Reusable blood coagulation detection device based on droplet microfluidic technology and use method

Abstract

The invention discloses a reusable blood coagulation detection device based on a droplet microfluidic technology and a use method, and belongs to the technical field of droplet microfluidics, and the device comprises a microfluidic chip, an injection pump and an optical detection device. The micro-fluidic chip is used for generating and capturing blood droplets and comprises a PDMS layer, a three-dimensional electrode and a glass substrate, the PDMS layer and the glass substrate are bonded, and the three-dimensional electrode is poured in a second flow channel of the PDMS layer. The injection pump is used for controlling constant flow and is connected with an inlet of the PDMS layer. The optical detection device is used for detecting the blood coagulation process in the micro-fluidic chip and comprises a light source, a first lens, a second lens and a photoelectric sensor. The liquid drops in the micro-fluidic chip are controlled by applying an external electric field, the blood liquid drops are captured and released, the blood coagulation process is monitored by adopting an optical method, and a new thought is provided for blood coagulation function detection. The chip is prevented from being blocked when blood is coagulated mainly in a mode of wrapping the blood by an oil phase, and possibility is provided for flushing and reusing the chip.

Description

Technical Field

[0001] The present invention belongs to the technical field of microfluidics, and relates to a reusable blood coagulation detection device and a usage method based on droplet microfluidics technology. Background Art

[0002] With the development of medical technology, blood purification technology plays an increasingly important role in the field of critical care medicine. The application of blood purification technology in the ICU can rescue critically ill patients with renal failure, liver failure, various poisonings and drug intoxications, severe pancreatitis, and severe systemic infections. During the blood purification process, it is very necessary to diagnose the bleeding risk and control thrombus formation. Therefore, it is necessary to quickly and accurately detect the blood coagulation process.

[0003] At present, there are still some deficiencies in commercially available blood coagulation detection instruments, such as large size of the measuring device, high equipment price, complex detection process, and the need for professional personnel to operate. Microfluidic chip technology has been widely used in blood coagulation detection due to its advantages of low cost, small sample volume, high throughput, and simple operation. Currently, the blood coagulation detection methods based on microfluidic chips can be mainly divided into coagulation methods, biochemical methods, immunoassay methods, etc. However, the flow channel size of the microfluidic chip is small, and the blood coagulation will block the flow channel and is difficult to clean. Therefore, most of the microfluidic chips used for blood coagulation detection are disposable chips.

[0004] Droplet microfluidics technology is a form of microfluidic chip technology, which has the advantages of small sample demand, fast mixing speed, high manipulation accuracy, etc., and is widely used in fields such as drug synthesis and release, single-cell analysis, molecular biology, and materials science. Droplet manipulation technology is the basic tool of droplet microfluidics. Droplet manipulation methods are mainly divided into droplet sorting, merging, splitting, mixing, and capturing. Droplet microfluidics technology can use a morphologically stable droplet as an independent microreactor. By using droplet microfluidics technology, blood and a coagulant can be encapsulated in an oil phase, so that blood coagulation is completed in the droplet, effectively avoiding the blockage of the microchannel caused by blood coagulation, and using droplet manipulation technology, the blood droplet is captured in a microcell, and the blood coagulation process is monitored by an optical method to realize the detection of the coagulation time. Summary of the Invention

[0005] Aiming at the deficiencies of the above-mentioned existing technologies, the present invention proposes a reusable blood coagulation detection device and a usage method based on droplet microfluidics technology. Blood and a coagulant are encapsulated in an oil phase to form a blood droplet, so that blood coagulation is completed in the oil phase, effectively avoiding the blockage of the microchannel by blood coagulation, realizing the rinsability of the microfluidic chip, using dielectrophoresis technology to realize the manipulation of droplets, and combining with an optical method to monitor the blood coagulation process, providing a new idea for realizing bedside blood coagulation function detection.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A reusable blood coagulation detection device based on droplet microfluidics technology. The reusable blood coagulation detection device mainly includes a microfluidic chip 1, an injection pump 2, and an optical detection device 3. The microfluidic chip 1 is used to generate and capture blood droplets, and includes a PDMS layer 4, a three-dimensional electrode 5, and a glass substrate 6. The PDMS layer 4 and the glass substrate 6 are bonded together by plasma treatment. The three-dimensional electrode 5 is cast in the flow channel two 12 of the PDMS layer 4. The injection pump 2 is used to control a constant flow rate. The injection pump 2 is connected to the inlet on the PDMS layer 4 to control the stable flow rate of the inlet. The optical detection device 3 is used to detect the blood coagulation process in the microfluidic chip 1, and includes a light source 7, a lens one 8, a lens two 9, and a photoelectric sensor 10. The light source 7 emits red light with a wavelength of 660 nm. After passing through the lens one 8, the light is focused above the droplet capture pool 18 of the microfluidic chip 1. After passing through the microfluidic chip 1, the lens two 9 focuses the light on the photoelectric sensor 10. The specific details of each part are as follows:

[0008] Furthermore, the PDMS layer 4 is provided with a concave flow channel one 11 for fluid flow and a flow channel two 12 for casting electrodes. The sizes of the flow channels are all at the micron level. Specifically:

[0009] The first flow channel 11 is used for droplet generation and fluid flow, and includes an oil phase inlet 13, a coagulant inlet 14, a whole blood inlet 15, a V-shaped flow channel 16, a main flow channel 17, a droplet capture pool 18, a side flow channel 19, and a liquid outlet 20. Specifically: The oil phase inlet 13 is connected to the forefront of the main flow channel 17. The coagulant inlet 14 and the whole blood inlet 15 are respectively connected to the two front ends of the V-shaped flow channel 16. The intersection of the V-shaped flow channel 16 is connected to the side of the front half of the main flow channel 17. The front half of the main flow channel 17 is relatively narrow and has the same size as the V-shaped flow channel 16. The width of the rear section of the main flow channel 17 is greater than that of the front half, and a section of it is concave-shaped. There is a droplet capture pool 18 in front of the concave shape of the rear section of the main flow channel 17. The upper end of the droplet capture pool 18 is connected to the side of the main flow channel 17. The side of the droplet capture pool 18 is connected to the side of the concave-shaped flow channel of the rear section of the main flow channel 17 through the side flow channel 19. The width of the side flow channel 19 is much smaller than that of the main flow channel 17. The end of the main flow channel 17 is connected to the liquid outlet 20. Among them, the coagulant flows into one side of the V-shaped flow channel 16 at the coagulant inlet 14, and the whole blood flows into the other side of the V-shaped flow channel 16 at the whole blood inlet 15. The coagulant and the whole blood are mixed at the intersection of the V-shaped flow channel 16. The oil phase flows into the main flow channel 17 at the oil phase inlet 13. At the intersection of the V-shaped flow channel 16 and the main flow channel 17, the oil wraps the mixed solution of blood and coagulant to form droplets. The droplets enter the main flow channel 17. Since the size of the main flow channel 17 is much larger than that of the side flow channel 19, its flow resistance is much smaller than that of the side flow channel 19. When the flow rate is small and no electric field is applied, the droplets will not enter the droplet capture pool 18 nor flow out from the side flow channel 19.

[0010] The second flow channel 12 is used for casting the three-dimensional electrode 5, which mainly includes two parts of structures and is symmetric about the droplet capture pool 18. The first part includes the pointed electrode first flow channel 21 and the flat electrode first flow channel 27, which are located on the side far from the droplet capture pool 18. The second part includes the pointed electrode second flow channel 24 and the flat electrode second flow channel 30, which are located on the side close to the droplet capture pool 18. Among them, the flat electrode first flow channel 27 and the flat electrode second flow channel 30 are adjacent to the main flow channel 17, and the pointed electrode first flow channel 21 and the pointed electrode second flow channel 24 are located on the outside. Specifically: both ends of the pointed electrode first flow channel 21 are respectively provided with a pointed electrode first injection hole 22 and a pointed electrode first outflow hole 23 and are communicated with each other. Both ends of the pointed electrode second flow channel 24 are respectively provided with a pointed electrode second injection hole 25 and a pointed electrode second outflow hole 26 and are communicated with each other. Both ends of the flat electrode first flow channel 27 are respectively provided with a flat electrode first injection hole 28 and a flat electrode first outflow hole 29 and are communicated with each other. Both ends of the flat electrode second flow channel 30 are respectively provided with a flat electrode second injection hole 31 and a flat electrode second outflow hole 32 and are communicated with each other. The main flow channel of the flat electrode first flow channel 27 is parallel to the main flow channel 17. The two ends of the main flow channel of the flat electrode first flow channel 27 are bent 45° towards the pointed electrode first flow channel 21 and are respectively communicated with the pointed electrode first injection hole 22 and the pointed electrode first outflow hole 23. The pointed electrode first flow channel 21 is perpendicular to the flat electrode first flow channel 27. The front end of the pointed electrode first flow channel 21 is V-shaped and has a tip. Both ends of the V-shaped channel at the front end of the pointed electrode first flow channel 21 are respectively connected to two straight channels perpendicular to the flat electrode first flow channel 27. The two straight channels of the pointed electrode first flow channel 21 are bent outward by 45° and are respectively communicated with the pointed electrode first injection hole 22 and the pointed electrode first outflow hole 23. The second part of the structure is symmetric about the droplet capture pool 18 with the first part of the structure.

[0011] Furthermore, the three-dimensional electrode 5 is formed by injecting molten solder into the second flow channel 12. Specifically: the solder is respectively injected into the second flow channel 12 from the pointed electrode first injection hole 22, the pointed electrode second injection hole 25, the flat electrode first injection hole 28, and the flat electrode second injection hole 31. After the pointed electrode first flow channel 21, the pointed electrode second flow channel 24, the flat electrode first flow channel 27, and the flat electrode second flow channel 30 are filled with solder, they respectively flow out from the pointed electrode first outflow hole 23, the pointed electrode second outflow hole 26, the flat electrode first outflow hole 29, and the flat electrode second outflow hole 32, and successively form the pointed electrode first 33, the pointed electrode second 34, the flat electrode first 35, and the flat electrode second 36. The pointed electrode first 33, the pointed electrode second 34, the flat electrode first 35, and the flat electrode second 36 are respectively arranged on both sides of the first flow channel 11. The pointed electrode first 33 and the flat electrode first 35 are on the same side, and the pointed electrode second 34 and the flat electrode second 36 are on the same side. The shape of the electrode is the same as the shape of the second flow channel 12.

[0012] Furthermore, the glass substrate 6 is ordinary glass that is smooth and dust-free.

[0013] Furthermore, the injection pump 2 is used to accurately control the flow rate of the input fluid, including channel one 37, channel two 38 and channel three 39. Specifically: channel one 37 is connected to the whole blood inlet 15, channel two 38 is connected to the coagulant inlet 14, and channel three 39 is connected to the oil phase inlet 13.

[0014] Furthermore, the optical detection device 3 infers the coagulation process by detecting the change of light transmittance during the blood coagulation process. The specific optical detection device 3 includes a light source 7, a lens 1 8, a lens 2 9, and a photoelectric sensor 10, wherein the light source 7 and the lens 1 8 are located on one side of the microfluidic chip 1, and the lens 2 9 and the photoelectric sensor 10 are located on the other side of the microfluidic chip 1, wherein the light source 7 emits 660nm red light to avoid the strong absorption peak of hemoglobin and reduce the interference of hemoglobin, and the light is focused on the droplet capture pool 18 through the lens 1 8 to detect the light transmittance of the captured blood droplets, and the lens 2 9 focuses the transmitted light on the photoelectric sensor 10. The basic principle of detection is: initially, the particles such as red blood cells and platelets in the liquid blood are evenly dispersed, with high light transmittance, and the light intensity received by the photoelectric sensor 10 is strong. As the blood coagulates, fibrinogen is converted into fibrin, forming a reticular structure and aggregating blood cells, resulting in an increase in sample turbidity, a gradual decrease in the intensity of the transmitted light, and a decrease in the light intensity received by the photoelectric sensor 10, so the coagulation process can be judged according to the change in light transmittance.

[0015] A process for manufacturing a reusable coagulation detection device based on droplet microfluidics technology comprises the following steps:

[0016] Step 1: Design and make the mask, use CAD to draw the shape of the flow channel, and process the film mask according to the drawn drawing.

[0017] Step 2: prepare a clean, dust-free glass sheet, wash the glass sheet thoroughly with deionized water, and dry the moisture on the glass sheet in an oven.

[0018] Step 3, make the adhesion layer between SU-8 and glass. Specifically: level the glue machine, put the glass sheet in the center of the glue machine tray, turn on the glue machine switch and the vacuum pump, ensure that the glue machine tray sucks the glass sheet tightly, pour an appropriate amount of RFJ-60 on the center of the glass sheet, adjust the speed of the glue machine, and fully expose and strengthen the adhesion layer after glue is completed. At this point, the adhesion layer between the adhesion layer of SU-8 and the glass is completed.

[0019] Step 4, making SU-8 mold, specifically: the first step is glue spreading, put the glass piece in the center of the glue spreader tray, turn on the glue spreader switch and the vacuum pump, ensure that the glue spreader tray sucks the glass piece tightly, pour an appropriate amount of SU-8 glue on the center of the glass piece, adjust the speed of the glue spreader, and complete the glue spreading; the second step is pre-baking, put the glass piece on the heating table, and dry it at 85-95℃ for about 90min; the third step is exposure, after the glass piece cools to room temperature, the mask and the glass piece are bonded together for exposure; the fourth step is post-baking, put the glass piece on the heating table, and bake it at 85-95℃ for 4min; the fifth step is development, put the glass piece in the developer and clean it for about 8-12min, then put it in a new developer to clean the residual photoresist, and then clean it with ethanol and deionized water in turn, and dry the glass piece after cleaning, so that the SU-8 mold is completed.

[0020] Step 5, PDMS layer 4 is prepared. Component A and component B of PDMS are mixed in a mass ratio of 10:1, and stirred thoroughly with a glass rod to make the mixture uniform. A vacuum oven is used to evacuate the air bubbles in the PDMS. The glass sheet is wrapped with tin foil on all sides, with the part with the SU-8 mold facing upward. The mixed PDMS with air bubbles fully removed is poured onto the SU-8 mold. The vacuum oven is adjusted to 85-95°C and baked for 1-2 hours until the PDMS is completely cured. The PDMS is demolded from the SU-8 and sliced. At this point, PDMS containing channel one 11 and channel two 12 can be obtained. Use a puncher to punch holes at the PDMS channel inlet and outlet to obtain oil phase inlet 13, coagulant inlet 14, whole blood inlet 15, liquid outlet 20, pointed electrode one injection hole 22, pointed electrode one outflow hole 23, pointed electrode two injection hole 25, pointed electrode two outflow hole 26, flat electrode one injection hole 28, flat electrode one outflow hole 29, flat electrode two injection hole 31, flat electrode two outflow hole 32. At this point, the PDMS layer 4 is completed.

[0021] Step 6, the PDMS layer 4 is bonded to the glass substrate 6. The surfaces of the PDMS layer 4 and the glass substrate 6 to be bonded are treated with oxygen plasma, and then aligned and pressed for bonding.

[0022] Step 7: Fabrication of the three-dimensional electrode 5. Place the bonded chip on a heating stage and preheat it at 140 °C for 2 - 3 min. Insert the low-melting solder wire into the injection holes of the pointed electrode 1 (hole 22), pointed electrode 2 (hole 25), flat electrode 1 (hole 28), and flat electrode 2 (hole 31). As the low-melting solder melts, slowly push the solder wire forward to completely fill the flow channels of the pointed electrode 1 (channel 21), pointed electrode 2 (channel 24), flat electrode 1 (channel 27), and flat electrode 2 (channel 30) with the low-melting solder until the molten solder flows out from the outflow holes of the pointed electrode 1 (hole 23), pointed electrode 2 (hole 26), flat electrode 1 (hole 29), and flat electrode 2 (hole 32). After the electrode injection is completed, remove the chip from the heating stage and wait for the solder electrode to cool and solidify. Thus, the fabrication of the three-dimensional electrode 5 is completed.

[0023] A method for using a reusable coagulation detection device based on droplet microfluidics technology, comprising the following steps:

[0024] Step 1: Generation of blood droplets. The coagulant flows into the V-shaped channel 16 through the coagulant inlet 14, and whole blood flows into the V-shaped channel 16 through the whole blood inlet 15. At the intersection of the V-shaped channel 16, the coagulant and whole blood are mixed. The oil phase enters the main channel 17 through the oil phase inlet 13, and at the intersection of the main channel 17 and the V-shaped channel 16, the oil wraps the mixed solution of blood and coagulant to form blood droplets, which enter the main channel 17.

[0025] Step 2: Capture of blood droplets. Without applying an electric field, the size of the main channel 17 is much larger than that of the side channel 19. Therefore, the flow resistance of the main channel 17 is much smaller than that of the side channel 19, and the blood droplets will not enter the droplet capture pool 18, let alone flow out from the side channel 19. The blood droplets flow directly through the main channel 17 to the liquid outlet 20. Apply a voltage of 300 - 600 V between the pointed electrode 2 (34) and the flat electrode 1 (35). At this time, the electric field strength at the tip of the pointed electrode 2 (34) is the largest. Since the dielectric constant of the blood droplet is much larger than that of the oil phase, the polarization phenomenon of the blood droplet is more obvious. Therefore, under the action of dielectrophoresis force, the blood droplet moves towards the place with a large electric field strength, that is, the blood droplet moves towards the direction of the pointed electrode 2 (34), and the blood droplet is controlled to enter the droplet capture pool 18. Thus, the capture of the blood droplet is completed.

[0026] Step 3: Detection of blood droplets. The light source 7 emits red light with a wavelength of 660 nm. The light passes through the lens 1 (8) and is focused above the droplet capture pool 18 to detect the light transmittance of the captured blood droplet. The lens 2 (9) focuses the transmitted light onto the photoelectric sensor 10. The light intensity received by the photoelectric sensor 10 decreases. Therefore, the coagulation process can be judged according to the change of the light transmittance.

[0027] Step 4, blood droplets are released. A voltage of 300-600V is applied to the pointed electrode 1 33 and the flat electrode 2 36. The electric field strength is the largest at the tip of the pointed electrode 1 33. Since the dielectric constant of the blood droplets is much larger than that of the oil phase, the polarization phenomenon of the blood droplets is more obvious. Therefore, under the action of the dielectrophoretic force, the blood droplets move to the place with large electric field strength, that is, the blood droplets move in the direction of the pointed electrode 1 33, and the blood leaves the droplet capture pool 18 and enters the main channel 17. At this point, the release of the blood droplets is completed.

[0028] Step 5, flushing the microfluidic chip. After completing the capture of blood droplets, the blood without the addition of the coagulant has not yet coagulated. At this time, the cleaning liquid is introduced into the main channel 17 from the oil phase inlet 13, the coagulant inlet 14 and the whole blood inlet 15, so as to realize the flushing of the microfluidic chip. Since the flow resistance of the main channel 17 is much smaller than that of the side channel 19, the cleaning liquid and the residual oil phase and blood droplets will not enter the droplet capture pool 18 and the side channel 19, and the captured blood droplets will not leave the droplet capture pool 18. The flushing liquid will directly flow out of the liquid outlet 20 after flushing away the residual liquid in the V-shaped channel 16 and the main channel 17, so that the detection of a single blood droplet can be realized while flushing the chip. After the blood test is completed, the blood droplets are released into the main channel 17. At this time, the cleaning liquid is again introduced into the main channel 17 from the oil phase inlet 13, the coagulant inlet 14 and the whole blood inlet 15. After flushing, the liquid is discharged through the liquid outlet 20. At this point, the coagulation test is completed.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention proposes a reusable coagulation detection device based on droplet microfluidics technology, which achieves the mixing of blood and anticoagulant through V-shaped flow channel 16 and wraps blood and coagulant in oil phase to form blood droplets, so that blood coagulation is completed in the oil phase, effectively avoiding blood coagulation blocking the microchannel, and providing the possibility of flushing and reusability of the chip. The present invention uses the method of applying an external electric field to manipulate the droplets in the microfluidic chip, realizes the capture and release of blood droplets, and uses optical method to monitor the blood coagulation process, providing a new idea for coagulation function detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a structural diagram of a reusable coagulation detection device based on droplet microfluidics technology of the present invention.

[0032] Figure 2 This is a structural diagram of a microfluidic chip of a reusable coagulation detection device based on droplet microfluidic technology of the present invention.

[0033] Figure 3 This is a structural diagram of an optical detection device of a reusable coagulation detection device based on droplet microfluidics technology of the present invention.

[0034] Figure 4 It is a top view of the PDMS layer of a reusable blood coagulation detection device based on droplet microfluidics technology of the present invention.

[0035] Figure 5 It is a schematic diagram of the PDMS flow channel of a reusable blood coagulation detection device based on droplet microfluidics technology of the present invention.

[0036] Figure 6 It is a schematic diagram of the electrode of a reusable blood coagulation detection device based on droplet microfluidics technology of the present invention.

[0037] Figure 7 It is a top view of the flow channel of a reusable blood coagulation detection device based on droplet microfluidics technology of the present invention.

[0038] Figure 8 It is an injection pump of a reusable blood coagulation detection device based on droplet microfluidics technology of the present invention.

[0039] Figure 9 It is a simulation diagram of droplet capture of a reusable blood coagulation detection device based on droplet microfluidics technology of the present invention, where a is the droplet flow trajectory without applying an electric field, b is the electric potential distribution diagram without applying an electric field, c is the droplet flow trajectory when capturing the droplet by applying an electric field, d is the electric potential distribution diagram when capturing the droplet by applying an electric field, e is the droplet flow trajectory when releasing the droplet by applying an electric field, and f is the electric potential distribution diagram when releasing the droplet by applying an electric field.

[0040] Description of reference numerals: 1 microfluidic chip; 2 injection pump; 3 optical detection device; 4 PDMS layer; 5 three-dimensional electrode; 6 glass substrate; 7 light source; 8 lens one; 9 lens two; 10 photoelectric sensor; 11 flow channel one; 12 flow channel two; 13 oil phase inlet; 14 coagulant inlet; 15 whole blood inlet; 16 V-shaped flow channel; 17 main flow channel; 18 droplet capture pool; 19 side flow channel; 20 liquid outlet; 21 pointed electrode one flow channel; 22 pointed electrode one injection hole; 23 pointed electrode one outflow hole; 24 pointed electrode two flow channel; 25 pointed electrode two injection hole; 26 pointed electrode two outflow hole; 27 flat electrode one flow channel; 28 flat electrode one injection hole; 29 flat electrode one outflow hole; 30 flat electrode two flow channel; 31 flat electrode two injection hole; 32 flat electrode two outflow hole; 33 pointed electrode one; 34 pointed electrode two; 35 flat electrode one; 36 flat electrode two; 37 channel one; 38 channel two; 39 channel three. Detailed implementation manners

[0041] The present invention will be further described in detail below in combination with examples of blood coagulation process detection, so that those skilled in the art can implement it with reference to the text of the specification.

[0042] Unless otherwise specified, the methods used in the following technical solutions are all conventional methods. The materials and reagents used in the following technical solutions can all be obtained through commercial channels.

[0043] A reusable blood coagulation detection device based on droplet microfluidics technology implemented in this example is characterized in that the reusable blood coagulation detection device mainly includes a microfluidic chip, an injection pump, and an optical detection device. The microfluidic chip mainly generates and captures blood droplets, including a PDMS layer 4, a three-dimensional electrode 5, and a glass substrate 6, where the PDMS layer 4 and the glass substrate 6 are bonded together by plasma treatment, and the three-dimensional electrode 5 is poured between the PDMS layer 4 and the glass substrate 6 through a second flow channel 12; the injection pump 2 is used to control a constant flow rate; the optical detection device is used to detect the blood coagulation process, including an optical sensor 5 and a processing module 6.

[0044] An inwardly concave first flow channel 11 for fluid flow and a second flow channel 12 for pouring electrodes are provided on the PDMS layer 4. The first flow channel 11 is used for droplet formation and fluid flow, including an oil phase inlet 13, a coagulant inlet 14, a whole blood inlet 15, a V-shaped flow channel 16, a main flow channel 17, a droplet capture pool 18, a side flow channel 19, and a liquid outlet 20. The coagulant flows into the V-shaped flow channel 16 at the coagulant inlet 14, and the whole blood flows into the V-shaped flow channel 16 at the whole blood inlet 15. At the intersection of the V-shaped flow channel 16, the coagulant and the whole blood are mixed. The oil phase enters the main flow channel 17 at the oil phase inlet 13, and at the intersection of the main flow channel 17 and the V-shaped flow channel 16, the oil-wrapped mixed solution of blood and coagulant forms blood droplets and enters the main flow channel 17. The size of the main flow channel 17 is much larger than that of the side flow channel 19. Therefore, the flow resistance of the main flow channel 17 is much smaller than that of the side flow channel 19. When the flow rate is small and no electric field is applied, the droplets will not enter the droplet capture pool 18 and will not flow out from the side flow channel 19; the second flow channel 12 is used to form the three-dimensional electrode 5, including a pointed electrode one flow channel 21, a pointed electrode one injection hole 22, a pointed electrode one outflow hole 23, a pointed electrode two flow channel 24, a pointed electrode two injection hole 25, a pointed electrode two outflow hole 26, a flat electrode one flow channel 27, a flat electrode one injection hole 28, a flat electrode one outflow hole 29, a flat electrode two flow channel 30, a flat electrode two injection hole 31, and a flat electrode two outflow hole 32.

[0045] The three-dimensional electrode 5 is formed by injecting molten solder into the second flow channel 12, including a pointed electrode one 33, a pointed electrode two 34, a flat electrode one 35, and a flat electrode two 36, where the shape of the pointed electrode is exactly the same as that of the second flow channel 12.

[0046] The glass substrate 6 is ordinary glass that is smooth and dust-free.

[0047] The injection pump 2 is used to accurately control the flow rate of the input fluid, and includes channel one 37, channel two 38 and channel three 39. Specifically, channel one 37 is connected to the whole blood inlet 15, channel two 38 is connected to the coagulant inlet 14, and channel three 39 is connected to the oil phase inlet 13.

[0048] The optical detection device 3 infers the coagulation process by detecting the change of transmittance during blood coagulation. Specifically, the light source 7 emits 660nm red light and focuses the light above the droplet capture pool 18 through the lens 1 8. The lens 2 9 focuses the light passing through the droplets onto the photoelectric sensor 10 to detect the transmittance of the captured blood droplets.

[0049] The processing process of a reusable coagulation detection device based on droplet microfluidics technology is as follows:

[0050] Step 1: Design and make the mask, use CAD to draw the shape of the flow channel, and process the film mask according to the drawn drawing.

[0051] Step 2: prepare a clean, dust-free glass sheet, wash the glass sheet thoroughly with deionized water, and dry the moisture on the glass sheet in an oven.

[0052] Step 3, make the adhesion layer between SU-8 and glass. Specifically: level the glue machine, put the glass sheet in the center of the glue machine tray, turn on the glue machine switch and the vacuum pump, ensure that the glue machine tray sucks the glass sheet tightly, pour an appropriate amount of RFJ-60 on the center of the glass sheet, adjust the speed of the glue machine, and fully expose and strengthen the adhesion layer after glue is completed. At this point, the adhesion layer between the adhesion layer of SU-8 and the glass is completed.

[0053] Step 4, making SU-8 mold, specifically: the first step is glue spreading, put the glass piece in the center of the glue spreader tray, turn on the glue spreader switch and the vacuum pump, ensure that the glue spreader tray sucks the glass piece tightly, pour an appropriate amount of SU-8 glue on the center of the glass piece, adjust the speed of the glue spreader, and complete the glue spreading; the second step is pre-baking, put the glass piece on the heating table, and dry it at 85-95℃ for about 90min; the third step is exposure, after the glass piece cools to room temperature, the mask and the glass piece are bonded together for exposure; the fourth step is post-baking, put the glass piece on the heating table, and bake it at 85-95℃ for 4min; the fifth step is development, put the glass piece in the developer and clean it for about 8-12min, then put it in a new developer to clean the residual photoresist, and then clean it with ethanol and deionized water in turn, and dry the glass piece after cleaning, so that the SU-8 mold is completed.

[0054] Step 5, PDMS layer 4 is prepared. Component A and component B of PDMS are mixed in a mass ratio of 10:1, and stirred thoroughly with a glass rod to make the mixture uniform. A vacuum oven is used to evacuate the air bubbles in the PDMS. The glass sheet is wrapped with tin foil on all sides, with the part with the SU-8 mold facing upward. The mixed PDMS with air bubbles fully removed is poured onto the SU-8 mold. The vacuum oven is adjusted to 85-95°C and baked for 1-2 hours until the PDMS is completely cured. The PDMS is demolded from the SU-8 and sliced. At this point, PDMS containing channel one 11 and channel two 12 can be obtained. Use a puncher to punch holes at the PDMS channel inlet and outlet to obtain oil phase inlet 13, coagulant inlet 14, whole blood inlet 15, liquid outlet 20, pointed electrode one injection hole 22, pointed electrode one outflow hole 23, pointed electrode two injection hole 25, pointed electrode two outflow hole 26, flat electrode one injection hole 28, flat electrode one outflow hole 29, flat electrode two injection hole 31, flat electrode two outflow hole 32. At this point, the PDMS layer 4 is completed.

[0055] Step 6, the PDMS layer 4 is bonded to the glass substrate 6. The surfaces of the PDMS layer 4 and the glass substrate 6 to be bonded are treated with oxygen plasma, and then aligned and pressed for bonding.

[0056] Step 7, production of three-dimensional electrode 5, place the bonded chip on the heating table, preheat at 140℃ for 2 to 3 minutes, insert the low-melting-point solder wire into the pointed electrode 1 injection hole 22, the pointed electrode 2 injection hole 25, the flat electrode 1 injection hole 28, and the flat electrode 2 injection hole 31. As the low-temperature solder melts, slowly push the solder wire forward to completely fill the pointed electrode 1 flow channel 21, the pointed electrode 2 flow channel 24, the flat electrode 1 flow channel 27, and the flat electrode 2 flow channel 30 with the low-temperature solder until the molten solder flows out from the pointed electrode 1 outflow hole 23, the pointed electrode 2 outflow hole 26, the flat electrode 1 outflow hole 29, and the flat electrode 2 outflow hole 32. After completing the electrode injection, remove the chip from the heating table and wait for the solder electrode to cool and solidify. At this point, the production of the three-dimensional electrode 5 is completed.

[0057] Based on the above system, a reusable coagulation detection device based on droplet microfluidics technology is specifically implemented as follows:

[0058] Step 1, select kaolin-cephalin suspension as coagulant, take 1ml of fresh rabbit whole blood and add it to the syringe in front of channel 1 37, take 1ml of coagulant and add it to the syringe in front of channel 2 38, take 1ml of colorless dimethyl silicone oil and add it to the syringe in front of channel 3 39, connect all pipes, check the air tightness of the device, and turn on the power of all equipment.

[0059] Step 2, regulate the input flow rate of each channel of the injection pump 2, including 3750 μl / min for channel 1, 3820 μl / min for channel 2, and 39100 μl / min for channel 3. Among them, channel 1 is connected to the whole blood inlet 15, channel 2 is connected to the coagulant inlet 14, and channel 3 is connected to the oil phase inlet 13.

[0060] Step 3, generate blood droplets. The coagulant flows into the V-shaped flow channel 16 at the coagulant inlet 14, and the whole blood flows into the V-shaped flow channel 16 at the whole blood inlet 15. At the intersection of the V-shaped flow channel 16, the coagulant and the whole blood are mixed. Dimethyl silicone oil enters the main flow channel 17 at the oil phase inlet 13. At the intersection of the main flow channel 17 and the V-shaped flow channel 16, the oil wraps the mixed solution of blood and coagulant to form blood droplets and enters the main flow channel 17.

[0061] Step 4, capture blood droplets. Without applying an electric field, due to the flow resistance of the main flow channel 17 being much smaller than that of the side flow channel 19, the blood droplets will neither enter the droplet capture pool 18 nor flow out from the side flow channel 19. The blood droplets flow through the main flow channel 17 towards the liquid outlet 20. Apply a voltage of 300 - 600 V between the pointed electrode 2 34 and the flat electrode 1 35. The electric field strength is the largest at the tip of the pointed electrode 2 34. Since the dielectric constant of the blood droplets is much larger than that of the oil phase, the polarization phenomenon of the blood droplets is more obvious. Therefore, under the action of the dielectrophoretic force, the blood droplets move towards the place with a large electric field strength, that is, the blood droplets move towards the direction of the pointed electrode 2 34, and the blood droplets enter the droplet capture pool 18. Thus, the capture of blood droplets is completed.

[0062] Step 5, detect blood droplets. The light source 7 emits red light with a wavelength of 660 nm. After passing through the lens 1 8, the light is focused above the droplet capture pool 18, and the lens 2 9 focuses the transmitted light onto the photoelectric sensor 10 to detect the change in light transmittance during the blood coagulation process to infer the coagulation process. Initially, the particles such as red blood cells and platelets in the liquid blood are evenly dispersed, and the light transmittance is relatively high. The light intensity received by the photoelectric sensor 10 is strong. As the blood coagulates, fibrinogen is converted into fibrin, forming a network structure and aggregating blood cells, resulting in an increase in the turbidity of the sample and a gradual decrease in the transmitted light intensity. The light intensity received by the photoelectric sensor 10 decreases. Therefore, the coagulation process can be judged based on the change in light transmittance.

[0063] Step 6, blood droplet release, a voltage of 300-600V is applied to the pointed electrode 1 33 and the flat electrode 2 36, the electric field strength at the tip of the pointed electrode 1 33 is the largest, because the dielectric constant of the blood droplets is much larger than that of the oil phase, the polarization phenomenon of the blood droplets is more obvious, so under the action of the dielectrophoretic force, the blood droplets move to the place with a large electric field strength, that is, the blood droplets move in the direction of the pointed electrode 1 33, and the blood leaves the droplet capture pool 18 and enters the main channel 17, and the blood droplet release is completed. The simulation data of droplet capture and release is shown in Figure 1. Figure 9 Shown

[0064] Step 7, flushing the microfluidic chip. After completing the capture of blood droplets, the blood without the addition of coagulant has not yet coagulated. At this time, the cleaning liquid is passed into the cross flow channel 9 from the oil phase inlet 13, the coagulant inlet 14 and the whole blood inlet 15 to achieve the flushing of the microfluidic chip. Since the flow resistance of the main channel 17 is much smaller than that of the side channel 19 and the size of the blood droplets is larger than that of the side channel 19, the cleaning liquid and the residual oil phase and blood droplets will not enter the droplet capture pool 18 and the side channel 19, and the blood droplets will not leave the droplet capture pool 18. The flushing liquid flushes away the residual liquid in the V-shaped flow channel 16 and the main channel 17 and flows out directly at the liquid outlet 20, so that the blood droplets can be detected while flushing the chip. After the blood test is completed, the blood droplets are released, and the cleaning liquid is again introduced into the main channel 17 from the oil phase inlet 13, the coagulant inlet 14 and the whole blood inlet 15 to achieve complete cleaning of the microfluidic chip. At this point, the coagulation test is completed, and the chip can be flushed and reused.

[0065] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A reusable coagulation detection device based on droplet microfluidics technology, characterized in that, The reusable blood coagulation detection device described above includes a microfluidic chip (1), an injection pump (2), and an optical detection device (3); the microfluidic chip (1) is used to generate and capture blood droplets, and includes a PDMS layer (4), a three-dimensional electrode (5), and a glass substrate (6), wherein the PDMS layer (4) and the glass substrate (6) are bonded together by plasma treatment, and the three-dimensional electrode (5) is cast in the flow channel two (12) of the PDMS layer (4); the injection pump (2) is used to control a constant flow rate, and the injection pump (2) is connected to the inlet on the PDMS layer (4) to control the stable inlet flow rate; the optical detection device (3) is used to detect the blood coagulation process in the microfluidic chip (1), and includes a light source (7), a first lens (8), a second lens (9), and a photoelectric sensor (10). The first lens (8) focuses the light emitted by the light source (7) above the droplet capture pool (18) of the microfluidic chip (1). After passing through the microfluidic chip (1), the second lens (9) focuses the light onto the photoelectric sensor (10).

2. The reusable blood coagulation detection device based on droplet microfluidics technology according to claim 1, wherein On the PDMS layer (4) described above, there are concave flow channels one (11) for fluid flow and flow channels two (12) for casting the three-dimensional electrode (5). The sizes of the flow channels are all in the micron level. The flow channel one (11) is used for droplet generation and fluid flow, and includes an oil phase inlet (13), a coagulant inlet (14), a whole blood inlet (15), a V-shaped flow channel (16), a main flow channel (17), a droplet capture pool (18), and a liquid outlet (20). Specifically: The oil phase inlet (13) is connected to the front end of the main flow channel (17). The coagulant inlet (14) and the whole blood inlet (15) are respectively connected to the two front ends of the V-shaped flow channel (16). The intersection of the V-shaped flow channel (16) is connected to the first half of the main flow channel (17). The second half of the main flow channel (17) is provided with a droplet capture pool (18) communicated therewith; the end of the main flow channel (17) is connected to the liquid outlet (20); The flow channel two (12) described above is used for casting the three-dimensional electrode (5), and mainly includes two parts of structures, and is symmetrical about the droplet capture pool (18). The first part includes a pointed electrode one flow channel (21) and a flat electrode one flow channel (27), which are located on the side far from the droplet capture pool (18). The second part includes a pointed electrode two flow channel (24) and a flat electrode two flow channel (30), which are located on the side close to the droplet capture pool (18); among them, the flat electrode one flow channel (27) and the flat electrode two flow channel (30) are adjacent to the main flow channel (17), and the pointed electrode one flow channel (21) and the pointed electrode two flow channel (24) are located on the outside; the second part of the structure is symmetrical to the first part of the structure about the droplet capture pool (18); The three-dimensional electrode (5) is formed by injecting molten solder into the flow channel two (12).

3. The reusable coagulation detection device based on droplet microfluidics technology according to claim 2, wherein In the flow channel one (11) described above: The width of the first half of the main runner (17) is smaller than that of the second half. The width of the first half of the main runner (17) is the same as that of the V-shaped runner (16). A concave-shaped runner is provided in the second half of the main runner (17). A droplet capture pool (18) is provided in front of the concave-shaped runner section. The upper end of the droplet capture pool (18) is connected to the side of the main runner (17). The side of the droplet capture pool (18) is connected to the side of the concave-shaped runner in the second half of the main runner (17) through a side runner (19). The width of the side runner (19) is smaller than that of the first half of the main runner (17).

4. The reusable coagulation detection device based on droplet microfluidics technology according to claim 2, wherein, In the runner two (12) described above: At both ends of the pointed electrode one runner (21), a pointed electrode one injection hole (22) and a pointed electrode one outflow hole (23) are respectively provided and are interconnected. At both ends of the pointed electrode two runner (24), a pointed electrode two injection hole (25) and a pointed electrode two outflow hole (26) are respectively provided and are interconnected. At both ends of the flat electrode one runner (27), a flat electrode one injection hole (28) and a flat electrode one outflow hole (29) are respectively provided and are interconnected. At both ends of the flat electrode two runner (30), a flat electrode two injection hole (31) and a flat electrode two outflow hole (32) are respectively provided and are interconnected. The main runner of the flat electrode one runner (27) is parallel to the main runner (17). The two ends of the main runner of the flat electrode one runner (27) are bent towards the pointed electrode one runner (21) and are respectively interconnected with the pointed electrode one injection hole (22) and the pointed electrode one outflow hole (23). The pointed electrode one runner (21) is perpendicular to the flat electrode one runner (27). The front end of the pointed electrode one runner (21) is V-shaped and has a tip. The two ends of the V-shaped channel at the front end of the pointed electrode one runner (21) are respectively connected to two straight channels perpendicular to the flat electrode one runner (27). The two straight channels of the pointed electrode one runner (21) are bent outwards and are respectively connected to the pointed electrode one injection hole (22) and the pointed electrode one outflow hole (23).

5. The reusable coagulation detection device based on droplet microfluidics technology according to claim 4, wherein, Specifically, for the three-dimensional electrode (5) described above: Solder is respectively injected into the runner two (12) from the pointed electrode one injection hole (22), the pointed electrode two injection hole (25), the flat electrode one injection hole (28), and the flat electrode two injection hole (31). After the solder fills the pointed electrode one runner (21), the pointed electrode two runner (24), the flat electrode one runner (27), and the flat electrode two runner (30), it flows out from the pointed electrode one outflow hole (23), the pointed electrode two outflow hole (26), the flat electrode one outflow hole (29), and the flat electrode two outflow hole (32) respectively, successively forming a pointed electrode one (33), a pointed electrode two (34), a flat electrode one (35), and a flat electrode two (36). The pointed electrode one (33), the pointed electrode two (34), the flat electrode one (35), and the flat electrode two (36) are respectively arranged on both sides of the runner one (11). The pointed electrode one (33) and the flat electrode one (35) are on the same side, and the pointed electrode two (34) and the flat electrode two (36) are on the same side. The shape of the electrode is the same as that of the runner two (12).

6. The reusable coagulation detection device based on droplet microfluidics technology according to claim 1, wherein The glass substrate (6) is smooth and dust-free ordinary glass.

7. The reusable coagulation detection device based on droplet microfluidics technology according to claim 2, wherein, The injection pump (2) is used to accurately control the flow rate of the input fluid, and includes channel one (37), channel two (38) and channel three (39). Specifically: channel one (37) is connected to the whole blood inlet (15), channel two (38) is connected to the coagulant inlet (14), and channel three (39) is connected to the oil phase inlet (13).

8. The reusable coagulation detection device based on droplet microfluidics technology according to claim 1, wherein The light source (7) emits 660nm red light.

9. A method for using the reusable coagulation detection device based on droplet microfluidics technology according to any one of claims 1-8, characterized in that, The following steps are involved: Step 1, blood droplets are generated, the coagulant flows into one side of the V-shaped flow channel (16) at the coagulant inlet (14), and the whole blood flows into the other side of the V-shaped flow channel (16) at the whole blood inlet (15), and the coagulant and the whole blood are mixed at the intersection of the V-shaped flow channel (16); the oil phase enters the main flow channel (17) at the oil phase inlet (13), and at the intersection of the main flow channel (17) and the V-shaped flow channel (16), the mixed solution of blood and coagulant wrapped in oil forms blood droplets and enters the main flow channel (17); Step 2, capturing blood droplets. When no electric field is applied, the size of the main channel (17) is larger than that of the side channel (19). The blood droplets will not enter the droplet capture pool (18) nor flow out of the side channel (19). The blood droplets will flow directly to the liquid outlet (20) through the main channel (17). A voltage is applied to the pointed electrode 2 (34) and the flat electrode 1 (35). At this time, the blood droplets move in the direction of the pointed electrode 2 (34), and the blood droplets are controlled to enter the droplet capture pool (18). At this point, the blood droplet capture is completed. Step 3, blood droplet detection, the light source (7) emits 660nm red light, which is focused above the droplet capture pool (18) through lens 1 (8), and the transmittance of the captured blood droplets is detected. Lens 2 (9) focuses the transmitted light onto the photoelectric sensor (10); the coagulation process is determined based on the change in transmittance; Step 4, blood droplet release, voltage is applied to the pointed electrode 1 (33) and the flat electrode 2 (36), at this time, the blood droplet moves in the direction of the pointed electrode 1 (33), the blood leaves the droplet capture pool (18), and enters the main flow channel (17), and the blood droplet release is completed; Step 5, flushing the microfluidic chip. After completing the capture of blood droplets, the blood without the addition of the coagulant has not yet coagulated. At this time, the cleaning liquid is introduced into the main channel (17) from the oil phase inlet (13), the coagulant inlet (14) and the whole blood inlet (15) to realize the flushing of the microfluidic chip. The flushing liquid flushes away the residual liquid in the V-shaped channel (16) and the main channel (17) and flows out directly at the liquid outlet (20), so that the single blood droplet can be detected while flushing the chip; after the blood test is completed, the blood droplets are released into the main channel (17). At this time, the cleaning liquid is again introduced into the main channel (17) from the oil phase inlet (13), the coagulant inlet (14) and the whole blood inlet (15). After flushing, the liquid is discharged through the liquid outlet (20). At this point, the coagulation test is completed.

10. The usage method of a reusable coagulation detection device based on droplet microfluidics technology according to claim 9, characterized in that, In the step 2, the voltage applied to the second pointed electrode (34) and the first flat electrode (35) is 300 - 600V; in the step 4, the voltage applied to the first pointed electrode (33) and the second flat electrode (36) is 300 - 600V.

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