Thromboelastography microfluidic chip and droplet formation method

Abstract

The application discloses a thromboelastography microfluidic chip and a droplet forming method, relates to the technical field of instant detection products, and discloses the thromboelastography microfluidic chip which comprises a cover sheet, an elastic film and a bottom sheet arranged in sequence from top to bottom, wherein the bottom sheet is provided with a flow channel and a plurality of groups of containing cavities; each containing cavity comprises a first reagent chamber, a second reagent chamber, a mixing chamber and a detection cavity which are sequentially connected in gas path through a second valve body, a third valve body and a fourth valve body; the first reagent chamber is connected in gas path with the flow channel through a first valve body; the elastic film is provided with a first gas-permeable through hole which is connected in gas path with the containing cavities and is located between the fourth valve body and the mixing chamber; and the cover sheet is provided with a first window, a second window, a third window, a fourth window, a mixing chamber window and a fifth window which is connected with the first gas-permeable through hole; the valve body control and pneumatic driving technology can ensure the full-process automation of sample quantification, activation, mixing and detection, and is suitable for the instant detection scene requirement.

Description

Technical Field

[0001] This invention relates to the field of point-of-care testing products, and in particular to a thromboelastography microfluidic chip and a droplet formation method. Background Technology

[0002] Thromboelastography (TEG), as a whole blood coagulation function assessment method, can comprehensively reflect the dynamic interactions of multiple aspects such as coagulation factors, platelets, and the fibrinolytic system by monitoring viscoelastic changes during the coagulation process in real time. Compared with traditional coagulation tests (such as PT and APTT), TEG provides a more comprehensive assessment of the actual coagulation environment in vivo, and is therefore widely used in clinical practice. However, traditional TEG equipment relies on large rotating mechanical structures and complex optical detection systems, resulting in limitations such as large size, complex operation, high testing costs, and large sample requirements, making it difficult to meet the application needs of real-time testing and resource-constrained scenarios.

[0003] Traditional thromboelastography equipment has significant limitations: First, it is complex to operate and susceptible to human error, relying on manual sample addition and multiple reagent addition steps, which easily introduces operational deviations. Second, it has low detection efficiency; for example, platelet aggregation function testing requires three to four separate tests using three to four channels, resulting in low throughput. Third, it requires large sample volumes; a single test requires 340 μL of sample, corresponding to a large amount of reagent and high cost.

[0004] For example, a microfluidic chip disclosed in Chinese invention patent application publication number CN117696136A integrates a traditional test cup into the chip. This results in a large sample volume requirement and high detection costs. After the chip is placed into the detection module, the cup lid needs to be installed on the detection module. After installation, the concentricity between the cup lid and the bottom of the cup is poor, affecting the detection results. In addition, this solution puts multiple reagents into a mixing chamber and uses magnetic beads for mixing, which violates the requirement that the sample needs to be activated by the first reagent and then mixed with the second reagent in the correct order, affecting the accuracy of the parameters.

[0005] For example, the device, cylinder, and method for hemostasis testing disclosed in Chinese invention patent application publication number CN105164528A employs a seven-layer chip bonding to form a flow channel, resulting in high material and processing costs due to the large number of layers. The sample holding structure needs to be connected to the chip body structure via a soft bridge to meet the resonant vibration requirements. This structure is difficult to process and bond, and the complex chip structure leads to poor stability of the soft bridge. Furthermore, the channel valve body allows for simultaneous opening and closing of liquid and gas, requiring 26 gas valves to drive the liquid, resulting in high control costs.

[0006] Therefore, how to provide a thromboelastography microfluidic chip and droplet formation method to at least partially improve the above-mentioned drawbacks is a technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide a thromboelastography microfluidic chip and a droplet formation method that can achieve high-precision and rapid detection with only a small amount of sample. It can ensure the full automation of sample quantification, activation, mixing and detection, reduce human intervention and adapt to the needs of real-time detection scenarios.

[0008] To achieve the above objectives, the present invention provides a thromboelastography microfluidic chip, comprising a cover, an elastic film, and a substrate arranged sequentially from top to bottom. The substrate has a flow channel and several sets of receiving cavities. Each receiving cavity includes a first reagent chamber connected to the flow channel via a first valve body, a second reagent chamber connected to the first reagent chamber via a second valve body, a mixing chamber connected to the second reagent chamber via a third valve body, and a detection cavity connected to the mixing chamber via a fourth valve body and in which a droplet generator is placed. The elastic film has a first ventilator connected to the receiving cavity via a gas path and located between the fourth valve body and the mixing chamber. The cover plate has a first window corresponding to the first valve body, a second window corresponding to the second valve body, a third window corresponding to the third valve body, a fourth window corresponding to the fourth valve body, a mixing chamber window corresponding to the mixing chamber, and a fifth window connected to the first vent hole. The first, second, third, and fourth windows are all used to connect to an external pneumatic device to form a positive pressure that causes the corresponding position of the elastic film to move downward, and when the corresponding position of the elastic film moves downward, it prevents the flow of liquid. The mixing chamber window and the fifth window are both connected to an external pneumatic device to form a positive pressure and a negative pressure that cause the liquid in the receiving cavity to flow in a directional manner.

[0009] In one possible implementation, the film also has a sample injection port for sample flow to the flow channel, and a waste liquid pool connected to the flow channel via a fifth valve body. The elastic film has a second vent hole connected to the waste liquid pool via a gas path and located above the gas path of the waste liquid pool. The cover has a sixth window corresponding to the fifth valve body and a pressure channel connected to the fifth window. The pressure channel is connected to the first and second vent holes. The sixth window is used to connect to an external pressure device to form a positive pressure that causes the corresponding position of the elastic film to move downward. When the corresponding position of the elastic film moves downward, the fifth valve body prevents liquid flow.

[0010] In one possible implementation, the elastic film has detection cavity through holes corresponding to the detection cavities, and the cover plate has detection cavity through grooves corresponding to the detection cavities. The detection cavity through holes are used for the droplet generator to pass through, so that the top of the droplet generator is accommodated in the detection cavity through groove. An upper seal is provided on the side of the detection cavity through groove away from the elastic film, and a lower seal is provided on the side of the detection cavity away from the elastic film. The upper and lower seals are used to seal both ends of the droplet generator in the height direction.

[0011] In one possible implementation, the droplet generator is interference-fitted with the detection chamber.

[0012] In one possible implementation, the cover sheet has two first positioning through holes, the elastic film has two second positioning through holes, and the bottom sheet has two third positioning through holes, with the first positioning through holes, the second positioning through holes, and the third positioning through holes corresponding one-to-one.

[0013] In one possible implementation, a first superhydrophobic patch is provided at the first vent hole, and a second superhydrophobic patch is provided at the second vent hole. The first and second superhydrophobic patches are used to prevent liquid from entering the air pressure channel.

[0014] In one possible implementation, the bottom of the waste liquid pool is positioned at a lower height than the bottom of the flow channel to prevent the sample and air bubbles in the waste liquid pool from flowing back.

[0015] In one possible implementation, the bottom of the mixing chamber is at a lower height than the bottom of the second reagent chamber.

[0016] In one possible implementation, the first window, second window, third window, fourth window, and fifth window all adopt stepped through holes.

[0017] Based on the above, this application also provides a droplet formation method applicable to any of the thromboelastography microfluidic chips. The droplet formation method includes: delivering a liquid to be tested into a flow channel; applying positive pressure at a second window and negative pressure at a fifth window, causing the liquid to fill a first reagent chamber and mix with the reagent in the first reagent chamber; applying positive pressure at a third window and negative pressure at a fifth window, causing the liquid to fill a second reagent chamber and mix with the reagent in the second reagent chamber; applying positive pressure at a fourth window and negative pressure at a fifth window, causing the liquid to fill a mixing chamber; alternating between positive and no pressure at the mixing chamber window and alternating between no pressure and positive pressure at the third window, causing the liquid to move back and forth between the mixing chamber and the second reagent chamber until it reacts completely with the reagent; applying positive pressure at the fourth window and negative pressure at the fifth window, causing the liquid to refill the mixing chamber; and applying positive pressure at the third window and positive pressure at the mixing chamber window to drive the liquid into a droplet generator for forming a test droplet.

[0018] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects: The thromboelastography microfluidic chip includes a cover, an elastic film, and a substrate arranged sequentially from top to bottom. The substrate is provided with a flow channel and several sets of receiving cavities. Each receiving cavity includes a first reagent chamber connected to the flow channel via a first valve body, a second reagent chamber connected to the first reagent chamber via a second valve body, a mixing chamber connected to the second reagent chamber via a third valve body, and a detection cavity connected to the mixing chamber via a fourth valve body and in which a droplet generator is placed. The elastic film is provided with... The receiving cavity is connected to the gas path and located between the fourth valve body and the mixing chamber. The cover plate has a first window corresponding to the first valve body, a second window corresponding to the second valve body, a third window corresponding to the third valve body, a fourth window corresponding to the fourth valve body, a mixing chamber window corresponding to the mixing chamber, and a fifth window connected to the first vent hole. The liquid to be tested is delivered into the flow channel. Positive pressure is applied to the second window and negative pressure is applied to the fifth window, causing the liquid to fill the first reagent chamber and mix with the reagent inside the first reagent chamber. Positive pressure is applied to the third window... Applying positive pressure and negative pressure at the fifth window fills the second reagent chamber with liquid and mixes it with the reagent inside. Applying positive pressure at the fourth window and negative pressure at the fifth window fills the mixing chamber with liquid. Alternating positive and negative pressure at the mixing chamber windows and at the third window causes the liquid to move back and forth between the mixing chamber and the second reagent chamber until it reacts completely with the reagent. This process generates vortex shear in the liquid, improving mixing efficiency. Applying positive pressure at the fourth window and negative pressure at the fifth window again fills the mixing chamber with liquid. Applying positive pressure at the third window and mixing... Positive pressure is applied to the window, driving liquid into the droplet generator to generate monodisperse droplets. Finally, impact vibration is applied to the entire thromboelastography microfluidic chip. After relevant detection of the droplets, thromboelastography data is obtained. With this setup, under the protection of a fully automated closed system, valve control and pneumatic drive technology are used to automatically generate droplets, impact vibration of the droplets to obtain relevant resonance data, and achieve the detection requirements. High-precision and rapid detection can be achieved with only a small amount of sample. It can ensure the full automation of sample quantification, activation, mixing and detection, reduce human intervention, and adapt to the needs of real-time detection scenarios. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0020] Figure 1This is a schematic diagram of the thromboelastography microfluidic chip provided in an embodiment of the present invention.

[0021] Figure 2 This is an exploded view of the thromboelastography microfluidic chip provided in an embodiment of the present invention.

[0022] Figure 3 This is a schematic diagram of the cover plate provided in an embodiment of the present invention.

[0023] Figure 4 This is a schematic diagram of the structure of the elastic film provided in an embodiment of the present invention.

[0024] Figure 5 This is a schematic diagram of the structure of the film provided in an embodiment of the present invention.

[0025] Figure 6 This is a schematic diagram of the structure of the film provided in an embodiment of the present invention from another perspective.

[0026] Figure 7 This is a partial cross-sectional view of the thromboelastography microfluidic chip provided in an embodiment of the present invention.

[0027] Figure 8 This is a schematic diagram of the droplet generator provided in an embodiment of the present invention.

[0028] Figure 9 This is a schematic diagram of the droplet generator provided in an embodiment of the present invention from another perspective.

[0029] The attached figures are labeled as follows.

[0030] 100-Cover plate, 101-First positioning through hole, 103-First window, 104-Mixing chamber window, 105-Detection cavity through groove, 106-Air pressure channel, 107-Second window, 108-Third window, 109-Fourth window, 110-Fifth window, 111-Sixth window.

[0031] 200 - Elastic film, 201 - Second positioning through hole, 202 - First venting through hole, 203 - Detection cavity through hole, 204 - Second venting through hole.

[0032] 300-Film, 301-Third positioning through hole, 302-Sample injection port, 303-Flow channel, 304-First reagent chamber, 305-Mixing chamber, 306-Detection chamber, 307-Waste liquid pool, 308-First valve body, 309-Second valve body, 310-Third valve body, 311-Fourth valve body, 312-Fifth valve body, 313-Second reagent chamber.

[0033] 410 - First superhydrophobic patch, 420 - Second superhydrophobic patch.

[0034] 500-Droplet Generator.

[0035] 600 - Top seal.

[0036] 700 - Lower seal. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0039] In the description of this invention, it should be understood that the terms "upper" and "lower" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the position or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations of this invention.

[0040] In order to clearly illustrate the technological innovation of this microfluidic chip in the field of sample testing, it should be specifically stated that the functional integration method of this invention (including the design of the cover plate 100 of the first window 103, the second window 107, the third window 108, the fourth window 109, the fifth window 110, the sixth window 111, the mixing chamber window 104, and the first positioning through hole 101, and the processing technology of the depth of each region of the receiving cavity in the substrate 300) and the assembly method (such as high-quality double-sided adhesive bonding process) are not limited to the parameter range described in the embodiments.

[0041] Without departing from the core technical features defined in this application (including but not limited to: the partitioned layout of the flow channel 303, the mixing chamber 305, and the waste liquid pool 307), those skilled in the art can adjust and optimize the implementation elements such as the selection of interlayer bonding materials and the depth gradient design of each region of the containment cavity.

[0042] In this application, the term "comprising" and its derivatives specifically refer to open technology protection for microfluidic chips with composite structures such as plexiglass and elastic film 200. The technical solutions covered by this application not only include the core components explicitly described in the embodiments (such as the first window 103, second window 107, third window 108, fourth window 109, fifth window 110, sixth window 111, mixing chamber window 104, and pressure channel 106 of the cover 100, the mixing chamber 305 of the substrate 300, and the detection chamber 306 for placing the droplet generator 500), but should also be interpreted as compatible with the following unexhaustive elements: sensor integration schemes for the liquid storage area and the detection area, or innovative implementation forms such as other interlayer encapsulation methods derived through some bonding processes.

[0043] The implementation details key structural parameters (including: thickness tolerance of cover plate 100 / substrate 300 ±0.1mm, aspect ratio of mixing chamber 305 1:1.76, and volume ratio of waste liquid tank 307 to detection area). Furthermore, conventional processes (such as PMMA laser engraving parameters, PDMS film molding and demolding procedures, and double-sided adhesive hot-pressing curing conditions) have been reasonably omitted based on well-known microfluidic technologies. It should be noted that technical details not fully described in the specification, such as interlayer alignment strategies (e.g., positioning hole tolerance control) and double-sided adhesive bonding strength testing methods, can be implemented using mature standards in the field.

[0044] The purpose of this invention is to provide a thromboelastography microfluidic chip and a droplet formation method that can achieve high-precision and rapid detection with only a small amount of sample. It can ensure the full automation of sample quantification, activation, mixing and detection, reduce human intervention and adapt to the needs of real-time detection scenarios.

[0045] Please see Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 and Figure 7To achieve the above objectives, the present invention provides a thromboelastography microfluidic chip, comprising, from top to bottom, a cover plate 100, an elastic film 200, and a substrate 300. The substrate 300 is provided with a flow channel 303 and several sets of receiving cavities. Each receiving cavity includes a first reagent chamber 304 connected to the flow channel 303 via a first valve body 308, a second reagent chamber 313 connected to the first reagent chamber 304 via a second valve body 309, a mixing chamber 305 connected to the second reagent chamber 313 via a third valve body 310, and a mixing chamber 305 connected to the mixing chamber 313 via a fourth valve body 311. The mixing chamber 305 is connected to the gas path and contains the detection chamber 306, which houses the droplet generator 500. Gas path connection means that gas is always allowed to pass through. The mixing chamber 305 is preferably a cylindrical chamber structure. The droplet generator 500 is used to generate monodisperse droplets with a capacity of 20 μL. After placing the droplet generator 500 in the detection chamber 306, it is necessary to ensure a tight fit between the droplet generator 500 and the liquid entry channel into the detection chamber 306. Preferably, the droplet generator 500 and the detection chamber 306 are interference-fitted to ensure accurate control during operation. Four sets of receiving chambers can be provided, each set used for... For the detection of the same or different indicators, the number of cavities can be adjusted according to actual needs. The elastic film 200 is provided with a first vent hole 202 that communicates with the cavities via an air passage and is located between the fourth valve body 311 and the mixing chamber 305. A first superhydrophobic patch 410 is provided at a corresponding position of the first vent hole 202 to prevent liquid from contacting the first vent hole 202. The cover plate 100 is provided with a first window 103 corresponding to the first valve body 308, a second window 107 corresponding to the second valve body 309, a third window 108 corresponding to the third valve body 310, and a third window 108 corresponding to the fourth valve body 309. The four valve bodies 311 correspond to the fourth window 109, the mixing chamber window 104 corresponds to the mixing chamber 305, and the fifth window 110 is connected to the first vent hole 202; the first window 103, the second window 107, the third window 108, and the fourth window 109 are all used to connect with an external pneumatic device to form a positive pressure that drives the elastic film 200 to move down to the corresponding position, and when the elastic film 200 moves down to the corresponding position, it prevents the flow of liquid. The mixing chamber window 104 and the fifth window 110 are both connected with an external pneumatic device to form a positive pressure and a negative pressure that drive the liquid in the containment cavity to flow in a directional manner.

[0046] It is understandable that gas path connection means allowing gas to pass through. For example, the first reagent chamber 304 is connected to the flow channel 303 through the first valve body 308, which means that the gas in the first reagent chamber 304 can enter the flow channel 303 through the first valve body 308, and the gas in the flow channel 303 can enter the first reagent chamber 304 through the first valve body 308. That is, the first valve body 308 always allows gas to pass through. The corresponding position of the elastic film 200 refers to the area in the elastic film 200 that can move down to fit against the top of the corresponding valve body to achieve the valve body restricting the flow of liquid. For example, when the first window 103 is under positive pressure, the area in the elastic film 200 between the first window 103 and the first valve body 308 can move down to fit against the top of the corresponding first valve body 308 to achieve the first valve body 308 restricting the flow of liquid.

[0047] It should be noted that each of the first window 103, each of the second window 107, each of the third window 108, each of the fourth window 109, each of the mixing chamber windows 104, and the fifth window 110 can be connected to an external pneumatic device through an independent pipe for individual control. Alternatively, all the first windows 103 can be connected to the external pneumatic device through the same first pipe for synchronous control of all the first valve bodies 308 to allow or block liquid flow. Similarly, all the second windows 107 can be connected to the external pneumatic device through the same second pipe, all the third windows 108 can be connected to the external pneumatic device through the same third pipe, all the fourth windows 109 can be connected to the external pneumatic device through the same fourth pipe, and all the mixing chamber windows 104 can be connected to the external pneumatic device through the same fifth pipe.

[0048] The cover plate 100 and the substrate 300 can be made of thermoplastic plastics such as plexiglass (transmittance ≥92%, haze ≤2%), and the elastic film 200 can be made of polydimethylsiloxane (elastic modulus 0.5-2MPa). The flow channel 303 has a width of 1mm and a depth of 1mm, guiding the sample through the valve body under negative pressure. The cover plate 100, elastic film 200 and substrate 300 can be bonded in various ways. The overall shape of the microfluidic chip is rectangular, with dimensions of 120.82mm × 63.2mm × 6mm. The cover plate 100 has a thickness of 2mm, the elastic film 200 has a thickness of 0.1mm, and the substrate 300 has a maximum thickness of 6mm. The substrate 300 includes a shell and a functional area with a receiving cavity, and the thickness of the functional area is 3.6mm. The first window 103, the second window 107, the third window 108, the fourth window 109, the fifth window 110, and the sixth window 111 all adopt stepped through holes (bottom diameter 1.5mm, top diameter 2mm), which facilitates cooperation with external pneumatic devices and increases the stress space of the elastic membrane 200, thus making operation easier. The aperture tolerance of the first window 103, the second window 107, the third window 108, the fourth window 109, the fifth window 110, and the sixth window 111 is ±0.05mm to facilitate a dynamic airtight connection with external pneumatic devices.

[0049] The thromboelastography microfluidic chip includes, from top to bottom, a cover plate 100, an elastic film 200, and a substrate 300. The substrate 300 has a flow channel 303 and several sets of receiving cavities. Each receiving cavity includes a first reagent chamber 304 connected to the flow channel 303 via a first valve body 308, a second reagent chamber 313 connected to the first reagent chamber 304 via a second valve body 309, a mixing chamber 305 connected to the second reagent chamber 313 via a third valve body 310, and a droplet generator 50 connected to the mixing chamber 305 via a fourth valve body 311. The detection chamber 306 of the 0 has an elastic diaphragm 200 with a first vent hole 202 that communicates with the receiving chamber via an air path and is located between the fourth valve body 311 and the mixing chamber 305. The cover plate 100 has a first window 103 corresponding to the first valve body 308, a second window 107 corresponding to the second valve body 309, a third window 108 corresponding to the third valve body 310, a fourth window 109 corresponding to the fourth valve body 311, a mixing chamber window 104 corresponding to the mixing chamber 305, and a fifth window 110 communicating with the first vent hole 202; the liquid to be tested is transported. The liquid flows into the flow channel 303; a positive pressure is applied to the second window 107 and a negative pressure is applied to the fifth window 110, so that the liquid fills the first reagent chamber 304 and mixes with the reagent in the first reagent chamber 304; a positive pressure is applied to the third window 108 and a negative pressure is applied to the fifth window 110, so that the liquid fills the second reagent chamber 313 and mixes with the reagent in the second reagent chamber 313; a positive pressure is applied to the fourth window 109 and a negative pressure is applied to the fifth window 110, so that the liquid fills the mixing chamber 305; the fourth window 109 is kept under positive pressure and the sixth window 111 is kept under positive pressure to close the liquid flow channel at its window position. Maintaining negative pressure at the third window 108 opens the liquid flow channel corresponding to the third window 108. Positive pressure is applied at the fifth window 110, and simultaneously, positive pressure is applied at the mixing chamber window 104, causing the liquid to flow back from the mixing chamber 305 into the second reagent chamber 313. Repeating these two steps multiple times allows the liquid to move back and forth between the second reagent chamber 313 and the mixing chamber 305, achieving homogenization of the liquid containing the sample and reagents. This process generates vortex shear in the liquid, improving mixing efficiency. Positive pressure is applied at the fourth window 109, and negative pressure is applied at the fifth window 110, causing the liquid to refill the mixing chamber 305.Negative pressure is applied to the fourth window 109, positive pressure to the third window 108, and positive pressure to the mixing chamber window 104, driving the liquid in the mixing chamber 305 into the droplet generator 500 to generate monodisperse droplets. Finally, impact vibration is applied to the entire thromboelastography microfluidic chip, and relevant thromboelastography data is obtained after relevant detection of the droplets. With this setup, under the protection of a fully automated closed system, valve control and pneumatic drive technology are used to automatically generate droplets, and impact vibration is applied to the droplets to obtain relevant resonance data, achieving the detection requirements. High-precision and rapid detection can be achieved with only a small amount of sample, ensuring full automation of sample quantification, activation, mixing, and detection, reducing human intervention, and adapting to the needs of real-time detection scenarios.

[0050] In one possible implementation, the film 300 further includes a sample injection port 302 for sample flow into the flow channel 303, and a waste liquid pool 307 connected to the flow channel 303 via a fifth valve body 312. The flow channel 303 includes an inlet, a first outlet, and a second outlet located between the inlet and the first outlet. The sample injection port 302 is connected to the inlet for sample flow into the flow channel 303. The first reagent chamber 304 is connected to the second outlet via a first valve body 308. The waste liquid pool 307 is connected to the first outlet via the fifth valve body 312 to accommodate excess sample flowing out along the flow channel 303. The bottom of the waste liquid pool 307 is lower than the bottom of the first outlet to prevent sample and air bubbles from flowing back into the waste liquid pool 307. The depth of both the detection chamber 306 and the waste liquid pool 307 is 2.5 mm. The bottom of the mixing chamber 305 is lower than the bottom of the second reagent chamber 313. The mixing chamber 305 has a depth of 1.76 mm. The elastic membrane 200 has a second vent 204 that connects to the waste liquid tank 307 via an air passage and is located above the air passage of the waste liquid tank 307. The cover 100 has a sixth window 111 corresponding to the fifth valve body 312 and a pressure channel 106 connected to the fifth window 110. The pressure channel 106 connects to the first vent 202 and the second vent 204. The sixth window 111 is used to connect to an external pressure device to form a positive pressure that causes the corresponding position of the elastic membrane 200 to move downwards. When the corresponding position of the elastic membrane 200 moves downwards, the fifth valve body 312 prevents liquid flow. A second superhydrophobic patch 420 is provided at the second vent 204. The first superhydrophobic patch 410 and the second superhydrophobic patch 420 are used for protection against... The process of preventing liquid from entering the pressure channel 106 and allowing excess sample to enter the waste liquid pool 307 is as follows: After the sample fills the first reagent chamber 304, positive pressure is applied at the first window 103 and the second window 107 to restrict the sample in the first reagent chamber 304 from flowing out of the first reagent chamber 304. Negative pressure is applied at the fifth window 110 to make the second vent 204 a negative pressure environment. No pressure or positive pressure is applied at the sixth window 111. Driven by the negative pressure environment of the second vent 204, excess sample in the sample injection port 302 and the flow channel 303 enters the waste liquid pool 307 for collection. Multiple cross baffles are set in the waste liquid pool 307 to fill the liquid from bottom to top sequentially and prevent liquid splashing and clogging of the second superhydrophobic patch 420 at the moment of start-up.

[0051] The first reagent chamber 304 is a quantitative chamber structure with a depth of 1.76 mm and a width of 2 mm, pre-filled with lyophilized reaction reagents (including but not limited to kaolin powder, batropine-activated factor XIII lyophilized powder, etc.). The second reagent chamber 313 is also a quantitative chamber structure with a depth of 1.76 mm and a width of 2 mm, pre-filled with lyophilized reaction reagents (including but not limited to calcium chloride lyophilized microspheres, adenosine diphosphate lyophilized microspheres, arachidonic acid lyophilized microspheres, etc.). Approximately 300 μL of blood sample can be directly injected into the sample injection port 302. The blood sample migrates along the flow channel 303 under the combined action of gravity, capillary effect, and negative pressure drive of the air pressure channel 106. The blood sample then enters the first reagent chamber 304, which is pre-embedded with relevant enzyme substrates. After the first reagent chamber 304 is filled, the remaining blood sample enters the waste liquid pool 307 under the negative pressure drive of the second vent hole 204. The sample in the first reagent chamber 304, driven by the negative pressure of the first vent 202 and the absence of positive pressure in the second window 107, allows the blood sample to enter the second reagent chamber 313 and mix with the reagents there. Then, driven by the negative pressure of the first vent 202 and the absence of positive pressure in the third window 108, the sample enters the mixing chamber 305. Positive pressure is applied to the fourth window 109 and the sixth window 111, sealing their respective liquid channels. Negative pressure is applied to the third window 108, opening its corresponding liquid channel. Positive pressure is applied to the fifth window 110, and simultaneously, positive pressure is applied to the mixing chamber window 104. The liquid flows back from the mixing chamber 305 to the second reagent chamber 313. This process is repeated multiple times, allowing the liquid to move back and forth between the second reagent chamber 313 and the mixing chamber 305, achieving homogenization of the liquid containing the sample and reagents. This process generates vortex shear in the liquid, improving mixing efficiency. Then, positive pressure is applied to the fourth window 109 and negative pressure to the fifth window 110, causing the liquid to refill the mixing chamber 305. Negative pressure is applied to the fourth window 109, positive pressure to the third window 108, and positive pressure to the mixing chamber window 104, driving the liquid in the mixing chamber 305 to migrate to the droplet generator 500 under pressure, ultimately forming droplets. Finally, an impact vibration is applied to the entire chip, and relevant data on thromboelastography are obtained after detecting the droplets. Under conditions such as the microflow channel 303 and the microvalve body, the fully automated, high-sensitivity, high-speed, and portable detection requirements of the microfluidic chip are ultimately achieved.

[0052] Please see Figure 8 and Figure 9In one possible implementation, the elastic film 200 has detection cavity through holes 203 corresponding to the detection cavities 306, the cover plate 100 has detection cavity through grooves 105 corresponding to the detection cavities 306, the fixed position of the droplet generator 500 has a baffle of a certain thickness, and the detection cavity 306 of the base plate 300 has a fully penetrating portion to accommodate the droplet generator 500. The detection cavity through holes 203 are used for the droplet generator 500 to pass through, so that the top of the droplet generator 500 is accommodated in the detection cavity through groove 105. The detection cavity through groove 105 is provided with an upper seal 600 on the side away from the elastic film 200, and a lower seal 700 is provided on the side of the detection cavity 306 away from the elastic film 200. The upper seal 600 and the lower seal 700 are used to seal the two ends of the droplet generator 500 in the height direction. The droplet generator 500 is an irregularly shaped cone with a square outer surface and a circular inner surface, and the diameter of its inner hole structure is determined by the required test droplet. The bottom part of the droplet generator 500, which is suspended from the droplet, is coated with a superhydrophobic material to maximize the droplet suspension.

[0053] In one possible implementation, the cover plate 100 is provided with two first positioning through holes 101, the elastic film 200 is provided with two second positioning through holes 201, and the substrate 300 is provided with two third positioning through holes 301. The first positioning through holes 101, the second positioning through holes 201, and the third positioning through holes 301 correspond one-to-one to ensure that the relative positions of the cover plate 100, the elastic film 200, and the substrate 300 are accurate, effectively ensuring the stability of the entire microfluidic chip. The diameter tolerance of the positioning holes is controlled within ±0.02mm to ensure that the interlayer alignment accuracy is ≤50μm.

[0054] The mating surfaces of the cover plate 100 and the elastic film 200 can be double-sided adhesive layers, connecting the cover plate 100 to the elastic film 200. The backing plate 300 can also be bonded to the elastic film 200 with double-sided adhesive. First, peel off the adhesive film from the backing plate 300. Align the elastic film 200 through the second positioning through-hole 201 and the third positioning through-hole 301, ensuring that the functional areas of the elastic film 200 correspond to the functional areas of the backing plate 300. Then, peel off the adhesive film from one side of the cover plate 100 and attach it to the backing plate 300 and the elastic film 200. Finally, bonding can be achieved through various methods, such as heat pressing or ultrasonic stitching.

[0055] During detection, after the blood sample reacts fully with the pre-embedded reagent to form droplets, an impact vibration is applied to the microfluidic chip, causing the sample to resonate. The resonant frequency changes with the coagulation state. When the capillary droplet amplitude is at its maximum, the applied vibration frequency can be equated to the blood sample resonant frequency. The silicon photodiode detects the current signal to record the resonant curve. The curve parameters are extracted to obtain the changes in the blood sample resonant frequency and coagulation function indicators. Compared with traditional thromboelastography equipment, this application has a smaller size and requires less sample. At the same time, it has the advantages of high sensitivity, low cost and mass production. It is suitable for point-of-care diagnostic scenarios such as emergency and anticoagulation therapy monitoring, and has high application value.

[0056] During detection, after the blood sample and pre-embedded reagent fully react to form droplets, an impact vibration is applied to the microfluidic chip, causing the sample to resonate. The resonant frequency changes with the coagulation state. When the capillary droplet amplitude is at its maximum, the applied vibration frequency can be equated to the blood sample resonant frequency. The silicon photodiode detects the current signal to record the resonant curve. The curve parameters are extracted to obtain the changes in the blood sample resonant frequency and coagulation function indicators. Compared with traditional thromboelastography detection, this application reduces the amount of sample and reagent used, can achieve fully automated operation, and has the advantages of high sensitivity, low cost, and mass production. It is suitable for point-of-care diagnostic scenarios such as emergency and anticoagulation therapy monitoring, and has high application value.

[0057] Based on the above, this application also provides a droplet formation method applicable to any of the thromboelastography microfluidic chips. The droplet formation method includes: delivering the liquid to be detected into the flow channel 303; applying positive pressure to the second window 107 and negative pressure to the fifth window 110, so that the liquid fills the first reagent chamber 304 and mixes with the reagent in the first reagent chamber 304; applying positive pressure to the third window 108 and negative pressure to the fifth window 110, so that the liquid fills the second reagent chamber 313 and mixes with the reagent in the second reagent chamber 313; applying positive pressure to the fourth window 109 and negative pressure to the fifth window 110, so that the liquid fills the mixing chamber 305; maintaining positive pressure on the fourth window 109 and positive pressure on the sixth window 111 to close the liquid flow channel at the window position. The liquid flow channel is opened by applying negative pressure to the third window 108 and positive pressure to the fifth window 110. At the same time, positive pressure is applied to the mixing chamber window 104, and the liquid flows back from the mixing chamber 305 to the second reagent chamber 313. The above two actions are repeated multiple times to make the liquid move back and forth between the second reagent chamber 313 and the mixing chamber 305, so as to achieve the mixing of the liquid containing the sample and reagent. This process can make the liquid generate vortex shear, which improves the mixing efficiency. Positive pressure is applied to the fourth window 109 and negative pressure is applied to the fifth window 110, so that the liquid refills the mixing chamber 305. Negative pressure is applied to the fourth window 109, positive pressure is applied to the third window 108, and positive pressure is applied to the mixing chamber window 104, which drives the liquid in the mixing chamber into the droplet generator 500 to generate monodisperse droplets. It should be noted that during the process of the sample entering the first reagent chamber 304, the second reagent chamber 313 and the mixing chamber 305, the fourth window 109 is always in a positive pressure environment, that is, the fourth valve 311 is in a closed state to prevent liquid flow; after the excess sample enters the waste liquid pool 307, the sixth window 111 is always in a positive pressure environment, that is, the fifth valve 312 is in a closed state to prevent liquid flow, so as to limit the backflow of the sample in the waste liquid pool 307.

[0058] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0059] This article uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the present invention.

Claims

1. A thromboelastography microfluidic chip, characterized in that, The device comprises, from top to bottom, a cover, an elastic film, and a base. The base has a flow channel and several sets of receiving cavities. Each receiving cavity includes a first reagent chamber connected to the flow channel via a first valve body, a second reagent chamber connected to the first reagent chamber via a second valve body, a mixing chamber connected to the second reagent chamber via a third valve body, and a detection cavity connected to the mixing chamber via a fourth valve body and containing a droplet generator. The elastic film has a first vent hole connected to the receiving cavity and located between the fourth valve body and the mixing chamber. The cover has a first window corresponding to the first valve body, a second window corresponding to the second valve body, a third window corresponding to the third valve body, a fourth window corresponding to the fourth valve body, a mixing chamber window corresponding to the mixing chamber, and a fifth window connected to the first vent hole. The cover plate also has a sample injection port for sample flow to the flow channel and a waste liquid pool connected to the flow channel via a fifth valve body. The elastic film has a second vent hole connected to the waste liquid pool via a gas path and located above the gas path of the waste liquid pool. The cover plate has a sixth window corresponding to the fifth valve body and a pressure channel connected to the fifth window. The pressure channel is connected to the first vent hole and the second vent hole. The first window, the second window, the third window, the fourth window, and the sixth window are all connected to an external pressure device to form a positive pressure that drives the corresponding position of the elastic film to move downward. When the corresponding position of the elastic film moves downward to fit against the top of the corresponding valve body, the corresponding valve body blocks the flow of liquid and always allows gas to pass through. The mixing chamber window and the fifth window are both connected to the external pressure device to form a positive pressure and a negative pressure that drive the liquid in the receiving cavity to flow in a directional manner. The droplet generator is an irregular cone-shaped body with a square outer edge and a circular inner edge. The bottom part of the droplet generator that hangs the droplet is coated with a superhydrophobic material to maximize the droplet hanging. During the test, after the blood sample reacts fully with the pre-embedded reagent to form droplets, an impact vibration is applied to the microfluidic chip to cause the sample to resonate. The resonant frequency changes with the coagulation state. When the capillary droplet amplitude is at its maximum, the applied vibration frequency is equal to the blood sample resonant frequency. The silicon photovoltaic cell detects the current signal to record the resonant curve. The curve parameters are extracted to obtain the changes in the blood sample resonant frequency and coagulation function indicators.

2. The thromboelastography microfluidic chip according to claim 1, characterized in that, The elastic film has detection cavity through holes corresponding to the detection cavities, and the cover plate has detection cavity through grooves corresponding to the detection cavities. The detection cavity through holes are used for the droplet generator to pass through, so that the top of the droplet generator is accommodated in the detection cavity through groove. The detection cavity through groove is provided with an upper seal on the side away from the elastic film, and the detection cavity is provided with a lower seal on the side away from the elastic film. The upper seal and the lower seal are used to seal both ends of the droplet generator in the height direction.

3. The thromboelastography microfluidic chip according to claim 1, characterized in that, The droplet generator is interference-fitted with the detection chamber.

4. The thromboelastography microfluidic chip according to claim 1, characterized in that, The cover plate has two first positioning through holes, the elastic film has two second positioning through holes, and the bottom plate has two third positioning through holes. The first positioning through holes, the second positioning through holes, and the third positioning through holes correspond one-to-one.

5. The thromboelastography microfluidic chip according to claim 1, characterized in that, A first superhydrophobic patch is provided at the first vent hole, and a second superhydrophobic patch is provided at the second vent hole. The first superhydrophobic patch and the second superhydrophobic patch are used to prevent liquid from entering the air pressure channel.

6. The thromboelastography microfluidic chip according to claim 1, characterized in that, The bottom of the waste liquid pool is positioned lower than the bottom of the flow channel to prevent the sample and air bubbles in the waste liquid pool from flowing back.

7. The thromboelastography microfluidic chip according to any one of claims 1-6, characterized in that, The bottom of the mixing chamber is at a lower height than the bottom of the second reagent chamber.

8. The thromboelastography microfluidic chip according to any one of claims 1-6, characterized in that, The first window, the second window, the third window, the fourth window, and the fifth window all use stepped through holes.

9. A droplet formation method, applicable to the thromboelastography microfluidic chip as described in any one of claims 1-8, characterized in that, The droplet formation method includes: The liquid to be tested is delivered into the flow channel; Positive pressure is applied to the second window and negative pressure is applied to the fifth window, so that the liquid fills the first reagent chamber and mixes with the reagent in the first reagent chamber; Positive pressure is applied to the third window and negative pressure is applied to the fifth window, so that the liquid fills the second reagent chamber and mixes with the reagent in the second reagent chamber; A positive pressure is applied to the fourth window and a negative pressure is applied to the fifth window, so that the liquid fills the mixing chamber; The liquid is alternately pressured with positive and negative pressure in the mixing chamber window and with negative and positive pressure in the third window, causing the liquid to move back and forth between the mixing chamber and the second reagent chamber until it reacts completely with the reagent. Positive pressure is applied to the fourth window and negative pressure is applied to the fifth window, so that the liquid refills the mixing chamber; Positive pressure is applied to the third window and the mixing chamber window to drive liquid into the droplet generator to form the droplet to be tested.

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