution blind hole is connected to the first main channel. The second channel structure includes a gas collection blind hole, a second main channel and a plurality of second branch channels distributed at equal intervals. The second branch channel array formed by the plurality of second branch channels and the gas collection blind hole are respectively located on both sides of the second main channel. One end of each second branch channel is connected to the second main channel, and the gas collection blind hole is connected to the second main channel. The first branch channels and the second branch channels are distributed alternately at intervals and are closely adjacent to each other;

[0011] The cover plate is hermetically connected to the substrate. The structure formed by the detection channel and the substrate in a sealed manner is the detection channel. The structure formed by the first channel structure and the second channel structure and the cover plate in a sealed manner is the negative pressure constant pressure valve. The inlet of the detection channel is communicated with the sample inlet hole, the outlet of the detection channel is communicated with the gas distribution blind hole, and the gas collection blind hole is communicated with the air extraction hole.

[0012] Both the first main channel and the first branch channels are linear. Each first branch channel is perpendicular to the first main channel, and the two outermost first branch channels are respectively connected to the two ends of the first main channel. The other end of each first branch channel is close to the second main channel;

[0013] Both the second main channel and the second branch channels are linear. Each second branch channel is perpendicular to the second main channel, and the two outermost second branch channels are respectively connected to the two ends of the second main channel. The other end of each second branch channel is close to the first main channel.

[0014] Both the first channel structure and the second channel structure are symmetric structures. The air distribution blind hole communicates with the exact middle position of the first main channel. The second channel structure further includes a plurality of connecting channels distributed in a fan shape. Each connecting channel and the air collecting blind hole are located on one side of the second main channel. One end of each connecting channel is connected to the second main channel, and the two outermost connecting channels communicate with the two ends of the second main channel. The other end of each connecting channel is connected to the air collecting blind hole.

[0015] The cross-section of the detection channel is square.

[0016] A blood coagulation detection device includes a microfluidic chip, a negative pressure mechanism, a support frame, a lifting mechanism, a camera module and a main control module. The microfluidic chip, the camera module and the main control module are all installed on the support frame. The microfluidic chip is horizontally arranged. The negative pressure mechanism includes a negative pressure generator, an air guide pipe and a suction cup. The two ends of the air guide pipe are respectively connected to the negative pressure generator and the suction cup. The fixed part of the lifting mechanism is installed on the support frame, and the suction cup is installed on the lifting part of the lifting mechanism. When the lifting part of the lifting mechanism descends to the lowest position, the suction cup covers the air extraction hole of the microfluidic chip. The camera module is located directly above the detection channel, and the camera module is electrically connected to the main control module.

[0017] The lifting mechanism includes a servo motor, a gear, a rack, a lifting rod and a limit sleeve. The servo motor is installed on the support frame, the gear is installed on the output end of the servo motor, the limit sleeve is fixed on the support frame, the lifting rod movably penetrates through the limit sleeve, and the lifting rod is in clearance fit with the limit sleeve. The upper end of the lifting rod is connected to the rack, and the suction cup is installed at the lower end of the lifting rod. The gear meshes with the rack.

[0018] The negative pressure mechanism further includes a suction cup seat. The suction cup seat is hollow. The suction cup is sleeved on the entrance of the suction cup seat, and the air guide pipe is connected to the outlet of the suction cup seat. The lower end of the lifting rod is fixedly connected to the top of the suction cup seat.

[0019] The negative pressure generator includes a linear module, a piston push rod, an airtight piston, a piston cylinder, a nozzle, a linkage seat and an engagement positioning plate. The linear module includes a slider, a screw rod and a motor. The slider is connected to the screw rod. The engagement positioning plate is fixed on the inner side of the motor housing. The piston cylinder penetrates through the engagement positioning plate and is fixedly connected to the engagement positioning plate. The airtight piston is located inside the piston cylinder and is hermetically and movably connected to the inner wall of the piston cylinder. The linkage seat is fixed on one side of the slider. The screw rod movably penetrates through the linkage seat. One end of the piston push rod is fixedly connected to the linkage seat, and the other end of the piston push rod is fixedly connected to the airtight piston. The nozzle is fixed on the end of the piston cylinder facing away from the linkage seat.

[0020] The described negative pressure generator further includes a limit reinforcement plate and a photoelectric switch. The limit reinforcement plate is located between the linkage seat and the connection positioning plate. The piston push rod fixedly penetrates through the limit reinforcement plate, and the screw rod movably penetrates through the limit reinforcement plate. The bottom of the support frame is fixedly attached to the top of the connection positioning plate. The photoelectric switch includes a photoelectric generator and a photoelectric receiver. The photoelectric generator is fixedly attached to the top of the limit reinforcement plate, and the photoelectric receiver is fixedly attached to the support frame.

[0021] The described camera module includes a zoom lens, an image sensor, and a fill light module. The zoom lens is installed on the support frame. The fill light module is installed at the front end of the lens housing of the zoom lens. The image sensor is installed at the rear end of the lens housing of the zoom lens. The image sensor and the fill light module are respectively electrically connected to the main control module.

[0022] A blood coagulation detection method includes the following steps:

[0023] S1. Turn on the servo motor, and the lifting rod starts to descend. When the lifting rod descends to a set distance, the suction cup covers the air extraction hole.

[0024] S2. Turn on the motor, and the piston push rod pulls the airtight piston to start air extraction to generate negative pressure. The suction cup tightly sucks the surface of the microfluidic chip. The suction cup is communicated with the air extraction hole of the microfluidic chip. When the piston push rod pulls the airtight piston to move to a set distance, the motor stops working.

[0025] S3. Inject the blood sample to be tested into the microfluidic chip from the sampling hole, and at the same time turn on the main control module and the camera module.

[0026] S4. The camera module starts to take real-time pictures of the detection channel. The camera module transmits the state pictures of the blood sample to be tested flowing in the detection channel obtained by real-time photography to the main control module. The main control module performs binary processing on the real-time obtained state pictures. The area where the blood sample to be tested has flowed through in the detection channel is displayed as black in the binary image, and the area where the blood sample to be tested has not flowed through in the detection channel is displayed as white in the binary image.

[0027] S5. The main control module obtains the real-time binary image according to the binary processing, calculates the real-time black ratio P1 and the real-time white ratio P2 in the real-time binary image. Then the main control module calculates the real-time chromaticity ratio P0 according to the following formula:

[0028] P0 = P1 / (P1 + P2) * 100% (1),

[0029] When the blood sample to be tested is at the sampling hole and has not entered the detection channel, the chromaticity ratio P0 is 0%. When the blood sample to be tested starts to move after entering the detection channel, the chromaticity ratio P0 starts to increase. The moment when the chromaticity ratio P0 starts to increase is the detection start point. Record the moment when the chromaticity ratio P0 starts to increase as T1.

[0030] S6. Taking time as the abscissa and the real-time chromaticity proportion P0 as the ordinate, plot the solidification curve;

[0031] S7. Name the obtained real-time chromaticity proportion data in sequence according to the time as D0, D1... D n , where n is an integer and n > 10. Starting from the 11th chromaticity proportion obtained, calculate the chromaticity proportion increment I m :

[0032] I m = D m - D m-1 (10 < m < n) (2),

[0033] where m is an integer;

[0034] S8. Calculate the chromaticity proportion increment multiple M m corresponding to each chromaticity proportion according to the chromaticity proportion increment I i , and the specific calculation formula is as follows:

[0035]

[0036] S9. Collect the blood samples of each person in the known population, make each collected blood sample into a standard blood sample, measure the PT' value of each standard blood sample, calculate the PT' mean value after the measurement is completed, and take the PT' mean value as the standard PT time;

[0037] S10. Taking time as the abscissa and the obtained chromaticity proportion increment multiple data as the ordinate, obtain the curve of the change of the chromaticity proportion increment multiple with time;

[0038] S11. Substitute the PT' mean value into the curve of the change of the chromaticity proportion increment multiple with time, and the chromaticity proportion increment multiple corresponding to the PT' mean value is the chromaticity proportion increment multiple calibration value M0;

[0039] S12. When it is detected that the chromaticity proportion increment multiple M i is greater than M0, this moment is the detection end point, record the moment when M i is greater than M0 as T2, and calculate the PT value of the solidification of the blood sample to be tested according to the following formula:

[0040] PT = T2 - T1 (4).

[0041] Compared with the prior art, the beneficial effects and advantages of the present invention are as follows:

[0042] 1. The microfluidic chip of the present invention is designed based on the principle of laminar flow formed when fluid flows in a narrow space. The design is ingenious. It includes a detection channel and a negative pressure constant pressure valve. Blood reacts with thrombin, gradually solidifying from a liquid state with better fluidity to a solid state. During the coagulation process, blood flows in the detection channel under the drive of negative pressure. The negative pressure constant pressure valve can control the driving force of negative pressure, avoiding excessive negative pressure from affecting the accuracy of the detection result.

[0043] 2. The negative pressure constant pressure valve of the microfluidic chip of the present invention can control the negative pressure driving force by adjusting the thickness of the partition wall between the first branch channel and the second branch channel, thereby achieving a stable, continuous, and appropriate driving force applied to the detection channel.

[0044] 3. The blood coagulation detection device of the present invention is based on the above-mentioned microfluidic chip. It has a simple and compact structure, is easy to carry, has a low manufacturing cost, and can be used in emergency departments, clinical departments, community medical institutions, physical examination centers, and individual families. It has important practical significance and can relieve the pressure on the medical system in underdeveloped areas and reduce the overuse of medical resources.

[0045] 4. The blood coagulation detection method of the present invention is based on the above-mentioned microfluidic chip. Different from the commonly used methods in the past, the present invention takes pictures of the blood sample during the coagulation and flow process in the detection channel and performs image processing, and finally obtains the prothrombin time (PT value). This method is novel, unique, simple, efficient, and easy to operate, and can quickly complete the determination of prothrombin time. Description of the Drawings

[0046] Figure 1 It is a schematic structural diagram of the microfluidic chip for blood coagulation detection.

[0047] Figure 2 It is an assembly diagram of the first channel structure and the second channel structure.

[0048] Figure 3 It is a schematic structural diagram of the blood coagulation detection device.

[0049] Figure 4 It is for Figure 3 the front view.

[0050] Figure 5 It is for Figure 3 the rear view.

[0051] Figure 6 It is a schematic structural diagram of the negative pressure generator.

[0052] Figure 7 It is a time-varying curve graph of the coagulation curve and the chromaticity ratio increment multiple obtained in Example 3.

[0053] Among them, 1 - cover plate, 2 - substrate, 3 - sample injection hole, 4 - air extraction hole, 5 - detection channel, 6 - air distribution blind hole, 7 - first main channel, 8 - first branch channel, 9 - gas collection blind hole, 10 - second main channel, 11 - second branch channel, 12 - connection channel, 13 - support frame, 14 - servo motor, 15 - gear, 16 - rack, 17 - lifting rod, 18 - limit sleeve, 19 - air duct, 20 - suction cup, 21 - suction cup seat, 22 - slider, 23 - screw, 24 - motor, 25 - piston push rod, 26 - airtight piston, 27 - piston cylinder, 28 - nozzle, 29 - linkage seat, 30 - connection positioning plate, 31 - limit reinforcement plate, 32 - photoelectric generator, 33 - photoelectric receiver, 34 - zoom lens, 35 - image sensor, 36 - light supplement module, 37 - microfluidic chip. Detailed implementation manners

[0054] The present invention will be described in detail below with reference to the accompanying drawings.

[0055] Embodiment 1

[0056] The structure of the blood coagulation detection microfluidic chip provided in this embodiment is as Figure 1 shown, including a cover plate 1 and a substrate 2. The material of the cover plate 1 is optical glass, and the material of the substrate 2 is polydimethylsiloxane (PDMS). PDMS is an organic polymer with high air permeability and a stable air permeability rate.

[0057] A detection channel 5 is formed on one surface of the cover plate 1. The detection channel 5 is serpentine, and the cross-section of the detection channel 5 is square. In this embodiment, the length of the detection channel 5 is 160 mm, the height of the cross-section of the detection channel 5 is 100 μm, and the width is 500 μm.

[0058] As Figure 2 shown, a first channel structure and a second channel structure are formed on one surface of the substrate 2. Both the first channel structure and the second channel structure are symmetric structures.

[0059] The first channel structure includes an air distribution blind hole 6, a first main channel 7, and a plurality of first branch channels 8 distributed at equal intervals. Both the first main channel 7 and the first branch channels 8 are linear. The first branch channel array formed by the plurality of first branch channels 8 and the air distribution blind hole 6 are respectively located on both sides of the first main channel 7. One end of each first branch channel 8 is connected to the first main channel 7. Each first branch channel 8 is perpendicular to the first main channel 7, and the two outermost first branch channels 8 are respectively connected to the two ends of the first main channel 7. The other end of each first branch channel 8 is close to the second main channel 11. The air distribution blind hole 6 is connected to the middle position of the first main channel 7.

[0060] The second channel structure includes a gas-collecting blind hole 9, a second main channel 10, second branch channels 11, and connecting channels 12. There are multiple second branch channels 11 and connecting channels 12. Both the second main channel 10 and the second branch channels 11 are linear. The multiple second branch channels 11 are evenly spaced, and the multiple connecting channels 12 are distributed in a fan shape. Each connecting channel 12 and the gas-collecting blind hole 9 are located on one side of the second main channel 10, and the array of second branch channels 11 is located on the other side of the second main channel 10. One end of each second branch channel 11 is connected to the second main channel 10. Each second branch channel 11 is perpendicular to the second main channel 10, and the two outermost second branch channels 11 are respectively connected to the two ends of the second main channel 10. The other end of each second branch channel 11 is close to the first main channel 7. One end of each connecting channel 12 is connected to the second main channel 10, and the two outermost connecting channels 12 communicate with the two ends of the second main channel 10. The other end of each connecting channel 12 is connected to the gas-collecting blind hole 9.

[0061] The first branch channels 7 and the second branch channels 11 are distributed at intervals alternately, and the first branch channels 7 and the second branch channels 12 are adjacent to each other.

[0062] The cover plate 1 is hermetically connected to the substrate 2. The structure formed by the detection channel 5 and the substrate 2 hermetically is the detection passage. The structures formed by the first channel structure and the second channel structure and the cover plate hermetically are the negative pressure constant pressure valves. The detection passage inlet communicates with the sampling hole 3, the detection passage outlet communicates with the air-distributing blind hole 9, and the gas-collecting blind hole 9 communicates with the air-extracting hole 4.

[0063] Here, the negative pressure constant pressure valve is designed by utilizing the stable gas-permeable property of PDMS. There is a PDMS partition wall with a certain thickness between the first branch channels 7 and the second branch channels 12. The two sides of the partition wall are gas channels. The gas-permeable rate of the negative pressure constant pressure valve is controlled by the thickness of the partition wall, that is, the pressure of the detection passage is controlled. By setting appropriate partition wall dimensions, a continuous and stable constant negative pressure driving force can be achieved in the detection passage.

[0064] Embodiment 2

[0065] The structure of the blood coagulation detection device provided in this embodiment is as Figure 3 、 Figure 4 and Figure 5 shown, and includes the microfluidic chip 37, the negative pressure mechanism, the support frame 13, the lifting mechanism, the camera module, and the main control module of Embodiment 1. Both the microfluidic chip 37 and the main control module are installed on the support frame 13, and the microfluidic chip 37 is horizontally arranged.

[0066] The lifting mechanism includes a steering gear 14, a gear 15, a rack 16, a lifting rod 17 and a limit sleeve 18. The steering gear 14 is installed on the support frame 13, and the gear 15 is installed on the output end of the steering gear 14. The limit sleeve 18 is fixed on the support frame 13, the lifting rod 17 movably penetrates through the limit sleeve 18, and the lifting rod 17 is in clearance fit with the limit sleeve 18. The upper end of the lifting rod 17 is connected to the rack 16, and the gear 15 meshes with the rack 16.

[0067] The negative pressure mechanism includes a negative pressure generator, a gas guide pipe 19, a suction cup 20 and a suction cup seat 21. The gas guide pipe 19 is a flexible pipe. The suction cup seat 21 is hollow, and the lower end of the lifting rod 17 is fixedly connected to the top of the suction cup seat 21. The suction cup 20 is sleeved on the inlet of the suction cup seat 21, and one end of the gas guide pipe 18 is connected to the outlet of the suction cup seat 21.

[0068] After the steering gear 14 is turned on, the steering gear 14 drives the gear 15 to rotate. The rotation of the gear 15 drives the rack 16 to move up and down. The up and down movement of the rack 16 drives the lifting rod 17 to lift and lower. When the lifting rod 17 descends to the lowest position, the suction cup 20 covers the air extraction hole 4 of the microfluidic chip 37.

[0069] As Figure 6 shown, the negative pressure generator includes a linear module, a piston push rod 25, an airtight piston 26, a piston cylinder 27, a nozzle 28, a linkage seat 29, an abutting positioning plate 30, a limit strengthening plate 31 and a photoelectric switch. The linear module includes a slider 22, a screw rod 23 and a motor 24, and the slider 22 is connected to the screw rod 23. The abutting positioning plate 30 is fixed on the inner side of the motor 24 housing, the linkage seat 29 is fixed on one side of the slider 22, the screw rod 26 movably penetrates through the linkage seat 29, and the abutting positioning plate 30 is directly opposite to the linkage seat 29. The piston cylinder 27 penetrates through the abutting positioning plate 30, and the piston cylinder 27 is fixedly connected to the abutting positioning plate 30. The airtight piston 26 is located inside the piston cylinder 27, and the airtight piston 26 is hermetically and movably connected to the inner wall of the piston cylinder 27. One end of the piston push rod 25 is fixedly connected to the linkage seat 29, and the other end of the piston push rod 25 is fixedly connected to the airtight piston 26. The nozzle 28 is fixed on the end of the piston cylinder 27 facing away from the linkage seat 29, and the other end of the gas guide pipe 19 is connected to the nozzle 28.

[0070] The limit strengthening plate 31 is located between the linkage seat 29 and the abutting positioning plate 30, and the bottom of the support frame 13 is fixed on the top of the abutting positioning plate 30. The piston push rod 25 fixedly penetrates through the limit strengthening plate 31, and the screw rod 23 movably penetrates through the limit strengthening plate 31. The photoelectric switch includes a photoelectric generator 32 and a photoelectric receiver 33. The photoelectric generator 32 is fixed on the top of the limit strengthening plate 31, and the photoelectric receiver 33 is fixed on the support frame 13.

[0071] After the motor is started, the slider 22 drives the linkage seat 29 to start moving away from the support frame 13. The linkage seat 29 drives the piston push rod 25 to start moving away from the support frame 13. The piston push rod 25 drives the airtight piston 26 to start moving, and starts to pump air and generate negative pressure. After the negative pressure is generated, the suction cup 20 tightly sucks the surface of the microfluidic chip 37. The suction cup 20 is hermetically connected to the air extraction hole 4 of the microfluidic chip 37, so as to generate a negative pressure driving force inside the microfluidic chip. When the detection is completed, the piston push rod 25 can be reset through the photoelectric switch.

[0072] The camera module includes a zoom lens 34 (M2812F14 - 3MP), an image sensor 35 (OV2640 200) and a supplementary light module 36. The supplementary light module uses 24 5050 - type LEDs integrated with WS2812 driving chips. The zoom lens 34 is installed on the support frame 13. The supplementary light module 36 is installed at the front end of the lens housing of the zoom lens 34. The image sensor 35 is installed at the rear end of the lens housing of the zoom lens 34. The image sensor 35 and the supplementary light module 36 are respectively electrically connected to the main control module.

[0073] Embodiment 3

[0074] The method for performing blood coagulation detection using the blood coagulation detection device of Embodiment 2 is as follows:

[0075] S1. Blood is collected by venous blood sampling and then filled into a plastic tube or siliconized glass tube containing 0.109 mol / L sodium citrate anticoagulant solution with a volume of 1 / 10. During this process, hemolysis and tissue fluid contamination should be avoided. When the blood sample is stored at -2 to 8°C, the measurement should not exceed 6 hours. When stored at 22 to 24°C, the measurement should not exceed 2 hours.

[0076] S2. Start the servo motor 14, and the lifting rod 17 starts to descend. When the lifting rod 17 descends to the set distance, the suction cup 20 covers the air extraction hole 4.

[0077] S3. Start the motor 24, and the piston push rod 25 pulls the airtight piston 26 to start pumping air to generate negative pressure. The suction cup 20 tightly sucks the surface of the microfluidic chip 37. The suction cup 20 is hermetically connected to the air extraction hole 4 of the microfluidic chip 37. When the piston push rod 25 pulls the airtight piston 26 to move to the set distance, the motor 24 stops working. At this time, the pressure is -50 mbar.

[0078] S4. Incubate the blood sample at 37°C for 3 minutes, mix the incubated blood sample evenly with 0.2 ml of thrombin reagent pre - warmed at 37°C to obtain a blood sample to be measured. The blood sample to be measured is injected into the microfluidic chip 37 from the injection hole 3. At the same time, start the main control module, the zoom lens, the image sensor and the supplementary light module.

[0079] S4. The zoom lens 34 starts real-time imaging of the detection channel, generally at a speed of 10 images per second, that is, the time interval for each imaging is 0.1 s. The image sensor 35 transmits the state picture of the blood sample to be measured flowing in the detection channel obtained by real-time imaging to the main control module. The main control module performs binary processing on the state picture obtained in real time. The area where the blood sample to be measured flows through in the detection channel is displayed as black in the binary image, and the area where the blood sample to be measured does not flow through in the detection channel is displayed as white in the binary image;

[0080] S5. The main control module calculates the real-time black proportion P1 and real-time white proportion P2 in the real-time binary image based on the binary processing. Then the main control module calculates the real-time chromaticity proportion P0 according to the following formula:

[0081] P0 = P1 / (P1 + P2) * 100% (1),

[0082] When the blood sample to be measured is at the sampling hole and has not entered the detection channel, the chromaticity proportion P0 is 0%. When the blood sample to be measured starts to move after entering the detection channel, the chromaticity proportion P0 starts to increase. The moment when the chromaticity proportion P0 starts to increase is the detection starting point, and the moment when the chromaticity proportion P0 starts to increase is recorded as T1;

[0083] S6. Taking time as the abscissa and real-time chromaticity proportion P0 as the ordinate, draw a coagulation curve, as Figure 7 shown;

[0084] S7. Name the obtained real-time chromaticity proportion data in sequence according to time as D0, D1... D n , where n is an integer and n > 10. Starting from the 11th chromaticity proportion obtained, calculate the chromaticity proportion increment I m :

[0085] I m = D m - D m - 1 (10 < m < n) (2),

[0086] where m is an integer;

[0087] S8. Calculate the chromaticity proportion increment multiple M m corresponding to each chromaticity proportion according to the chromaticity proportion increment I i , and the specific calculation formula is as follows:

[0088]

[0089] S9. Collect the blood samples of each person in a known population (requiring that the individuals in the population are in good health, aged between 20 and 40 years old, and the number of individuals is greater than 20). Make each collected blood sample into a standard blood sample, measure the PT' value of each standard blood sample. After the measurement is completed, calculate the average PT' value. The calculated average PT' value is 13.5 s. Take the average PT' value as the standard PT time;

[0090] S10. Use time as the abscissa and the obtained chromaticity ratio increment multiple data as the ordinate to obtain the time change curve of the chromaticity ratio increment multiple, as Figure 7 shown;

[0091] S11. Substitute the average PT' value into the time change curve of the chromaticity ratio increment multiple. The chromaticity ratio increment multiple corresponding to the average PT' value is the chromaticity ratio increment multiple calibration value M0. Here, the chromaticity ratio increment multiple calibration value M0 is 3.4 times;

[0092] S12. When it is detected that M i is greater than M0, this moment is the detection end point. Record the moment when M i is greater than M0 as T2. The PT value of the blood sample to be tested when it coagulates is calculated according to the following formula:

[0093] PT = T2 - T1 (4).

Claims

1. A microfluidic chip for blood coagulation detection, characterized in that: The described microfluidic chip is provided with a sampling hole, an air extraction hole, a detection channel and a negative pressure constant pressure valve. The microfluidic chip includes a cover plate made of optical glass material and a substrate made of a breathable material. A detection channel is formed on one surface of the cover plate, and the detection channel is serpentine; One surface of the substrate is provided with a first channel structure and a second channel structure. The first channel structure includes a gas distribution blind hole, a first main channel and a plurality of first branch channels distributed at equal intervals. The first branch channel array formed by the plurality of first branch channels and the gas distribution blind hole are respectively located on both sides of the first main channel. One end of each first branch channel is communicated with the first main channel, and the gas distribution blind hole is communicated with the first main channel. The second channel structure includes a gas collection blind hole, a second main channel and a plurality of second branch channels distributed at equal intervals. The second branch channel array formed by the plurality of second branch channels and the gas collection blind hole are respectively located on both sides of the second main channel. One end of each second branch channel is communicated with the second main channel, and the gas collection blind hole is communicated with the second main channel. The first branch channels and the second branch channels are distributed alternately at intervals and are closely adjacent to each other; The cover plate is hermetically connected to the substrate. The structure formed by hermetically sealing the detection channel and the substrate is the detection channel, and the structure formed by hermetically sealing the first channel structure and the second channel structure and the cover plate is the negative pressure constant pressure valve. The detection channel inlet is communicated with the sampling hole, the detection channel outlet is communicated with the gas distribution blind hole, and the gas collection blind hole is communicated with the air extraction hole.

2. The blood coagulation detection microfluidic chip according to claim 1, wherein: The first main channel and the first branch channels are both linear. Each first branch channel is perpendicular to the first main channel, and the two outermost first branch channels are respectively connected to the two ends of the first main channel. The other end of each first branch channel is close to the second main channel; The second main channel and the second branch channels are both linear. Each second branch channel is perpendicular to the second main channel, and the two outermost second branch channels are respectively connected to the two ends of the second main channel. The other end of each second branch channel is close to the first main channel.

3. The hemagglutination detection microfluidic chip according to claim 2, wherein: The first channel structure and the second channel structure are both symmetric structures. The gas distribution blind hole is communicated with the middle position of the first main channel. The second channel structure further includes a plurality of connecting channels distributed in a fan shape. Each connecting channel and the gas collection blind hole are both located on one side of the second main channel. One end of each connecting channel is connected to the second main channel, and the two outermost connecting channels are communicated with the two ends of the second main channel. The other end of each connecting channel is connected to the gas collection blind hole.

4. The microfluidic chip for blood coagulation detection according to claim 1, wherein: The cross section of the detection channel is square.

5. A blood coagulation detection device, characterized in that: It includes the microfluidic chip, a negative pressure mechanism, a support frame, a lifting mechanism, a camera module and a main control module according to claims 1-4. The microfluidic chip, the camera module and the main control module are all installed on the support frame. The microfluidic chip is horizontally arranged. The negative pressure mechanism includes a negative pressure generator, a gas guide pipe and a suction cup. The two ends of the gas guide pipe are respectively connected to the negative pressure generator and the suction cup. The fixed part of the lifting mechanism is installed on the support frame, and the suction cup is installed on the lifting part of the lifting mechanism. When the lifting part of the lifting mechanism descends to the lowest position, the suction cup covers the air extraction hole of the microfluidic chip. The camera module is located directly above the detection channel, and the camera module is electrically connected to the main control module.

6. The blood coagulation detection device according to claim 5, characterized in that: The lifting mechanism described above includes a servo motor, a gear, a rack, a lifting rod, and a limit sleeve. The servo motor is installed on the support frame, the gear is installed on the output end of the servo motor, the limit sleeve is fixed to the support frame, the lifting rod movably penetrates through the limit sleeve, the lifting rod is in clearance fit with the limit sleeve, the upper end of the lifting rod is connected to the rack, the suction cup is installed on the lower end of the lifting rod, and the gear meshes with the rack.

7. The blood coagulation detection device according to claim 6, wherein: The negative pressure mechanism further includes a suction cup seat. The suction cup seat is hollow, the suction cup is sleeved on the inlet of the suction cup seat, the air guide pipe is connected to the outlet of the suction cup seat, and the lower end of the lifting rod is fixedly connected to the top of the suction cup seat.

8. The blood coagulation detection device according to claim 5, wherein: The negative pressure generator includes a linear module, a piston push rod, an airtight piston, a piston cylinder, a nozzle, a linkage seat, and an engagement positioning plate. The linear module includes a slider, a screw rod, and a motor. The slider is connected to the screw rod. The engagement positioning plate is fixed to the inner side of the motor housing. The piston cylinder penetrates through the engagement positioning plate and is fixedly connected to the engagement positioning plate. The airtight piston is located inside the piston cylinder and is hermetically and movably connected to the inner wall of the piston cylinder. The linkage seat is fixed to one side of the slider. The screw rod movably penetrates through the linkage seat. One end of the piston push rod is fixedly connected to the linkage seat, and the other end of the piston push rod is fixedly connected to the airtight piston. The nozzle is fixed to the end of the piston cylinder facing away from the linkage seat.

9. The blood coagulation detection device according to claim 8, wherein: The negative pressure generator further includes a limit reinforcement plate and a photoelectric switch. The limit reinforcement plate is located between the linkage seat and the engagement positioning plate. The piston push rod fixedly penetrates through the limit reinforcement plate, and the screw rod movably penetrates through the limit reinforcement plate. The bottom of the support frame is fixed to the top of the engagement positioning plate. The photoelectric switch includes a photoelectric generator and a photoelectric receiver. The photoelectric generator is fixed to the top of the limit reinforcement plate, and the photoelectric receiver is fixed to the support frame.

10. The blood coagulation detection device according to claim 5, wherein: The camera module includes a zoom lens, an image sensor, and a supplementary light module. The zoom lens is installed on the support frame. The supplementary light module is installed at the front end of the lens housing of the zoom lens. The image sensor is installed at the rear end of the lens housing of the zoom lens. The image sensor and the supplementary light module are respectively electrically connected to the main control module.

11. A blood coagulation detection method, characterized in that It includes the following steps: S1. Turn on the servo motor, and the lifting rod starts to descend. When the lifting rod descends to a set distance, the suction cup covers the air extraction hole. S2. Turn on the motor, the piston push rod pulls the airtight piston to start air extraction to generate negative pressure, the suction cup tightly sucks the surface of the microfluidic chip, the suction cup is communicated with the air extraction hole of the microfluidic chip. When the piston push rod pulls the airtight piston to move to a set distance, the motor stops working. S3. Inject the blood sample to be tested into the microfluidic chip from the sample injection hole, and at the same time turn on the main control module and the camera module. S4. The camera module starts to take real-time pictures of the detection channel. The camera module transmits the state pictures of the blood sample to be tested flowing in the detection channel obtained by real-time shooting to the main control module. The main control module performs binary processing on the state pictures obtained in real time. The area where the blood sample to be tested flows through in the detection channel is displayed as black in the binary image, and the area where the blood sample to be tested does not flow through in the detection channel is displayed as white in the binary image. S5. The main control module calculates the real-time black proportion P1 and the real-time white proportion P2 in the real-time binary image according to the binary processing to obtain the real-time binary image. The main control module then calculates the real-time chromaticity proportion P0 according to the following formula: P0 = P1 / (P1 + P2) * 100% (1), When the colorimetric proportion P0 of the blood sample to be measured at the moment when it is located in the sample injection hole and has not entered the detection channel is 0%, when the blood sample to be measured starts to move after entering the detection channel, the colorimetric proportion P0 starts to increase. The moment when the colorimetric proportion P0 starts to increase is the detection starting point, and record the moment when the colorimetric proportion P0 starts to increase as T1; S6. With time as the abscissa and the real-time colorimetric proportion P0 as the ordinate, plot the coagulation curve; S7. Name the obtained real-time chromaticity ratio data as D0, D1... D in sequence according to the time order. n , where n is an integer and n > 10. Starting from the 11th chromaticity ratio obtained, calculate the chromaticity ratio increment I of each chromaticity ratio. m : I m = D m - D m-1 (10 < m < n)(2), where m is an integer; S8. Calculate the chromaticity proportion increment multiple M corresponding to each chromaticity proportion according to the chromaticity proportion increment I m The specific calculation formula is as follows: i ​ S9. Collect blood samples from each person in the known population, prepare each collected blood sample into a standard blood sample, and measure the PT value of each standard blood sample. After the measurement is completed, calculate the PT mean value and use the PT mean value as the standard PT time. ′ Value, and after the measurement is completed, calculate the PT ′ Mean value, and use the PT ′ Mean value as the standard PT time; S10. With time as the abscissa and the obtained colorimetric proportion increment multiple data as the ordinate, obtain the curve of the change of the colorimetric proportion increment multiple with time; S11. Substitute the PT ′ average value into the curve of the increment multiple of the chromaticity ratio over time, and the increment multiple of the chromaticity ratio corresponding to the PT ′ average value is the calibration value M0 of the increment multiple of the chromaticity ratio; S12. When the increment multiple M of the chromaticity ratio is detected i is greater than M0, this moment is the detection end point, and record M i The moment greater than M0 is T2, and the PT value of the blood sample to be tested for coagulation is calculated according to the following formula: PT = T2 - T1 (4).

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