Four-item fan-shaped detection disc for thrombus

Abstract

The invention discloses a thrombus four-item fan-shaped detection disc which comprises a disc body with a fan-shaped structure, and a sample adding groove, a separation groove connected with the sample adding groove and a quantitative unit connected with the separation groove which are sequentially arranged on the disc body from the rotating center of the disc body to the peripheral direction, the four detection units are connected with the quantitative unit, the at least one second waste liquid tank is respectively connected with the four detection units, a group of freeze-drying ball combinations for detecting four markers of thrombus are respectively preset in the four detection units, and each group of freeze-drying ball combinations comprises antibody-coated magnetic bead freeze-drying balls and enzyme-labeled antibody freeze-drying balls; the magnetic bead freeze-drying ball is pre-arranged in the detection groove, and the enzyme-labeled antibody freeze-drying ball is pre-arranged in the first injection groove. The detection disc is matched with detection equipment to realize the functions of whole blood separation, plasma quantification, freeze-drying dissolution, reagent mixing, rotary incubation, centrifugal cleaning and the like, four set results of thrombus are obtained through single whole blood, the detection sensitivity is high, the results are accurate and reliable, and the repeatability is good.

Description

Technical Field

[0001] This invention relates to the field of biochemical detection technology, and in particular to a thrombosis four-item sector-shaped detection disc. Background Technology

[0002] Thrombosis is a disease with insidious onset and sudden onset, resulting in a high mortality rate. Whether it's arterial thrombosis, venous thrombosis, or microvascular thrombosis, it poses a significant threat to human health. Multiple studies have shown that early detection and diagnosis of high-risk individuals in various clinical departments can enable early intervention and treatment, effectively reducing the harm of thrombosis to human health. Existing research indicates that when the body is in a pre-thrombotic state, changes have already occurred in the vascular endothelium, coagulation, and fibrinolytic systems. TAT (thrombin-antithrombin III complex), PIC (plasmin-α2 plasmin inhibitor complex), TM (thrombomodulin), and tPAI·C (tissue plasminogen activator-plasminogen activator inhibitor-1 complex) are effective indicators reflecting early changes in the body's vascular endothelium, coagulation, and fibrinolytic systems. They are suitable for early diagnosis, risk assessment, and treatment efficacy evaluation of high-risk individuals in various clinical departments, as well as for thrombosis risk screening in healthy individuals.

[0003] Currently, chemiluminescence immunoassay is widely used in the detection of four thrombotic markers due to its simple operation, high specificity, high sensitivity, and short detection time. In chemiluminescence immunoassay, magnetic microparticle immunoassay technology utilizes magnetic solid-phase microparticles of a specific particle size synthesized from polymer materials as carriers. These microparticles are coated with various immunologically active substances, such as antibodies or antigens with specific affinity, through methods such as physical adsorption and chemical coupling. It features fast separation speed, high efficiency, good reproducibility, simple operation, and no impact on the biological characteristics and functions of the separated cells or other biological materials. Under the influence of an external magnetic field, these microparticles can move directionally, allowing for the separation, concentration, or purification of certain specific components. However, at present, chemiluminescence immunoassay generally uses liquid reagents for detection, which requires refrigeration, resulting in higher transportation and storage costs.

[0004] With the development of detection technology, microfluidic chip technology has been widely used in many fields due to its advantages such as high throughput, integration, convenience, ease of operation, and low cost. How to combine microfluidic chip technology with chemiluminescence immunoassay to achieve faster detection of the four thrombosis markers is a pressing technical problem that needs to be solved in this field. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, the present invention aims to provide a thrombosis four-item sector-shaped detection disk, which, when used with detection equipment, can realize functions such as whole blood separation, plasma quantification, lyophilization and dissolution, reagent mixing, rotation incubation, and centrifugation washing. It can obtain the results of the four-item thrombosis package from a single whole blood sample, with high detection sensitivity, accurate and reliable results, and good repeatability.

[0006] To solve the above problems, the solution adopted by the present invention is as follows: This invention includes a thrombosis four-marker sector-shaped detection disk, comprising a sector-shaped disk body, a sample loading groove arranged sequentially on the disk body from the center of rotation outwards, a separation groove connected to the sample loading groove, a quantitative unit connected to the separation groove, four detection units connected to the quantitative unit, and at least one second waste liquid tank connected to each of the four detection units. Each detection unit includes a detection groove and an injection groove assembly. The detection groove is connected to the second waste liquid tank. The injection groove assembly includes a first injection groove and a second injection groove connected to the detection groove, and the injection groove assembly is located between the quantitative unit and the detection groove. Each of the four detection units contains a set of lyophilized beads for detecting four thrombosis markers. Each set of lyophilized beads includes antibody-coated magnetic bead lyophilized beads and enzyme-labeled antibody lyophilized beads. The magnetic bead lyophilized beads are pre-placed in the detection groove, and the enzyme-labeled antibody lyophilized beads are pre-placed in the first injection groove.

[0007] Preferably, the freeze-dried pellet combinations are TAT freeze-dried pellet combination, PIC freeze-dried pellet combination, TM freeze-dried pellet combination, and tPAI·C freeze-dried pellet combination.

[0008] Preferably, a limiting threshold is provided in the first injection groove to restrict the movement of the enzyme-labeled antibody lyophilized bulb.

[0009] Preferably, the top of the second waste liquid tank is provided with a connection end that connects to the third microchannel, and guide grooves are provided on both sides of the tank. The distance from the top of the guide groove to the rotation center of the disc is less than the distance from the connection end to the rotation center of the disc.

[0010] Preferably, a first accommodating cavity and a second accommodating cavity are sequentially arranged in the second waste liquid tank along the rotation center of the disc towards the outer periphery, and the depth of the first accommodating cavity is greater than the depth of the second accommodating cavity.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: In this invention, the detection disc, in conjunction with the detection equipment, can achieve functions such as whole blood separation, plasma quantification, lyophilization and dissolution, reagent mixing, rotary incubation, and centrifugal washing. It obtains a four-item thrombosis test result from a single whole blood sample, exhibiting high sensitivity, accurate and reliable results, and good repeatability. By pre-packaging the lyophilized bulbs in the detection disc, transportation and consumable costs can be effectively reduced. The detection disc adopts a fan-shaped structure, with multiple discs combined into a circular disc, allowing for simultaneous detection of multiple targets, greatly improving disc utilization and reducing waste. By combining the detection disc with lyophilized bulb reagents, the properties of the detection disc and reagent as consumables and main raw materials, respectively, can be changed. Combining the two products creates a composite consumable, improving user convenience. Furthermore, the package discs can be customized to meet specific customer needs. Attached Figure Description

[0012] Figure 1 This is a schematic diagram of the structure of the thrombosis four-item sector-shaped detection disc in a preferred embodiment of the present invention; Figure 2 for Figure 1 A schematic diagram of the structure of the first injection tank; Figure 3 This is a schematic diagram of the structure of the second waste liquid tank in a specific embodiment; The reference numerals in the attached drawings are as follows: 1 is the disc body, 10 is the positioning groove, 11 is the first positioning groove, 12 is the second positioning groove, 13 is the lever, 21 is the sample loading groove, 22 is the separation groove, 23 is the first microchannel, 241 is the diversion channel, 242 is the first waste liquid tank, 243 is the quantitative tank, 244 is the first vent, 25 is the second microchannel, 261 is the detection tank, 262 is the first injection tank, 2621 is the limiting threshold, 263 is the second injection tank, 27 is the third microchannel, 28 is the second waste liquid tank, 281 is the second vent, 282 is the connecting end, 283 is the guide groove, 284 is the first accommodating cavity, 285 is the second accommodating cavity, 30 is the antibody-coated magnetic bead lyophilized bulb, and 31 is the enzyme-labeled antibody lyophilized bulb. Detailed Implementation

[0013] The present invention will be further described in detail below with reference to the accompanying drawings.

[0014] Reference Figure 1This is a schematic diagram of a thrombosis four-item sector-shaped detection disk according to a preferred embodiment of the present invention. The thrombosis four-item sector-shaped detection disk includes a sector-shaped disk body 1, and a sample loading groove 21 for adding whole blood samples arranged sequentially on the disk body 1 from the rotation center outwards. A separation groove 22 for separating whole blood samples is connected to the sample loading groove 21. A quantitative unit for quantitatively separating plasma is connected to the separation groove 22. Four detection units are connected to the quantitative unit, and at least one second waste liquid tank 28 for collecting waste liquid is connected to each of the four detection units. The detection unit includes a detection tank 261 and an injection tank combination for lyophilization dissolution, reagent mixing, rotation incubation, centrifugal washing, and detection. The tank 261 is connected to the second waste liquid tank 28. The injection tank assembly includes a first injection tank 262 and a second injection tank 263, which are respectively connected to the detection tank 261. The injection tank assembly is located between the quantitative unit and the detection tank 261. The separation tank 22 is connected to the quantitative unit through the first microfluidic channel 23. The first microfluidic channel 23 is used to separate the whole blood sample and control the flow of plasma in the whole blood sample to the quantitative unit. The quantitative unit is connected to the detection tank 261 through the second microfluidic channel 25. The second microfluidic channel 25 is used to control the flow of a quantitative amount of plasma into the detection tank 261. The detection tank 261 is connected to the second waste liquid tank 28 through the third microfluidic channel 27. The third microfluidic channel 27 is used to control the flow of waste liquid into the second waste liquid tank 28. Each of the four detection units contains a pre-set set of lyophilized pellets for detecting four thrombosis markers. Each set includes antibody-coated magnetic bead lyophilized pellets 30 and enzyme-labeled antibody lyophilized pellets 31. The magnetic bead lyophilized pellets 30 are pre-placed in the detection tank 261, and the enzyme-labeled antibody lyophilized pellets 31 are pre-placed in the first injection tank 262. The disc adopts a fan-shaped structure, and multiple discs are combined into a circular disc, which can simultaneously detect multiple targets, greatly improving the utilization rate of the discs and reducing disc waste. The detection disc, in conjunction with the detection equipment, can realize functions such as whole blood separation, plasma quantification, lyophilization and dissolution, reagent mixing, rotation incubation, centrifugation and washing. The results of the four thrombosis markers can be obtained from a single whole blood sample, with high detection sensitivity, accurate and reliable results, and good repeatability. By pre-packaging the lyophilized pellets in the detection disc, the cost of transportation and consumables can be reduced.

[0015] Specifically, this application uses the following lyophilized bead combinations for detecting four thrombosis markers: TAT lyophilized bead combination (including TAT antibody-coated magnetic bead lyophilized beads and alkaline phosphatase-labeled TAT antibody lyophilized beads), PIC lyophilized bead combination (including PIC antibody-coated magnetic bead lyophilized beads and alkaline phosphatase-labeled PIC antibody lyophilized beads), TM lyophilized bead combination (including TM antibody-coated magnetic bead lyophilized beads and alkaline phosphatase-labeled TM antibody lyophilized beads), and tPAI·C lyophilized bead combination (including tPAI·C antibody-coated magnetic bead lyophilized beads and alkaline phosphatase-labeled tPAI·C antibody lyophilized beads).

[0016] A semi-permeable membrane can be installed in the first microchannel 23 to separate the sample in the separation tank 22 during centrifugation, and the plasma enters the quantitative unit through the first microchannel; a microfluidic valve can be installed in the second microchannel 25 to enhance the control of the plasma flow direction during centrifugation; a microfluidic valve can be installed in the third microchannel 27 to enhance the control of the waste liquid flow direction during centrifugation.

[0017] Specifically, the central angle of the disk body 1 is 90°, and a first positioning groove 11 and a second positioning groove 12 are provided on the outer periphery of the disk body 1. The first positioning groove 11 and the second positioning groove 12 have different sizes and are used for positioning during disk installation. In this case, the positioning groove 10 is preferably set to an arc shape or a U shape to facilitate installation and disassembly.

[0018] In another specific embodiment, a positioning groove 10 is provided at the center of the disk body 1. In one specific embodiment, the positioning groove 10 is selected as a W-shaped or wave-shaped structure to facilitate positioning during disk installation.

[0019] Specifically, a paddle 13 is provided on the outer periphery of the disc body 1 to facilitate the disassembly of the testing disc.

[0020] For details, please refer to again. Figure 1 The quantitative unit includes a diversion channel 241 for diverting plasma, a first waste liquid tank 242 for receiving excess plasma, and four quantitative tanks 243 for quantitatively measuring plasma entering the detection tank 27. One end of the diversion channel 241 is connected to the separation tank 22 via a first microchannel 23, and the other end is connected to the first waste liquid tank 242. The four quantitative tanks 243 are connected to the diversion channel 241 and are connected to the detection tank 261 via a second microchannel 25. Furthermore, the diversion channel 241 is approximately arc-shaped, and the distance from the end near the first microchannel 23 to the rotation center of the disk 1 is no greater than the distance from the end near the first waste liquid tank 242 to the rotation center of the disk 1, facilitating the collection of excess plasma into the first waste liquid tank 242.

[0021] Specifically, the first waste liquid tank 242 is connected to a first vent hole 244, and the second waste liquid tank 28 is connected to a second vent hole 281. The vent hole structure can adjust the air pressure inside the plate 1, which facilitates the flow of plasma or waste liquid inside the plate 1 and makes it easier for them to be collected.

[0022] Specifically, refer to Figure 2 A limiting threshold 2621 is provided in the first injection groove 262 to restrict the movement of the enzyme-labeled antibody lyophilized ball 31. The limiting threshold 2621 and the first injection groove 262 cooperate to form a accommodating space for the enzyme-labeled antibody lyophilized ball 31, thereby fixing the position of the enzyme-labeled antibody lyophilized ball 31 to a certain extent.

[0023] Specifically, refer to Figure 3In one specific embodiment, the top of the second waste liquid tank 28 is provided with a connecting end 282 that connects to the third microchannel 27. Guide channels 283 are respectively provided on both sides of the connecting end 282. The distance from the top of the guide channel 283 to the rotation center of the disk 1 is less than the distance from the connecting end 282 to the rotation center of the disk 1. The guide channels 283 can guide the solution in the second waste liquid tank 28 during forward and reverse rotation mixing, preventing the solution from flowing back into the third microchannel 27.

[0024] Refer again Figure 3 Within the second waste liquid tank 28, a first receiving cavity 284 and a second receiving cavity 285 are sequentially arranged along the outer periphery from the rotation center of the disk body 1. The depth of the first receiving cavity 284 is greater than the depth of the second receiving cavity 285. Compared to the first receiving cavity 283, the second receiving cavity 284 has a greater surface tension on the solution in the second waste liquid tank 28, which can better ensure that the solution remains in the second receiving cavity 284 and prevent the solution in the second waste liquid tank 28 from flowing back into the third microfluidic channel 27 during forward and reverse rotation mixing. At this time, in order to ensure better dissolution of the enzyme-labeled antibody lyophilized beads 31, the enzyme-labeled antibody lyophilized beads 31 are pre-placed in the second receiving cavity 284.

[0025] The following is about Figure 1 The working principle of the four-item sector-shaped test panel for thrombosis will be further explained: The first step is to add a whole blood sample to the sample compartment of the testing tray; The second step involves centrifugation, during which the whole blood sample enters the separation tank, and the plasma in the whole blood sample is separated into the diversion channel via the first microfluidic channel. The third step is to continue centrifugation. After the plasma fills the quantitative tank, the excess plasma enters the first waste liquid tank. The fourth step is to adjust the centrifugation speed so that the plasma in the quantitative tank breaks through the second microchannel and enters the detection tank, which initially dissolves the antibody-coated lyophilized magnetic beads in the detection tank. Step 5: Stop centrifugation, add activation solution to the second injection tank, and centrifuge again to allow the activation solution to enter the detection tank and completely dissolve the lyophilized beads. Then, continuously change the centrifugation direction by rotating in both directions, and may also use heating to help mix and incubate the plasma with the antibody-coated magnetic beads, obtaining a mixture containing the magnetic bead-antibody-antigen intermediate. Adjust the centrifugation speed to allow the waste liquid in the mixture in the detection tank to break through the third microchannel and enter the second waste liquid tank. At the same time, with the help of an external magnetic field, the magnetic bead-antibody-antigen intermediate is retained in the detection tank. Stop centrifugation, add washing solution to the second injection tank, and centrifuge again to allow the washing solution to enter the detection tank. Then, continuously change the centrifugation direction by rotating in both directions to thoroughly clean the magnetic bead-antibody-antigen intermediate. Adjust the centrifugation speed again to allow the washing solution containing impurities to break through the third microchannel and enter the second waste liquid tank. At the same time, with the help of an external magnetic field, the cleaned high-purity magnetic bead-antibody-antigen intermediate is retained in the detection tank. Step 6: Stop centrifugation, add activation solution to the first injection tank to completely dissolve the lyophilized enzyme-labeled antibody beads; centrifuge again to allow the activated enzyme-labeled antibody solution to enter the detection tank, and then continuously change the centrifugation direction by rotating in both directions, and may also assist in heating the detection tank to achieve mixing and incubation of the magnetic beads-antibody-antigen intermediate and enzyme-labeled antibody, obtaining a mixture containing antigen-antibody sandwich conjugate; Step 7: Adjust the centrifugation speed to allow the waste liquid in the mixture in the detection tank to break through the third microchannel and enter the second waste liquid tank. At the same time, with the help of an external magnetic field, the antigen-antibody sandwich conjugate is retained in the detection tank. Stop centrifugation, add cleaning solution to the injection tank, and centrifuge again to allow the cleaning solution to enter the detection tank. Then, continuously change the centrifugation direction and achieve thorough cleaning of the antigen-antibody sandwich conjugate by rotating in both directions. Adjust the centrifugation speed again to allow the cleaning solution containing impurities to break through the third microchannel and enter the second waste liquid tank. At the same time, with the help of an external magnetic field, the cleaned high-purity antigen-antibody sandwich conjugate is retained in the detection tank. Step 8: Stop centrifugation, add the luminescent substrate to the second injection tank, centrifuge again to allow the luminescent substrate to enter the detection tank, and achieve the mixing and reaction of the antigen-antibody sandwich conjugate and the luminescent substrate by rotating in both directions and by heating the detection tank. After the reaction is completed, the luminescence values ​​in the four detection tanks can be detected and analyzed simultaneously by the detection equipment.

[0026] The embodiments described herein are preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape, and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A sector-shaped detection disc for four thrombosis parameters, characterized in that, The device includes a fan-shaped disk body, a sample loading groove arranged sequentially on the disk body from the center of rotation outwards, a separation groove connected to the sample loading groove, a quantitative unit connected to the separation groove, four detection units connected to the quantitative unit, and at least one second waste liquid tank connected to each of the four detection units. Each detection unit includes a detection groove and an injection groove assembly. The detection groove is connected to the second waste liquid tank. The injection groove assembly includes a first injection groove and a second injection groove connected to the detection groove. The injection groove assembly is located between the quantitative unit and the detection groove. Each of the four detection units is pre-loaded with a set of lyophilized beads for detecting four thrombosis markers. Each set of lyophilized beads includes antibody-coated magnetic bead lyophilized beads and enzyme-labeled antibody lyophilized beads. The magnetic bead lyophilized pellets are pre-placed in the detection cell, and the enzyme-labeled antibody lyophilized pellets are pre-placed in the first injection cell.

2. The thrombosis four-item sector-shaped detection disc as described in claim 1, characterized in that, The freeze-dried pellet combinations are TAT freeze-dried pellet combination, PIC freeze-dried pellet combination, TM freeze-dried pellet combination and tPAI·C freeze-dried pellet combination.

3. The thrombosis four-item sector-shaped detection disc as described in claim 1, characterized in that, The first injection tank is equipped with a limiting threshold that restricts the movement of enzyme-labeled antibody lyophilized pellets.

4. The thrombosis four-item sector-shaped detection disc as described in claim 1, characterized in that, The top of the second waste liquid tank is provided with a connection end that connects to the third microchannel, and guide grooves are provided on both sides of the tank. The distance from the top of the guide groove to the center of rotation of the disc is less than the distance from the connection end to the center of rotation of the disc.

5. The thrombosis four-item sector-shaped detection disc as described in claim 1, characterized in that, The second waste liquid tank has a first accommodating cavity and a second accommodating cavity arranged sequentially along the rotation center of the disc towards the outer periphery, and the depth of the first accommodating cavity is greater than the depth of the second accommodating cavity.

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