Microfluidic detection chip for tuberculosis specific cellular immunity

Abstract

The invention provides a microfluidic detection chip for tuberculosis specific cellular immunity, which belongs to the technical field of mycobacterium tuberculosis detection, and is a closed chip mainly formed by sequentially assembling a chip bottom plate sealing plate, a chip base body and a chip top sealing plate from bottom to top, and the chip is provided with a plurality of sample detection units, the kit can be used for completing IGRA detection of negative control, positive control and sample control of 1-8 to-be-detected samples at the same time. Besides, the invention further provides an assembly process of the centrifugal microfluidic detection chip for tuberculosis specific cellular immunity, a method for detecting chip IGRA by using the chip and application of the chip IGRA, the total coincidence rate of the chip IGRA and the traditional IGRA detection result is 100%, and the chip IGRA can be used for detecting a very small amount of to-be-detected samples, so that the detection cost is reduced, and the detection efficiency is improved. In addition, the flux and the detection speed of IGRA detection are improved, and the method has an important application prospect in IGRA detection of a large number of clinical samples.

Description

Technical Field

[0001] The present invention relates to the technical field of Mycobacterium tuberculosis detection, and particularly to a microfluidic detection chip for specific cellular immunity of tuberculosis established by combining the detection of specific cellular immune responses of Mycobacterium tuberculosis and centrifugal microfluidic detection. Background Art

[0002] Tuberculosis is caused by infection with a single pathogen, Mycobacterium tuberculosis (MBT). Since 2007, tuberculosis infection has been a serious global public health problem. Currently, the Interferon-Gamma Release Assay (IGRA) detects the level of IFN-γ produced by stimulating peripheral blood with Mycobacterium tuberculosis antigen. Because IGRA has high detection specificity and is not interfered by BCG vaccination and most NTMs; compared with the traditional tuberculin skin test (TST), IGRA has a faster detection speed; and IGRA has a single judgment threshold, which helps to standardize the detection results. Therefore, IGRA is commonly used for the auxiliary diagnosis of latent tuberculosis infection (LTBI) and active tuberculosis, and is recommended by the WHO for the auxiliary diagnosis of tuberculosis infection. However, IGRA detection includes multiple steps such as sample collection, centrifugation, plasma separation, antigen stimulation, cell culture, and cytokine detection. These steps rely on a variety of large-scale professional equipment (including centrifuges, constant temperature cell culture incubators, and fluorescence analyzers), etc., and also require precise operation and strict time control, which increases the complexity of IGRA operation. In addition, IGRA has high requirements for operators, which limits its wide application. For example, after the operator obtains the sample to be tested, a pipette is needed to slowly aspirate the plasma supernatant after centrifugation to complete plasma separation, and it is easy to aspirate impurities such as red blood cells during this process; then it is also necessary to manually mix the plasma with the diluent for culture and aspirate a quantitative volume of diluted plasma for detection. These manual processing steps for blood samples are not only time-consuming but also prone to introducing human errors, thus affecting the accuracy of the final IGRA detection results.

[0003] Microfluidic technology can be divided into three main types according to the principle of liquid manipulation: self-powered type, cartridge type, and centrifugal type. Among them, centrifugal microfluidics (Lab-on-a-Disc, abbreviated as LOAD), also known as the laboratory on a chip, integrates basic operations such as sample pretreatment, sample separation and extraction, reagent supply, metering, aliquoting, valves, mixing, culturing, washing, analysis, or preparation on a small disc. By driving the liquid flow through centrifugal force to perform related liquid processing, it can avoid problems such as air bubble introduction, uneven mixing, and low parallel processing ability existing in the self-powered type and cartridge type. It has the advantages of miniaturization, high throughput, integration, automation, fast detection, low sample volume requirement, closed type to reduce pollution, and portability to enable point-of-care testing. Among them, valve operation is the key to achieving precise liquid manipulation in centrifugal microfluidics, mainly relying on non-contact valves, including passive valves (also known as self-actuating valves) that do not require external energy input and active valves that need to be driven by exogenous factors such as pressure, magnetism, electricity, etc.; passive valves such as capillary valves, hydrophobic valves, siphon valves, etc. are easily affected by various factors, resulting in unstable control; active valves such as pneumatic valves, electromagnetic valves, electric valves, etc., but need to embed additional driving structures into the microfluidic chip, which will increase the number of chip layers. Summary of the Invention

[0004] The object of the present invention is to overcome the defects of the existing interferon-γ release assay (IGRA), and provide a centrifugal microfluidic detection chip for tuberculous specific cellular immunity that is simple and convenient for detection operation, and can be used for IGRA detection.

[0005] The object of the present invention also lies in providing a centrifugal microfluidic detection chip for tuberculous specific cellular immunity, which can simultaneously complete the detection of three tubes of negative control (N), positive control (P), and sample control (T) at one time.

[0006] The object of the present invention also lies in combining centrifugal microfluidics with tuberculous specific cellular immunity to prepare a brand-new centrifugal microfluidic detection chip for tuberculous specific cellular immunity, avoiding possible human errors introduced by traditional IGRA and improving the accuracy and precision of detection.

[0007] To achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention provides a centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity, which is a sealed chip mainly formed by sequentially assembling three layers of structures from bottom to top: a chip bottom sealing plate 1, a chip substrate 2, and a chip top sealing plate 5. The chip is provided with detection units for multiple samples, preferably 3 - 10, to adapt to the detection of multiple samples to be tested, and can be used to at least simultaneously complete the IGRA detection of negative control, positive control, and sample controls of 1 - 8 samples to be tested. The number, position, and related structures of the sample detection units 22 on the chip substrate 2 and the single detection units 17 on the chip top sealing plate correspond to each other; among them, each sample detection unit 22 on the chip substrate 2 includes a strip slot 34 for fixing the chromatographic strip 3, 9 chambers that can accommodate gas or liquid, and channels for gas-liquid communication between the strip slot 34 and the 9-chamber structure. The 9 chambers are respectively 2 chambers, namely the sample enrichment preparation chamber 23 and the red blood cell enrichment chamber 26, belonging to the single-chamber centrifugal sedimentation structure 51, a pressure buffer chamber 49 for buffering the air pressure in the single-chamber centrifugal sedimentation structure 51, 2 quantitative chambers, namely the plasma quantification chamber 45 and the red blood cell collection chamber 42, forming a concentric collinear interconnection structure 52, a waste liquid collection chamber 44 for collecting excess liquid, a diluent chamber 28 for diluting the sample to be tested, a desiccant chamber 32 for humidity adjustment, and a mixing chamber 35 for mixing the diluent and purified plasma; among them, the single-chamber centrifugal sedimentation structure 51 includes a sample enrichment preparation chamber 23, a plasma extraction inlet 24, a fence structure 25, and a red blood cell enrichment chamber 26; the concentric collinear interconnection structure 52 includes a plasma quantification chamber 45, a three-way left stepped channel 40, a three-way middle liquid inlet stepped channel 41, a red blood cell collection chamber 42, and a three-way right exhaust stepped channel 43; the channels include a plasma transfer channel 48 that connects the plasma extraction inlet 24 and the puncture valve fixed stepped hole 47 and promotes the liquid to flow in from the plasma extraction inlet 24 and out from the puncture valve fixed stepped hole 47; a puncture valve 4 is fixedly installed on the puncture valve fixed stepped hole 47, and the puncture valve 4 is a three-layer structure composed of two upper and lower nylon gaskets 11 and an intermediate aluminum foil sacrificial layer 10, belonging to an active valve that does not require additional driving structure cooperation; the single detection unit 17 on the chip top sealing plate includes a strip detection window 18 for observing the IGRA detection result of the chromatographic strip 3 and 6 gas-liquid interaction hole positions. The 6 gas-liquid interaction hole positions are respectively a sample addition hole 13 and an exhaust hole 19 for gas-liquid interaction with the single-chamber centrifugal sedimentation structure 51, a diluent chamber liquid injection hole 15 and a diluent chamber exhaust hole 16 for gas-liquid interaction with the diluent chamber 28, a pressure buffer hole 20 for buffering the air pressure in the pressure buffer chamber 49, and a puncture hole 21 located above the axis of the puncture valve 4.

[0008] According to a preferred embodiment of the present invention, in the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity, chip positioning structures 12 with the same shape and pin positioning holes 14 at corresponding positions are provided on the three-layer structures of the chip bottom sealing plate 1, the chip substrate 2, and the chip top sealing plate 5.

[0009] According to a preferred embodiment of the present invention, in the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity, the paper strip detection window 18 is sealed with a pressure-sensitive encapsulation adhesive 6 made of a transparent material.

[0010] According to a preferred embodiment of the present invention, in the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity, the coaxial positions of the puncture valve 4 from top to bottom are as follows: an airbag 7, a puncture airbag fixing plate 9, a puncture hole 21, a nylon gasket 11, an aluminum foil sacrificial layer 10, and a nylon gasket 11; the buffer airbag fixing plate 8 is installed on the chip top sealing plate 5, and the airbag 7 is installed on the buffer airbag fixing plate 8. Therefore, the coaxial positions from top to bottom are: an airbag 7, a buffer airbag fixing plate 8, a pneumatic buffer hole 20, and a pneumatic buffer chamber 49. The airbag 7 installed on the puncture airbag fixing plate 9 and the airbag 7 installed on the buffer airbag fixing plate 8 are both preferably made of an elastic material with a high elastic modulus.

[0011] According to a preferred embodiment of the present invention, in the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity, magnetic beads 53 for promoting the stirring and mixing of the solution are pre-placed in the mixing chamber 35.

[0012] According to a preferred embodiment of the present invention, in the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity, when only the matrix solution is pre-loaded in the test sample enrichment preparation chamber 23, the corresponding sample detection unit is marked as a negative control; if both the matrix solution and a tuberculosis non-specific stimulating antigen are pre-loaded in the test sample enrichment preparation chamber 23, the corresponding sample detection unit is marked as a positive control; if both the matrix solution and a Mycobacterium tuberculosis-specific antigen are pre-loaded in the test sample enrichment preparation chamber 23, the corresponding sample detection unit is marked as a sample control.

[0013] Second aspect, the present invention provides an assembly process of a centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity, including: First step: Preparation work; Second step: Assembly of the chip bottom plate sealing plate 1, installing pins in the pin positioning holes 14 to form positioning columns; Third step: Assembly of the chip substrate 2: Through the positioning columns formed by installing the chip bottom plate sealing plate 1, lock and fix the chip substrate 2 with the chip bottom plate sealing plate 1, and the exposed positioning columns are still visible after fixation; Fourth step: Assembly of the chip top sealing plate 5: Through the positioning columns exposed after assembling the chip substrate 2, lock and fix the chip top sealing plate 5 with the chip substrate 2; At the central axis position of the puncture hole 21, sequentially assemble the puncture airbag fixing plate 9 and the airbag 7 from bottom to top; At the central axis position of the air pressure buffer hole 20, sequentially assemble the buffer airbag fixing plate 8 and the airbag 7 from bottom to top; At the opening position of the strip detection window 18 on the chip top sealing plate 5, use pressure-sensitive encapsulation glue 6 for sealing.

[0014] Third aspect, the present invention provides a detection process of chip IGRA using the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity, including: Detection preparation: Equilibrate the chip at room temperature for at least 20 minutes; Sampling and sealing: Inject the sample to be tested and the diluent into the corresponding single-chamber centrifugal sedimentation structure 51 and the diluent chamber 28 respectively from the sampling hole 13 and the diluent chamber injection hole 15, and complete the sealing of the holes after adding the liquid; Incubation: Place the chip in a constant temperature environment of 37°C and incubate for 22 ± 4 hours; Chip IGRA detection: Complete the separation of plasma in the sample to be tested, liquid mixing and chip IGRA detection through 5 centrifugation operations of the chip on the servo motor, and observe the detection result of chip IGRA at the strip detection window 18; Judgment of the chip IGRA detection result: Judge the positive and negative of the detection line and the quality control line on the chromatography strip 3 by naked eyes; or perform optimization processing and semi-quantitative analysis on the intensity value of the fluorescence signal obtained by quantitative detection with a fluorescence scanner.

[0015] According to a preferred embodiment of the present invention, in the detection process of chip IGRA using the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity, for the 5 centrifugations in the chip IGRA detection, the chip is fixed on the flange block of the servo motor and then driven by the servo motor to perform centrifugation. Among them, it is accelerated to a high-speed rotation state of no higher than 3000 rpm at an acceleration of no higher than 500 rpm / s, and the centrifugation effect is no higher than 6 minutes.

[0016] Fourth aspect, the present invention provides a detection application of chip IGRA using the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity.

[0017] According to a preferred embodiment of the present invention, in the detection application of chip IGRA, based on the intensity values of the fluorescence signals obtained by quantitative detection with a fluorescence scanner, after optimization processing, by calculating the baseline and integral area, semi-quantitative analysis of the IGRA detection results can be achieved.

[0018] Advantages of the present invention: The present invention provides a microfluidic detection chip for tubercle-specific cellular immunity. The chip mainly forms a three-layer structure through a chip bottom plate sealing plate, a chip matrix, and a chip top plate sealing plate. Structures such as chromatography strips and puncture valves are provided on the chip matrix, and structures such as pressure-sensitive encapsulation glue, airbags, buffer airbag fixing plates, and puncture airbag fixing plates are provided on the chip top plate sealing plate. The chip body designs innovative structures such as a single-chamber centrifugal sedimentation structure, a concentric center co-linear interconnection structure, a mixing chamber, and a strip detection chamber, which can automatically complete key steps such as whole blood incubation, red blood cell separation, reagent mixing, and strip detection. The single-chamber centrifugal sedimentation structure of the chip uses centrifugal force to separate plasma and red blood cells, and the plasma enters the concentric center co-linear interconnection structure through the plasma transfer channel to further separate pure plasma. In the mixing chamber, the plasma is mixed with the diluent to form a diluted plasma sample. Finally, the diluted plasma is transferred to the strip card slot by siphon action to complete strip chromatography detection. The chip also designs a normally closed valve that is actuated. The opening and closing of the valve are controlled by applying pressure to the airbag to ensure precise control and on-demand release of the fluid. To improve the mixing efficiency, magnetic beads can be placed in the mixing chamber, and the Euler force generated by the acceleration and deceleration of the servo motor is used to stir the reagents, enabling sufficient contact between the components of the reagents in the chamber, thereby improving the detection sensitivity. In addition, obstacles can be integrated in the mixing chamber to cooperate with the Euler force generated by the acceleration and deceleration of the servo motor to further shorten the mixing time. The centrifugal microfluidic detection chip for tubercle-specific cellular immunity of the present invention focuses on the simplicity and automation of operation, has a high-throughput detection function, can simultaneously detect the three tubes of negative control N, positive control P, and sample control T at one time, and realizes on-site rapid point-of-care testing (POCT) of "sample in - result out". By using the centrifugal microfluidic detection chip for tubercle-specific cellular immunity of the present invention to perform IGRA detection, the entire detection process only needs to rely on an intelligent servo motor (such as a centrifugal microfluidic system) that integrates centrifugation and temperature control functions to complete, without relying on multiple independent devices such as centrifuges, constant temperature cell culture boxes, and fluorescence analyzers, significantly improving the detection throughput and operation convenience, and also realizing fully automatic, high-throughput, "sample in - result out" type IGRA detection. Moreover, the centrifugal microfluidic detection chip for tubercle-specific cellular immunity of the present invention can improve the accuracy and reliability of IGRA detection and is applicable to the auxiliary diagnosis of latent tuberculosis infection (LTBI) and active tuberculosis.

[0019] The present invention also provides a centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity, which can perform high-throughput parallel detection on multiple samples to be tested. Specifically, the chip can at least distribute one sample to be tested into three independent detection units for detecting the negative control, positive control, and sample control of the IGRA experiment respectively. Therefore, the simultaneous detection of these three types of independent control tubes, namely negative control (N), positive control (P), and sample control (T), can be carried out on one chip. And the traditional IGRA and the chip IGRA of the present invention are used to perform parallel comparative detection on 3 negative samples of Mycobacterium tuberculosis and simulated positive samples respectively. The detection results preliminarily verify that its negative coincidence rate, positive coincidence rate, and total coincidence rate can reach 100%.

[0020] The present invention designs an IGRA tuberculosis detection chip based on centrifugal microfluidics, which is fully enclosed and has a self-ventilation function. During the experimental process after sample addition and sealing, the chip is always in a sealed state, and the gas and liquid inside it can reach a balanced state in the closed space. Among them, the piercing valve is an active valve that does not require additional driving structure cooperation. The liquid flow is realized by squeezing the airbag operation. This operation method will not damage the sealing of the chip. The air pressure expansion buffer inside the chip is realized by a deformable airbag. Through this design, the chip realizes the simplicity and automation of operation under sealed conditions, and can automatically complete key steps such as whole blood incubation, red blood cell separation, reagent mixing, and strip detection. This "sample in - result out" design has a higher function integration degree, making on-site rapid and instant detection possible, being able to complete the detection task more efficiently, reducing manual operation links, reducing human errors, and improving the accuracy and reliability of detection.

[0021] Regarding valves, generally, active valves will increase the number of chip layers due to the supporting driving structure. In order to achieve precise control and on-demand release of liquid, the present invention uses an active normally closed valve - the piercing valve. The design of the piercing valve makes clever use of the differences in material physical properties. Through modular piercing valve and other structures, not only the efficient integration of different functions is realized, but also the complexity of chip manufacturing and the number of chip layers are not increased. The modular design enables the piercing valve to be flexibly combined with other functional modules, enhancing the scalability and adaptability of the chip.

[0022] The centrifugal microfluidic chip for IGRA tuberculosis detection of the present invention has a unique two-stage red blood cell filtration structure. The first stage is a single-chamber centrifugal sedimentation structure, and the second stage is a concentric-center collinear interconnection structure. Among them, the first stage is completed in the single-chamber centrifugal sedimentation structure. By adding a fence structure in the single-chamber centrifugal sedimentation structure, the agglomerated red blood cells can be piled up to the lower layer through the fence structure, and the partition of red blood cells and plasma is realized through the physical structure, thereby reducing the influence of the agglomerated red blood cells on the plasma transfer; and the outlet is set at the position where the stratification of red blood cells and plasma is the most obvious in the single-chamber centrifugal sedimentation structure, thereby minimizing the "fluid-structure coupling effect" of plasma and red blood cells. The second stage is completed in the concentric-center collinear interconnection structure. By controlling the centrifugal force generated by the rotation angular velocity direction and magnitude of the servo motor, the density difference of different components in the blood sample, and the microfluidic technology, the direction of the inertial force received by the liquid in the concentric-center collinear interconnection structure is controlled, so that the liquid containing more red blood cells preferentially flows into the red blood cell collection chamber through the inlet stepped channel in the three-way fork, and the gas in the red blood cell collection chamber is discharged through the right exhaust stepped channel in the three-way fork. Therefore, the red blood cell collection chamber with a blind hole structure is preferentially filled with the liquid containing more red blood cells, and the liquid relatively containing fewer red blood cells (equivalent to pure plasma) is subsequently quantitatively filled into the plasma quantitative chamber, further reducing the possibility of red blood cells flowing indirectly into the chromatography strip through the plasma siphon channel. In addition, there is also a potential third-stage red blood cell filtration structure, which is mainly completed in the mixing chamber. Through the optimized settings of the shape of the mixing chamber, the lower chamber under the siphon valve inlet, and the position of the liquid outlet, after the above two-stage red blood cell filtration structure, the plasma is basically pure, and no red blood cells can be observed with the naked eye in the mixing chamber. However, it cannot be excluded that in very rare cases, a very small number of red blood cells have entered the mixing chamber. Under the action of centrifugal force, the very small number of red blood cells will sink into the lower chamber under the siphon valve inlet, and the only liquid outlet in the mixing chamber can only flow out the liquid in the space above the liquid level of the lower chamber under the siphon valve inlet (the upper chamber of the siphon valve inlet) to the chromatography strip. Therefore, the optimized design of the mixing chamber structure, that is, the position of the liquid outlet, realizes the third enrichment of red blood cells and can further prevent the very small number of red blood cells that have flowed into the mixing chamber from flowing to the chromatography strip and affecting the detection accuracy. In summary, it can be seen that: the chip can realize the multi-stage enrichment of red blood cells, the separation and purification of plasma, and the quantitative extraction without using a red blood cell filter membrane and a cell separation solution.

[0023] When using the chip of the present invention for chip IGRA detection, by adding a diluent and pre-installing magnetic beads in the mixing chamber, it can help solve the problem that the plasma viscosity is not conducive to flowing on the chromatographic strip, avoiding the poor detection effect of the mixed liquid on the chromatographic strip due to the liquid viscosity, and thus improving the accuracy of the chip IGRA detection results. In chip IGRA detection, the inertial force generated by 5 centrifugations is used to improve the enrichment, directional flow, and liquid mixing efficiency, effectively avoiding problems such as interference from red blood cells in the whole blood sample to be tested or poor chromatographic strip effect caused by uneven mixing of plasma and diluent, and enhancing the reliability of the detection results.

[0024] The centrifugal microfluidic detection chip for tubercle-specific cellular immunity of the present invention has an outer shape of a flat short cylindrical shape with a thickness of about 1 cm and a diameter not exceeding 10 cm. It is overall small and compact, and adopts a modular design, making the IGRA operation simple and convenient, and can be used for the auxiliary detection of Mycobacterium tuberculosis infection. In the modular design of the chip of the present invention, especially the single-chamber centrifugal sedimentation structure, the coaxial connection structure of the centers, the 9-chamber structure including the mixing chamber and the strip detection chamber, the puncture valve structure of the normally closed active valve, etc. cooperate with each other, and automatically execute key steps such as whole blood incubation, red blood cell separation, reagent mixing, and strip detection with the assistance of centrifugal force, integrating whole blood incubation - blood separation - reagent mixing - strip detection on a closed chip. During the experiment, only the blood sample (such as whole blood) and the reagent need to be loaded and injected respectively from the sample injection hole of the chip and the liquid injection hole of the diluent chamber, and then no additional manual operation is required. The chip automatically completes gas-liquid replacement in a closed space according to the optimized path, realizes the detection experiment of the release level of interferon-γ after stimulation by tubercle-specific antigen in vitro, the detection result is displayed by an immunofluorescent strip, and the detection result can also be quantitatively analyzed, that is, it can quickly and automatically achieve the "sample in - result out" type of point-of-care testing on-site. The detection results are divided into 3 types: positive, negative, and indeterminate. Brief Description of the Drawings

[0025] Figure 1 is an exploded view of the centrifugal microfluidic detection chip for tubercle-specific cellular immunity of the present invention; Figure 2 is an exploded view of a partial structure on the chip top sealing plate of the centrifugal microfluidic detection chip for tubercle-specific cellular immunity of the present invention; Figure 3 is a top view of the chip top sealing plate of the centrifugal microfluidic detection chip for tubercle-specific cellular immunity of the present invention; Figure 4 is the state of the centrifugal microfluidic detection chip for tubercle-specific cellular immunity of the present invention when the puncture valve is closed Figure 3 and is a longitudinal sectional view at the a-a angle; Figure 5 It is an enlarged view of a single detection unit on the chip top sealing plate of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention; Figure 6 It is a top view of the chip substrate of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention; Figure 7 It is a diagram of various pore positions set at the corresponding positions on the chip top sealing plate of a single detection unit of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention, looking down on the chip substrate structure; Figure 8 It is a schematic diagram of the key structure of a single sample detection unit on the chip substrate of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention, looking down on the chip substrate; Figure 9 It is a design principle diagram of the radial stack formed by centrifuging a blood sample by the single-chamber centrifugal sedimentation structure in a single sample detection unit on the chip substrate of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention; Figure 10 It is a partial enlarged view of the single-chamber centrifugal sedimentation structure in a single sample detection unit on the chip substrate of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention; Figure 11 It is the axis line of the single-chamber centrifugal sedimentation structure in a single sample detection unit on the chip substrate of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention when the puncture valve is in the open state Figure 10 longitudinal sectional view of b-b in; Figure 12 It is a partial enlarged view of the concentric collinear interconnection structure in a single sample detection unit on the chip substrate of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention, looking down on the chip substrate; Figure 13 It is a design principle diagram of the transfer of diluted plasma by the mixing chamber in a single sample detection unit on the chip substrate of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention; Figure 14 It is a partial enlarged view of the mixing chamber structure in a single sample detection unit on the chip substrate of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention; Figure 15 It is 4 steps for data analysis of IGRA detection of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention; Step 1 is to obtain the original data of the fluorescence signal, Step 2 is filtering and normalization analysis, Step 3 is peak-finding algorithm analysis, and Step 4 is the calculation of the baseline and integral area; Figure 16The following is a comparison of the results of performing IGRA tests on 3 negative samples of Mycobacterium tuberculosis using traditional IGRA and the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention. Here, 1-3 represent the 3 negative samples of Mycobacterium tuberculosis respectively, A is traditional IGRA, and B is the chip IGRA of the present invention; Figure 17 The following is a comparison of the results of performing IGRA tests on simulated tuberculosis-positive samples 3 times using traditional IGRA and the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention. Here, 1-3 represent the 3 repeated tests respectively, A is traditional IGRA, and B is the chip IGRA of the present invention; Figures 1 - 14 Among them: 1. Chip bottom sealing plate, 2. Chip matrix, 3. Chromatography strip, 4. Puncture valve, 5. Chip top sealing plate, 6. Pressure-sensitive encapsulation adhesive, 7. Airbag, 8. Buffer airbag fixing plate, 9. Puncture airbag fixing plate, 10. Aluminum foil sacrificial layer, 11. Nylon gasket, 12. Chip positioning structure, 13. Sampling hole, 14. Pin positioning hole, 15. Diluent chamber filling hole, 16. Diluent chamber exhaust hole, 17. Single detection unit of the chip top sealing plate, 18. Strip detection window, 19. Exhaust hole, 20. Air pressure buffer hole, 21. Puncture hole, 22. Sample detection unit, 23. Preparation chamber for enriching the sample to be tested, 24. Plasma extraction inlet, 25. Fence structure, 26. Red blood cell enrichment chamber, 27. Internal air circulation channel, 28. Diluent chamber, 29. Diluent transfer siphon channel, 30. First capillary valve, 31. First connection channel of the desiccant chamber, 32. Desiccant chamber, 33. Second connection channel of the desiccant chamber, 34. Strip card slot, 35. Mixing chamber, 36. Plasma siphon channel, 37. Mixed liquid siphon channel, 38. Second capillary valve, 39. Mixed liquid serpentine channel, 40. Left stepped channel of the three-way, 41. Middle liquid inlet stepped channel of the three-way, 42. Red blood cell collection chamber, 43. Right exhaust stepped channel of the three-way, 44. Waste liquid collection chamber, 45. Plasma quantification chamber, 46. Annular flow channel, 47. Fixed stepped hole for the puncture valve, 48. Plasma transfer channel, 49. Air pressure buffer chamber, 50. Air pressure buffer chamber connection channel, 51. Single-chamber centrifugal sedimentation structure, 52. Concentric line interconnection structure, 53. Magnetic beads, 54. Plasma layer, 55. White blood cell layer, 56. Red blood cell layer, 57. Red blood cell particles, 58. Centrifugal force, 59. Centrifugal force component in the far-centrifugal direction, 60. Lower chamber of the siphon valve inlet, 61. Upper chamber of the siphon valve inlet. Detailed implementation manners

[0026] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings. To clearly and completely illustrate the specific exemplary embodiments of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention, many specific details are described to comprehensively complete and understand the general inventive concept of the present invention. However, in other cases, one or more well-known embodiments can also be implemented without these specific details.

[0027] The present invention claims to protect a centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity. In the present invention, "detection" specifically refers to the detection of IGRA-specific cellular immunity occurring on the chromatographic strip of the chip for the sample to be tested, belonging to the field of in vitro diagnostic reagents. An IGRA immunofluorescent chromatographic strip is installed on the strip card slot of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention, which can be used to perform IGRA detection on the sample to be tested. During the chip detection process, a centrifugal function that can preferably be used to fix the chip and drive the chip to complete high-speed rotation within 5000 rpm and an intelligent servo motor that can complete the constant temperature function at 37°C are preferred. This servo motor is a small device independently developed by the laboratory of the applicant of the present invention.

[0028] The following will be combined with the attached Figures 1 - 17 A detailed description will be given of the structure, function, assembly of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention, as well as the specific process and application of performing IGRA detection using this chip.

[0029] Example 1 Structure of the Centrifugal Microfluidic Detection Chip for Tuberculosis-Specific Cellular Immunity The centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention is disc-shaped. Along the circumferential direction of the chip, a plurality of detection units for samples are uniformly arranged, preferably 3-10, to adapt to the detection of multiple samples to be tested. At least 1 detection unit on the chip needs to be set as a negative control (N), and at least 1 detection unit needs to be set as a positive control (P). The other detection units are used to detect different sample controls (T). Therefore, the number of detection units that can simultaneously detect 1-8 samples to be tested (n≦8) can be 3, 4, 5, 6, 7, 8, 9, 10; or at least 1 detection unit is shared as a negative control (N), and for each sample to be tested during simultaneous detection, 1 is separately set as a positive control (Pn) for separate verification and 1 is set as a sample control (Tn) for detection. Therefore, the number of detection units that can simultaneously detect 1-4 samples to be tested (n≦4) can be 3, 5, 7, 9; or for each sample to be tested, a complete set of control tubes (Nn, Pn, Tn) is separately set. Therefore, the number of detection units that can simultaneously detect 1-3 samples to be tested (n≦3) is a multiple of three, that is, the number of detection units can be 3, 6, 9. It can be seen that the number of detection units is at least 3. To clearly illustrate the structure of each detection unit, the attached drawings of the present invention are mainly drawn taking the setting of 3 detection units as an example. The 3 detection units can simultaneously complete a three-tube control experiment including a negative control (N), a positive control (P), and a sample control (T) when performing IGRA detection on 1 sample to be tested. Among them, the sample control (T) is sometimes also called a test control (T). The following description will be given by taking the setting of 3 detection units as an example for illustration.

[0030] The present invention provides a centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity. Figure 1 It is an exploded schematic diagram of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention. Figure 2 It is an exploded schematic diagram of a partial structure on the top sealing plate 5 of the chip. Figure 1 And Figure 2 As can be seen from the combination, the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention includes three main layers of structures, which are the chip bottom sealing plate 1, the chip substrate 2, and the chip top sealing plate 5 from bottom to top. The chromatography strip 3 and the puncture valve 4 are arranged on the chip substrate 2, and the upper and lower parts of the chip substrate 2 are sealed and protected by the chip bottom sealing plate 1 and the chip top sealing plate 5 respectively. Among them, the number of the chromatography strip 3 and the puncture valve 4 is set to be the same according to the number of detection units of the chip. Figure 1 And Figure 2 In the case of setting 3 detection units, the number of the chromatography strip 3 and the puncture valve 4 is 3 each. Figure 1 And Figure 2It can also be seen that on the upper part of the top sealing plate 5 of the chip, there are also provided pressure-sensitive encapsulation adhesives 6, buffer airbag fixing plates 8 and puncture airbag fixing plates 9 that are the same in number as the detection units. On the upper parts of the buffer airbag fixing plate 8 and the puncture airbag fixing plate 9, airbags 7 are respectively provided. Therefore, the number of airbags 7 is twice the number of detection units.

[0031] Specifically, the centrifugal microfluidic detection chip for tubercle-specific cellular immunity of the present invention is a flat short cylindrical shape with a thickness of about 1 cm. Among them, the preparation materials of the chip bottom sealing plate 1, the chip substrate 2 and the chip top sealing plate 5 can all be polymer materials with good biocompatibility, preferably poly(methyl methacrylate) (PMMA). In addition to good biocompatibility, PMMA is also a polymer with excellent light transmittance (90%-92%); the chip bottom sealing plate 1 is preferably processed and formed by laser etching technology, and the chip substrate 2 and the chip top sealing plate 5 are both preferably formed by micro milling technology. As Figure 1 shown, when the number of detection units is 3, the shape of the chip bottom sealing plate 1 is a cylinder with a diameter of 60-80 mm and a thickness of 0.5-2 mm, preferably a cylinder with a diameter of 72 mm and a thickness of 1 mm; the shape of the chip substrate 2 is a cylinder with a diameter of 100-140 mm and a thickness of 5-8 mm, preferably a cylinder with a diameter of 120 mm and a thickness of 6 mm; Figure 1 As shown, the chip substrate 2 shown is provided with three sample detection units 22 at an angle of 120° with the center of the circle as the axis. Therefore, the three sample detection units 22 can perform IGRA detection and can at least simultaneously complete the control detection of the three detection tubes that must be set for the negative control (N), positive control (P) and sample control (T). When the chip top sealing plate 5 is prepared with PMMA, the shape of the chip top sealing plate 5 is a cylinder with a diameter of 100-140 mm and a thickness of 0.5-2 mm. The chip top sealing plate 5 is preferably a cylinder with the same diameter as the chip substrate 2. For example, the chip top sealing plate 5 is preferably a cylinder with a diameter of 120 mm and a thickness of 1 mm.

[0032] In addition, when preparing the top sealing plate 5 of the chip, although the preferred PMMA has excellent light transmittance, if the detection result of the chromatographic strip 3 on the chip substrate 2 is directly observed through PMMA with a thickness of 1 mm, PMMA will still have a certain impact on the fluorescence signal on the chromatographic strip 3: one is to weaken the fluorescence signal. Since PMMA will absorb and scatter part of the fluorescence signal, the intensity of the effective signal detected is reduced; the other is that under light of certain specific wavelengths, the absorption characteristics of PMMA will hinder the transmission of the fluorescence signal, that is, the light transmittance of PMMA is limited and not completely absorption-free. Therefore, PMMA will introduce a certain amount of background noise. When this background noise is superimposed on the target fluorescence signal, it will directly reduce the signal-to-noise ratio of the detected and observed fluorescence signal, thereby affecting the accuracy of the fluorescence curve. Therefore, when preparing the top sealing plate 5 of the chip, a strip detection window 18 needs to be processed above the strip card slot 34 for placing the chromatographic strip 3 on the chip substrate 2 at the corresponding position of the top sealing plate 5 of the chip by means of hollowing and grooving, forming an opening for observing the detection result on the chromatographic strip 3 without passing through PMMA; further, in order to ensure the sealing of the chip, at the opening position of the strip detection window 18 on the top sealing plate 5 of the chip, a pressure-sensitive encapsulating adhesive 6 with a thickness of 0.05-0.2 mm is used for sealing. The preferred material of the pressure-sensitive encapsulating adhesive 6 is a polypropylene film. Compared with PMMA, this polypropylene film will neither weaken the fluorescence signal nor hinder the acquisition of the fluorescence signal due to the absorption of fluorescence.

[0033] Figure 3 This is a top view of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention. It can be clearly seen that pin positioning holes 14 are provided on the top sealing plate 5 of the chip. Figure 6 This is a top view of the chip substrate 2 of the present invention. It can be seen that the chip substrate 2 is also provided with pin positioning holes 14. In fact, pin positioning holes 14 are also provided on the bottom sealing plate 1 of the chip. The two layers of structures of the top sealing plate 5 and the chip substrate 2 are fixed by the cooperation of the pin positioning holes 14 at the corresponding positions. Preferably, a combination of fixing fittings such as positioning fixing pins, interference fit pins, split pins with split pins, retaining rings with split pins, and threaded pins with nuts is used for fixing. After fixing, positioning columns are formed at the corresponding positions, which can effectively prevent the chip from shifting or rotating during the preparation and assembly process, thereby improving the assembly accuracy and reliability of the chip. In addition, a longitudinal sectional view of the puncture valve in the closed state is shown from the a-a angle of the puncture airbag fixing plate 9 as Figure 4 shown. Figure 4 This is the structure of the puncture valve 4 in the complete state, that is, the structure of the puncture valve in the closed state. Figure 4 Combined Figure 1 、 Figure 5It can be seen that: the airbag 7 is fixedly installed on the puncture airbag fixing plate 9, the puncture airbag fixing plate 9 is fixedly installed on the chip top sealing plate 5, the chip top sealing plate 5 is provided with a puncture hole 21, and below the puncture hole 21 is the puncture valve 4 shown by a dotted line. The puncture valve 4 is composed of two upper and lower nylon gaskets 11 and an intermediate aluminum foil sacrificial layer 10. The sectional structure of the coaxial position where the puncture valve 4 is located from top to bottom is successively: the airbag 7, the puncture airbag fixing plate 9, the puncture hole 21, the nylon gasket 11, the aluminum foil sacrificial layer 10, and the nylon gasket 11. The puncture hole 21 is coaxially aligned with the puncture valve 4 up and down, so as to ensure that the force applied at the position of the puncture hole 21 on the chip top sealing plate 5 can be accurately transmitted to the aluminum foil sacrificial layer 10 of the lower puncture valve 4.

[0034] Regarding the related structure of the puncture valve 4, the airbag 7 can seal and protect the puncture hole 21. The airbag 7 is an elastic material with a high elastic modulus, preferably silicone rubber, natural rubber or polyurethane thermoplastic elastomer; the puncture airbag fixing plate 9 is used to fix the airbag 7 to ensure that the airbag 7 can stably apply a force during operation. When a force is applied along the vertical direction of the axis, the force can be transmitted to the airbag 7 and the puncture valve 4 at a fixed point; the puncture valve 4 is a three-layer structure composed of two upper and lower nylon gaskets 11 and an intermediate aluminum foil sacrificial layer 10. That is, the material of the puncture valve 4 is preferably nylon and aluminum foil, or it can be replaced by two polytetrafluoroethylene gaskets and an intermediate stainless steel sacrificial layer to improve durability. Whether it is nylon with aluminum foil or polytetrafluoroethylene with stainless steel, the puncture valve 4 utilizes the physical properties of the materials to control the opening and closing of the puncture valve through an actively applied external mechanical force, belonging to an active valve that does not require additional drive structure cooperation. The puncture valve 4 is usually Figure 4 in the closed state shown, which can effectively prevent the unexpected flow of fluid. When it is necessary to open the channel, only need to squeeze the airbag 7 above the puncture hole 21. After the airbag 7 and the aluminum foil sacrificial layer 10 are compressed, their deformation degrees are different. The aluminum foil sacrificial layer 10 with poor elasticity will be preferably damaged, while the airbag 7 will not be damaged due to its elasticity. This material combination ensures that this active valve can not only be opened after the application of force, but also ensure the overall sealing of the microfluidic chip while opening.

[0035] Figure 5 It is a partial enlarged view of a single detection unit 17 on the chip top sealing plate. It can be seen that a chip positioning structure 12 is provided at the center of the circle on the chip top sealing plate 5. Combining Figure 1 It can be seen that the chip positioning structures 12 with the same shape are provided at the centers of the circles of the three layers of the chip bottom sealing plate 1, the chip substrate 2, and the chip top sealing plate 5. Figure 5It can be seen that the chip positioning structure 12 is a combination of a circular shape and a flat keyway shape. The flat keyway preferably has round heads at both ends. The shape of this combination is suitable for fixing the chip of the present invention on the flange block of the servo motor, ensuring that not only can the chip of the present invention rotate coaxially with the servo motor, but also the chip will not shift during rotation, so as to ensure the stability and accuracy of rotation.

[0036] Figure 5 The dotted line specifically shows a partial enlarged view of a single detection unit 17 on the top sealing plate of the chip. It can be seen that on the top sealing plate 5 of the chip, three identical single detection units 17 on the top sealing plate of the chip are distributed at an angle of 120° with the center of the circle as the center. The single detection unit 17 on the top sealing plate of the chip includes a pin positioning hole 14, a strip detection window 18, and 6 hole positions for gas-liquid interaction. The pin positioning hole 14 is used to prevent the chip from shifting or rotating during the bonding process, thereby improving the assembly accuracy and reliability of the chip; the strip detection window 18 is located above the strip card slot 34 on the chip substrate 2, and the chromatographic strip 3 is placed in the strip card slot 34 in a positioned manner, so the detection line and quality control line of the lower chromatographic strip 3 can be observed from the strip detection window 18; the strip detection window 18 can be sealed with a pressure-sensitive encapsulation adhesive 6 made of a transparent material, thereby ensuring the tightness of the entire chip; the pressure-sensitive encapsulation adhesive 6 is preferably a transparent material with a thickness of 0.1 mm.

[0037] Figure 7 This is a top view of the chip substrate 2 of the present invention showing various hole position diagrams corresponding to the positions on the top sealing plate of the chip, specifically showing the hole position structure for gas-liquid interaction. Figure 5 and Figure 7It can be seen that the holes for gas-liquid interaction include: a sample addition hole 13, an exhaust hole 19, a diluent chamber injection hole 15, a diluent chamber exhaust hole 16, an air pressure buffer hole 20 and a puncture hole 21; the sample addition hole 13 and the exhaust hole 19 are gas-liquid interaction holes of the sample enrichment preparation chamber 23 to be tested, and are respectively used for sample addition and exhaust of the sample enrichment preparation chamber 23 to be tested; in terms of position, the sample addition hole 13 and the blood sample chamber exhaust hole 19 are symmetrically distributed at both ends of the upper layer in the vertical direction of the single-chamber centrifugal sedimentation structure 51, and the sample to be tested can flow to the single-chamber centrifugal sedimentation structure 51 after being added from the sample addition hole 13, and the gas of the entire single-chamber centrifugal sedimentation structure 51 can also be discharged through the exhaust hole 19 on the upper layer of the sample enrichment preparation chamber 23 to be tested; then a seal is used to seal the sample addition hole 13 and the exhaust hole 19 respectively. The hole 19 is sealed, and the sealing member is preferably a single-sided adhesive or a polyester film (PET film); the diluent chamber injection hole 15 and the diluent chamber exhaust hole 16 are gas-liquid interactive holes of the diluent chamber 28, which are respectively used for adding diluent to the diluent chamber 28 and exhausting gas; in terms of position, the diluent chamber injection hole 15 and the diluent chamber exhaust hole 16 are symmetrically distributed at the two ends of the upper layer in the vertical direction of the diluent chamber 28, and the diluent is added to the diluent chamber 28 from the diluent chamber injection hole 15 for storage, and the gas in the diluent chamber 28 is discharged from the diluent chamber exhaust hole 16. After the liquid addition is completed, a sealing operation similar to the sample addition hole 13 and the exhaust hole 19 is adopted: preferably, the two hole positions of the diluent chamber injection hole 15 and the diluent chamber exhaust hole 16 are sealed. The air pressure buffer hole 20 is located above the air pressure buffer chamber 49 and is sealed and protected by an airbag 7 on the outside. The air pressure buffer hole 20 can make the air pressure in the single-chamber centrifugal sedimentation structure 51 be buffered and adjusted through the airbag 7; the puncture hole 21 is coaxially aligned with the puncture valve 4, and the force applied at the position of the puncture hole 21 on the sealing plate 5 on the top of the chip can be accurately and point-located to the aluminum foil sacrificial layer 10 of the puncture valve 4, thereby opening the active valve like the puncture valve 4.

[0038] Figure 8 The key structure of a single sample detection unit 22 on the chip substrate 2 of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention is shown, and it can be seen that each sample detection unit 22 includes a paper strip card slot 34, and 9 chambers and channels directly or indirectly connected thereto, and the chromatography paper strip 3 is positioned and fixed in the paper strip card slot 34. The liquid addition and detection operations of each sample detection unit 22 are the same. Figure 9 It is a radial stacking diagram formed by centrifuging a blood sample by a single chamber centrifugal sedimentation structure 51 in a single sample detection unit 22 on the chip substrate 2. Figure 10 It is a partial enlarged view of a single-chamber centrifugal sedimentation structure 51 in a single sample detection unit 22 on a single chip substrate on the chip substrate 2; Figure 8 , Figure 9 and Figure 10As can be seen from the combination, the preparation chamber 23 for enriching the sample to be tested, the fence structure 25, the erythrocyte enrichment chamber 26 and the plasma extraction inlet 24 located on the preparation chamber 23 for enriching the sample to be tested together form Figure 9 , Figure 10 the single-chamber centrifugal sedimentation structure 51 shown by the dashed line in [Figure]. The optional shape of the single-chamber centrifugal sedimentation structure 51 is designed as a shape that can achieve uniform distribution of centrifugal force, such as a sector, an ellipse, a square, a circle, etc., and is preferably Figures 9 - 10 the inverted sector ring shown in [Figure], and this shape can achieve uniform distribution of centrifugal force. The single-chamber centrifugal sedimentation structure 51 can also be used as a chamber for incubating whole blood samples for 22 ± 4 hours when the sample to be tested is whole blood.

[0039] Since whole blood is mainly composed of plasma and erythrocytes, where plasma accounts for (50% - 55%) of the volume of whole blood, while erythrocytes only account for 45% of the volume of whole blood. The IGRA detection marker is γ-interferon (IFN-γ) secreted by immune cells, and the molecular weight of IFN-γ is relatively small, usually 17,100 daltons (Da). After centrifuging the whole blood sample, cells and larger particles will precipitate to the bottom, while IFN-γ with a smaller molecular weight will remain in the supernatant where the plasma layer is located. Detecting the centrifugally collected supernatant can effectively avoid the interference of erythrocytes in the whole blood sample and improve the accuracy of detecting the content of γ-interferon. Specifically, when the sample to be tested is whole blood, the sample to be tested is first incubated in the single-chamber centrifugal sedimentation structure 51 for 22 ± 4 hours, and then centrifuged after incubation. The mass densities of the components in whole blood are different. Under the action of the centrifugal field of rotation, as the rotation time increases, in the direction along the radius of the center of the circle, from low to high are plasma, white blood cells, and erythrocytes in turn. Therefore, a radial stack structure composed of pure plasma supernatant and erythrocyte particles at the bottom of the container will be formed in the chamber of the single-chamber centrifugal sedimentation structure 51 as Figure 9 shown.

[0040] Specifically, under the action of the centrifugal force brought by the servo motor, the radial stack-like stratification formed in the single-chamber centrifugal sedimentation structure 51 from the proximal end to the distal end is that the upper layer is the supernatant of pure plasma, and the lower layer is particles with a larger molecular weight such as white blood cells and erythrocytes. The stratification from the proximal end to the distal end is as Figure 9Shown in sequence are: a plasma layer 54, a white blood cell layer 55, and a red blood cell layer 56. Since the layered state between the plasma layer 54, the white blood cell layer 55, and the red blood cell layer 56 is no longer maintained after the servo motor stops rotating, the formed stack-like structure will also no longer exist. Therefore, the present invention uses a physical separation method to ensure the continuous maintenance of the layered state by adding a filter-like structure in the single-chamber centrifugal sedimentation structure 51. The filter-like structure preferably provided in the single-chamber centrifugal sedimentation structure in the present invention is a fence structure 25, and the pore diameter of the fence structure 25 is 7-10 microns. Therefore, the fence structure 25 allows red blood cells (6-8 microns) to pass through and prohibits white blood cells (10-20 microns) from passing through. The stepped filter channels are arranged equidistantly around the center of the circle by the fence structure 25, providing sufficient interaction space for the red blood cells to settle downward to the red blood cell enrichment chamber 26, and prompting the red blood cells to smoothly enter the red blood cell enrichment chamber 26.

[0041] When the sample to be tested enters the IGAR detection after the sample addition and incubation operations are completed on the chip, the sample to be tested stimulated by incubation is first preliminarily enriched in the single-chamber centrifugal sedimentation structure 51. That is, under the action of the centrifugal force generated by the high-speed rotation of the chip driven by the servo motor, the red blood cells will gradually be collected into the red blood cell enrichment chamber 26, and the plasma and white blood cells in the whole blood are enriched in the sample to be tested enrichment preparation chamber 23. Through the physical separation of the fence structure 25, different components in the whole blood sample are preliminarily layered and enriched, that is, the red blood cells settle to the red blood cell enrichment chamber 26, the white blood cells are intercepted in the layer where the fence structure 25 is located, and the uppermost layer is the plasma layer containing gamma interferon. At this time, the puncture valve 4 is in Figure 4 the closed state shown and will not release the sample in the single-chamber centrifugal sedimentation structure 51 into the next-level chamber through the plasma extraction inlet 24.

[0042] Among them, the plasma extraction inlet 24 is arranged at the central position where the sample to be tested enrichment preparation chamber 23 and the fence structure 25 meet. It can also be said that it is located at the impact interface between the preliminarily layered and enriched plasma layer and the red blood cells, that is, at the liquid level junction of the upper purified plasma and the lower red blood cells, and it is ensured that when the liquid level height in the single-chamber centrifugal sedimentation structure 51 is lower than the plasma extraction inlet 24, the liquid will not continue to be transferred through the plasma extraction inlet 24, that is, the liquid cannot flow in or out through the plasma extraction inlet 24. Regarding the volume, since the detection volume of the sample to be tested is preferably 1 mL, the volume of the single-chamber centrifugal sedimentation structure 51 should be not less than 1 mL. Among them, the main accommodation chambers in the single-chamber centrifugal sedimentation structure 51 include the sample to be tested enrichment preparation chamber 23 and the red blood cell enrichment chamber 26. The volume of the sample to be tested enrichment preparation chamber 23 is set to 650 ± 50 μL, and the volume of the red blood cell enrichment chamber 26 is set to 500 ± 50 μL; preferably, the volume of the sample to be tested enrichment preparation chamber 23 is 613 μL, and the volume of the red blood cell enrichment chamber 26 is 450 μL.

[0043] Figure 10 It is also shown that the plasma extraction inlet 24 on the single-chamber centrifugal sedimentation structure 51 and the center point of the lower puncture valve fixing stepped hole 47 are located on the same axis b-b of the chip. Figure 11 Specifically, a longitudinal sectional view of the axis b-b is shown. It can be seen that when the puncture valve 4 is in the open state, in a single sample detection unit 22 on the chip substrate 2, the flow direction of the liquid in the plasma transfer channel 48 is as Figure 10 indicated by the arrow in the figure. Figure 10 and Figure 11 Combined, it can be seen that the plasma transfer channel 48 is located at the bottom of the chip substrate 2. The plasma extraction inlet 24 and the puncture valve fixing stepped hole 47 are connected through the plasma transfer channel 48, and the liquid flows in from the plasma extraction inlet 24, passes through the plasma transfer channel 48, and then flows out from the puncture valve fixing stepped hole 47. Specifically, the puncture valve fixing stepped hole 47 is a stepped hole. Figure 10 A first stepped large hole is formed between the outermost circle and the middle circle of the puncture valve fixing stepped hole 47 in the figure as the fixing hole for installing the puncture valve 4, with a depth of 1-2 mm, preferably 1.5 mm; when the puncture valve 4 is in the open state, the first stepped large hole communicates with the annular flow channel 46, and the liquid flows out from the first stepped large hole of the puncture valve fixing stepped hole 47 and then flows in the annular flow channel 46; a second stepped middle hole extending downward is formed between the middle circle and the innermost circle as the reserved space for the residual aluminum foil after the aluminum foil sacrificial layer 10 of the puncture valve 4 is punctured, preventing the residual aluminum foil from blocking the liquid flow channel and ensuring the smooth flow of the liquid. The second stepped middle hole has a smaller diameter and shallower depth than the first stepped large hole, and is drilled and extended downward with the stepped surface of the first stepped large hole. The depth of the second stepped middle hole is 0.5-1.5 mm, preferably 1 mm; a third stepped small hole formed inside the innermost circle serves as the main liquid outflow hole and extends downward to penetrate through to the plasma transfer channel 48. The plasma transfer channel 48 is 1-3 mm long, the inner diameter of the plasma transfer channel 48 is 15-200 microns, and the plasma transfer channel 48 is 10-20 microns away from the top of the chip substrate 2, that is, the depth of the plasma transfer channel 48 is 10-20 microns. Since Figure 11 The open state of the puncture valve 4 is shown, that is, the aluminum foil sacrificial layer 10 in the middle of the puncture valve 4 has been damaged and penetrates through the puncture hole 21 on the top sealing plate 5 of the chip. Although the puncture valve 4 can be penetrated through with the puncture hole 21 when it is open, there is still the sealing protection of the airbag 7 installed in a sealed manner in the coaxial direction of the puncture hole 21 and the puncture valve 4 on the top sealing plate 5 of the chip. Therefore, when the puncture valve 4 is in the open state, the chip is still in a sealed environment.

[0044] Figure 8 Each sample detection unit 22 in the figure includes 9 chambers that can accommodate gas or liquid. In addition to the above Figures 9 - 10The two chambers, namely the preparation chamber 23 for enriching the sample to be tested and the erythrocyte enrichment chamber 26, in the single-chamber centrifugal sedimentation structure 51 shown also include a pneumatic buffer chamber 49 for buffering the air pressure in the single-chamber centrifugal sedimentation structure 51, two quantitative chambers, namely the plasma quantitative chamber 45 and the erythrocyte collection chamber 42, that form a concentric collinear interconnection structure 52, a waste liquid collection chamber 44 for collecting excess liquid, a diluent chamber 28 for diluting the sample to be tested, a desiccant chamber 32 for humidity adjustment, and a mixing chamber 35 for mixing the diluent and the purified plasma. Figure 8 It can be seen that the liquid of the sample to be tested passes through different chambers and various channels for gas-liquid interaction between different chambers, and finally the diluted plasma is fully mixed evenly in the mixing chamber 36 and then transferred to the chromatographic strip 3 at the position of the strip card slot 34 by siphon for IGRA detection.

[0045] Among them, the pneumatic buffer chamber 49 is connected to the single-chamber centrifugal sedimentation structure 51 through a pneumatic buffer chamber connection channel 50. The pneumatic buffer chamber 49 of the chip substrate 2 is opposite to the position of the pneumatic buffer hole 20 on the upper chip top seal plate 5. At the position of the pneumatic buffer hole 20, a buffer airbag fixing plate 8 is installed on the chip top seal plate 5, and the airbag 7 is further installed on the buffer airbag fixing plate 8. Therefore, in this vertical direction, from top to bottom in sequence are: the airbag 7, the buffer airbag fixing plate 8, the pneumatic buffer hole 20, and the pneumatic buffer chamber 49. Thus, the air pressure change in the single-chamber centrifugal sedimentation structure 51 can be transmitted to the pneumatic buffer chamber 49 through the pneumatic buffer chamber connection channel 50. The air pressure received in the pneumatic buffer chamber 49 can also be transmitted to the upper layer of the chip to the airbag 7, and finally buffered by the airbag 7 made of an elastic material. Regarding the flow of gas, each sample detection unit 22 also has a separate circulation channel for ensuring the free flow of air, namely the internal air circulation channel 27. Figure 8 It can be seen that the chambers directly connected in sequence from left to right by the internal air circulation channel 27 include the diluent chamber 28, the mixing chamber 35, the two quantitative chambers, namely the plasma quantitative chamber 45 and the erythrocyte collection chamber 42, in the concentric collinear interconnection structure 52, and the waste liquid collection chamber 44. Since the flow of liquid between each chamber is not in a vacuum state, it is necessary for the existing gas in the already constructed closed space to circulate continuously to assist in driving the flow of liquid. Therefore, the internal air circulation channel 27 ensures the gas-liquid replacement in the closed space where the chip is located.

[0046] Figure 12 It is a partial enlarged view of the concentric collinear interconnection structure 52 in a single sample detection unit 22 on the chip substrate 2. Combining Figure 8 、 Figure 11 and Figure 12 it can be seen that: Since Figure 12The concentric - center - collinear interconnection structure 52 shown by the dashed line in the figure includes two chambers, namely a plasma quantification chamber 45 and a red blood cell collection chamber 42, and three stepped channels that connect the two chambers: a three - branch left stepped channel 40, a three - branch middle liquid - inlet stepped channel 41, and a three - branch right exhaust stepped channel 43. Since the centers of the two chambers are located on the same axis line, and the three stepped channels are located in the middle of the two chambers and connect them, the structure composed of the two chambers and the three stepped channels is called the concentric - center - collinear interconnection structure 52. Among them, the three stepped channels are also called "three - branch stepped channels" or "concentric - center - collinear interconnection stepped channels". The synergistic effect of the three stepped channels can provide sufficient interaction space for the formation and layered transfer of the interface between plasma and red blood cells. The concentric - center - collinear interconnection structure 52 is connected to the mixing chamber 35 through a plasma siphon channel 36. Among them, the inlet of the plasma siphon channel 36 communicates with the three - branch left stepped channel 40 in the concentric - center - collinear interconnection structure 52, which is conducive to extracting the pure plasma sample quantitatively collected in the plasma quantification chamber 45 into the mixing chamber 35 through the siphon effect of the plasma siphon channel 36. Since the inlet of the plasma siphon channel 36 is located below the plasma quantification chamber 45, the siphon effect only collects and transfers the plasma in the upper plasma quantification chamber 45, and can also avoid collecting the blood sample containing red blood cells in the lower red blood cell collection chamber 42.

[0047] Regarding the concentric - center - collinear interconnection structure 52, specifically: Among the two chambers, the volume of the plasma quantification chamber 45 is 100 - 300 μL, and the volume of the red blood cell collection chamber 42 is 100 - 400 μL. The specific volume can be customized according to the requirements of the IGRA reaction system. Among them, the volume of the plasma quantification chamber 45 is preferably 100 μL, and the volume of the red blood cell collection chamber 42 is preferably 100 μL; there are size differences among the three stepped channels. The diameters of the two side channels, namely the three - branch left stepped channel 40 and the three - branch right exhaust stepped channel 43, are 0.2 - 1 mm, and the diameter of the middle three - branch middle liquid - inlet stepped channel 41 is slightly larger than that of the side channels. The selectable diameter of the channel is 0.5 - 2 mm. In particular, it is necessary to ensure that the diameter of the three - branch right exhaust stepped channel 43 is significantly smaller than that of the three - branch middle liquid - inlet stepped channel 41. The adjacent three - branch middle liquid - inlet stepped channel 41 and three - branch right exhaust stepped channel 43 form such an obvious difference in channel diameters, which can reduce the resistance of the liquid flowing through the three - branch middle liquid - inlet stepped channel 41. For example, agglomerated red blood cells increase the viscosity of the liquid, and the wider - diameter three - branch middle liquid - inlet stepped channel 41 can ensure that the red - blood - cell - containing liquid with different viscosities preferentially flows through the three - branch middle liquid - inlet stepped channel 41 and into the red blood cell collection chamber 42.

[0048] The specific diameter of the channels can be custom-designed according to the specific requirements of the IGRA reaction system. Among them, the diameter difference between the three-way left stepped channel 40 and the three-way right exhaust stepped channel is not significant, and the same 0.4 mm can be preferably selected. The preferred diameter of the three-way middle liquid inlet stepped channel 41 is 1 mm. Since the plasma siphon channel 36 communicates with the three-way left stepped channel 40, the diameter difference between the plasma siphon channel 36 and the three-way left stepped channel 40 is also not significant. The diameter of the plasma siphon channel 36 is 0.2 - 1 mm, preferably 0.4 mm. In particular, after the inlet of the plasma siphon channel 36 communicates with the three-way left stepped channel 40, a certain angle is formed, that is, the angle range of the elevation angle θ formed by the plasma siphon channel 36 and the perpendicular line of the three-way left stepped channel 40 is 10° to 45°. Within this angle range, combined with the adjustment of the length of the plasma siphon channel 36, it is ensured that when the servo motor drives the chip to rotate, the centrifugal forces generated on the transfer siphon channel 36 and the plasma metering chamber 45 at the same centrifugal radius are the same, that is, the liquid levels of the plasma in the transfer siphon channel 36 and the plasma metering chamber 45 are maintained on the same centrifugal radius. Even if some red blood cell particles 57 accidentally enter and stay in the plasma siphon channel 36 in advance, within the angle range of the elevation angle θ, the red blood cell particles 57 in the plasma siphon channel 36 will also receive the action of the centrifugal force component 59 along the far center direction of the centrifugal force 58. Under the action of this centrifugal force component 59, it can promote the red blood cell particles 57 therein to settle into the red blood cell collection chamber 42, thereby not only avoiding the blockage caused by the accumulation of red blood cell particles 57 in the plasma siphon channel 36, but also avoiding the transfer of red blood cell particles 57 into the mixing chamber 35 through the plasma siphon channel 36.

[0049] Therefore, the design of the three-way middle liquid inlet stepped channel 41 with a larger diameter and smaller resistance, in coordination with the angular design of maintaining a certain elevation angle θ between the plasma siphon channel 36 and the three-way left stepped channel 40, can not only ensure that the liquid of red blood cells preferentially flows into the red blood cell collection chamber 42 through the three-way middle liquid inlet stepped channel 41 smoothly, but also ensure that the red blood cell particles 57 that accidentally enter the plasma siphon channel 36 in advance flow into the red blood cell collection chamber 42 smoothly under the action of the external centrifugal force component 59. In addition, it can more effectively ensure that the gas generated during the liquid flow process can be discharged smoothly through the three-way right exhaust stepped channel 43 with a smaller channel diameter, ensuring free gas-liquid replacement of the liquid and gas in the concentric collinear interconnection structure 52. The concentric collinear interconnection structure 52 can also control the angular velocity direction and magnitude of the servo motor, and then control the flow directions of different stratified blood samples according to their respective mass characteristics and the inertial forces they are subjected to. It can be seen that the mutual cooperation among the three stepped channels in the concentric collinear interconnection structure 52 not only ensures the smooth connection of the liquid between the two chambers, but also the difference in diameters among the three stepped channels and the angular design of the elevation angle θ between the plasma siphon channel 36 and the three-way left stepped channel 40 enable each of the three stepped channels to undertake specific functions.

[0050] Regarding the waste liquid collection chamber 44. Since the single-chamber centrifugal sedimentation structure 51 and the concentric collinear interconnection structure 52 of the present invention are connected through the plasma transfer channel 48 and the annular flow channel 46 on the chip substrate 2. Among them, the plasma extraction inlet 24 of the single-chamber centrifugal sedimentation structure 51 is connected to the puncture valve fixed stepped hole 47 through the plasma transfer channel 48. Also, because the position of the puncture valve fixed stepped hole 47 above the plasma transfer channel 48 can be connected to the annular flow channel 46, and there is an arc-shaped drop channel away from the center direction at the connection between the puncture valve fixed stepped hole 47 and the annular flow channel 46, the distance between the annular main body of the annular flow channel 46 and the center (i.e., the radius of the annular flow channel 46) is greater than the distance between the puncture valve fixed stepped hole 47 and the center. Therefore, under the action of centrifugal force, the liquid in the single-chamber centrifugal sedimentation structure 51 can first flow from the plasma extraction inlet 24 through the plasma transfer channel 48 to the position of the puncture valve fixed stepped hole 47, as Figure 12As shown, after the puncture valve 4 is damaged by an external force and is in an open state, after the liquid flows out of the puncture valve fixed stepped hole 47 through the plasma transfer channel 48, it can further start from the puncture valve fixed stepped hole 47 and flow through the annular flow channel 46 communicating with the puncture valve fixed stepped hole 47 to the plasma quantitative chamber 45 and the red blood cell collection chamber 42 of the concentric collinear interconnection structure 52. Among them, the red blood cell collection chamber 42 is a blind hole structure. In the preliminary experiment, only 2 stepped channels were designed (equivalent to the middle liquid inlet stepped channel 41 and the right exhaust stepped channel 43 of the three-way, and the inlet of the plasma siphon channel 36 is directly communicated with the middle liquid inlet stepped channel 41 of the three-way). As a result, it was found through a stroboscope in the chip model that the gas-liquid replacement was not smooth and there was a jamming phenomenon; through optimization, the Figure 12 gas-liquid replacement channel structure with 3 stepped channels shown in the present invention can avoid the phenomenon of liquid flowing in and blocking the exhaust channel, improve the gas-liquid replacement efficiency of the red blood cell collection chamber 42, and ensure the smooth inflow of liquid and the free discharge of gas.

[0051] The position of the annular flow channel 46 is set above the plasma quantitative chamber 45 and the waste liquid collection chamber 44, and these two chambers are connected from above. And because the volumes of both the plasma quantitative chamber 45 and the red blood cell collection chamber 42 are quantitatively set, for example, the volumes are preferably both 100 μL. When the liquid flowing into the concentric collinear interconnection structure 52 from the single-chamber centrifugal sedimentation structure 51 exceeds a certain range of the volumes of the two chambers and the liquid level of the excess liquid is higher than the annular flow channel 46, it can flow into the waste liquid collection chamber 44 through the annular flow channel 46, so as to ensure that the plasma quantitative chamber 45 can be filled with the required quantitative volume (such as preferably 100 μL) of pure plasma. It can be seen that the waste liquid collection chamber 44 is mainly due to the specific setting of the annular flow channel 46 and the quantitative setting of the two chambers in the concentric collinear interconnection structure 52. From another perspective, setting the waste liquid collection chamber 44 and the annular flow channel 46 can ensure that the plasma quantitative chamber 45 in the single-chamber centrifugal sedimentation structure 51 can quantitatively retain the pure plasma solution.

[0052] Regarding the diluent chamber 28, since the plasma retained in the plasma quantification chamber 45 is pure plasma, generally the viscosity of this plasma is relatively high. If it is directly transferred to the chromatography strip 3, it is not only difficult to flow smoothly, but also may cause false negative test results due to the "hook effect" caused by high concentration, making it difficult to ensure the accuracy of the IGRA test using the chromatography strip 3. Therefore, the diluent can be used to dilute the plasma with relatively high viscosity to reduce the viscosity of the plasma to be tested and ensure its smooth flow and migration on the chromatography strip 3. Generally, a serial dilution of the plasma concentration can be performed, that is, diluted by two times. If the quantification volume of the plasma quantification chamber 45 is preferably 100 μL, the volume of the diluent chamber 28 can be correspondingly set to not less than 100 μL; the diluent chamber 28 is connected to the mixing chamber 35 through the diluent transfer siphon channel 29, and the diluent can be guided from the diluent chamber 28 to the mixing chamber 35 by siphon action. In addition, in order to prevent the diluent from filling the diluent transfer siphon channel 29 due to capillary action at the initial stage of the detection, a first capillary valve 30 is provided at a certain part of the diluent transfer siphon channel 29. The function of this first capillary valve 30 is to provide a certain resistance. Since the total energy will increase when the liquid expands at the solid-liquid interface, it can make the gradually increasing energy of the flowing liquid gradually overcome the resistance obstacle of the first capillary valve 30 during the expansion process. The diluent transfer siphon channel 29 needs to overcome the resistance of the capillary valve during operation. For example, when the servo motor drives the chip of the present invention to rotate, when a certain critical rotational speed is reached, the diluent can be transferred to the mixing chamber. For example, when centrifuging and preliminarily enriching the sample to be tested, under the action of centrifugal force, the diluent can flow through the diluent transfer siphon channel 29 and break through the resistance of the first capillary valve 30, and then gradually be transferred to the mixing chamber 35 under siphon action.

[0053] Regarding the desiccant chamber 32, a solid desiccant is stored in the desiccant chamber 32, which can absorb and remove gaseous or liquid water molecules, and is used to reduce the humidity in the chamber environment connected to the desiccant chamber 32. One end of the desiccant chamber 32 is connected to the mixing chamber 35 through the first desiccant chamber connection channel 31, and the other end of the desiccant chamber 32 is connected to the chamber where the strip card slot 34 is located through the second desiccant chamber connection channel 33. Therefore, the desiccant chamber 32 can be used to maintain the humidity in the two closed chamber environments of the mixing chamber 35 and the strip card slot 34 where the chromatography strip 3 is located. In particular, in the single-chamber centrifugal sedimentation structure 51 of the chip of the present invention, the whole blood sample to be tested will undergo an incubation process at 37°C, and water mist will be formed in the entire closed chamber during the incubation process. In particular, water droplets will accumulate on the inner surface of the pressure-sensitive encapsulation adhesive 6 of the chip top sealing plate 5 above the chamber where the strip card slot 34 is located. If there are too many water droplets, they will drip onto the interference chromatography strip 3, thereby affecting the intensity of the fluorescence signals on the test line and the quality control line, and thus affecting the accuracy of the detection. Therefore, by providing a desiccant chamber 32 communicating with the two chambers of the mixing chamber 35 and the strip card slot 34 in the last two steps of liquid flow to mainly absorb the moisture in these two closed chambers, the best reaction humidity condition in the final IGRA detection environment can be ensured, thereby improving the accuracy of the IGRA detection.

[0054] Regarding the mixing chamber 35, the mixing chamber 35 is a chamber where the plasma of the sample to be tested and the diluent are mixed to obtain diluted plasma. For plasma with a relatively high viscosity, magnetic beads 53 can be added as a dynamic "stirrer" in the mixing chamber 35. It will not only be affected by the centrifugal force and the Euler force generated by the acceleration and deceleration of the servo motor, but also be affected by the Coriolis force generated by the movement under the action of the centrifugal force. Therefore, the plasma and the diluent in the mixing chamber 35 are preferably fully contacted and mixed under the combined action of the magnetic beads and inertial forces such as centrifugal force, Euler force, and Coriolis force. Among them, the centrifugal force is an inertial force introduced in a rotating reference frame, "simulating" the tendency of an object to fly out tangentially due to inertia, its direction is along the radial direction away from the axis of rotation, and its magnitude depends on the angular velocity, the mass of the object, and its distance from the axis of rotation; the Euler force is an inertial force generated in a non-uniformly rotating (the angular acceleration of rotation is not zero) reference frame, and its direction is perpendicular to the plane determined by the angular acceleration and the position vector of the object (that is, a deflecting force perpendicular to the radial direction during the uniform acceleration process), and its magnitude depends on the angular acceleration, the mass of the object, and its distance from the axis of rotation; the Coriolis force is an inertial force that an object is subjected to due to linear motion in a rotating reference frame, and its direction is perpendicular to the plane determined by the angular velocity vector and the relative velocity vector of the object, and its magnitude depends on the angular velocity, the mass of the object, and its relative velocity. In addition, other mixing methods that are beneficial to the mixing of plasma and diluent can also be selected in the mixing chamber 35, such as ultrasonic, mechanical stirring, vortex oscillation and other mixing methods.

[0055] Figure 13 The schematic diagram of the design of the mixing chamber 35 for transferring the diluted plasma obtained after mixing is shown in FIG. Figure 14 This is a partial enlarged view of the mixing chamber structure. Figure 13 and Figure 14 The shape of the mixing chamber 35 is a variant of the fan-shaped ring shown in the single-chamber centrifugal sedimentation structure 51, called an "asymmetric double-arc trapezoid": unlike the fan-shaped ring, which has parallel concentric arcs at the upper and lower ends, the upper and lower ends of the "asymmetric double-arc trapezoid" mixing chamber 35 are not absolutely parallel arcs, and the two sides are wide on one side and narrow on the other side, and they gradually narrow from wide to narrow. Therefore, under the centrifugal force, the liquid flowing into the mixing chamber 35 will theoretically have radial stacking layers similar to those formed in the single-chamber centrifugal sedimentation structure 51. The difference is that due to the mixing The only liquid outlet of the chamber 35 is located at the lower end of the relatively narrow side of the "asymmetric double-arc trapezoid". The mixing chamber 35 is connected to the mixed liquid siphon channel 37 through the liquid outlet position. The arc formed by the lower edge of the liquid outlet position and the upper end of the "asymmetric double-arc trapezoid" can form a concentric arc. Therefore, when the liquid continues to flow into the mixing chamber 35, the liquid level in the mixing chamber 35 gradually rises. When the liquid level is lower than the liquid outlet position, the stacking layers formed by the inflowing liquid are concentrated on the wider side of the bottom of the mixing chamber 35, that is, gradually filling the mixing chamber 35. Figure 12 As shown in the figure, the lower cavity of the siphon valve inlet is at the bottom cavity of the siphon valve inlet; when the liquid level gradually rises to or reaches the lower edge of the liquid outlet position, the liquid at the connection point between the liquid outlet of the mixing chamber 35 and the mixed liquid siphon channel 37 is affected by gravity and flows to the mixed liquid siphon channel 37. As the liquid flows to the mixed liquid siphon channel 37, the liquid outlet position is filled with liquid and a negative pressure is generated. Under the action of atmospheric pressure, the liquid in the mixing chamber 35 continues to replenish and flow to the mixed liquid siphon channel 37, and flows into the chromatography paper strip 3 placed on the paper strip slot 34 through the mixed liquid siphon channel 37. As long as the liquid in the mixing chamber 35 is located at Figure 13 When the liquid in the mixing chamber 35 is at the position of the upper chamber 61 of the siphon valve inlet, the liquid will continue to flow through the liquid outlet until all the liquid in the upper chamber 61 of the siphon valve inlet is transferred out by the siphon principle. When the liquid in the mixing chamber 35 is only retained to the volume of the lower chamber 60 of the siphon valve inlet, that is, when the liquid level drops below the liquid outlet position of the mixing chamber 35, the siphon phenomenon is destroyed and the siphon flow of the liquid will also stop. The arc between the liquid stack layers in the mixing chamber 35 is the theoretical stratification line of the two virtual chambers, the lower chamber 60 of the siphon valve inlet and the upper chamber 61 of the siphon valve inlet.

[0056] Regarding the inlet design of the mixing chamber 35, the inlets for gas-liquid interaction with the mixing chamber 35 are all located at the upper end of the mixing chamber 35, including: the inlet for transferring diluent into the mixing chamber 35 from the diluent chamber 28 through the diluent transfer siphon channel 29; the inlet for transferring the quantified plasma into the mixing chamber 35 from the plasma quantification chamber 45 of the concentric collinear interconnection structure 52 through the plasma siphon channel 36; the inlet for communicating with the desiccant chamber 32 through the second connection channel 33 of the desiccant chamber and capable of maintaining the humidity of the mixing chamber 35; and the inlet for the internal air circulation channel 27 that closes the free circulation of air inside the chip. Figure 8 It can be seen that the inlets located at the upper end of the mixing chamber 35 are arranged in sequence from left to right as the second connection channel 33 of the desiccant chamber, the diluent transfer siphon channel 29, the internal air circulation channel 27, and the inlet of the plasma siphon channel 36.

[0057] It can be seen that after the plasma and diluent flowing into the mixing chamber 35 are mixed, the mixed solution is quantitatively transferred to the chromatography strip 3 for IGRA detection by using the siphon principle. Among them, regarding the volume of the mixing chamber 35, if the customized volume of the plasma quantification chamber 45 is 100 μL, the volume of the corresponding diluent chamber 28 is generally customized to be 100 μL, and the theoretical total volume of the diluted plasma finally flowing into the mixing chamber 35 and obtained by mixing is 200 μL. Since only the liquid level of the fan-shaped annular liquid in the upper chamber 61 of the siphon valve inlet is higher than the arc (or stratification line), it can flow out through the mixed solution siphon channel 37. For example, the customized volume of the lower chamber 60 of the siphon valve inlet is preferably 130 μL, and the remaining liquid in the upper chamber 61 of the siphon valve inlet is about 70 μL, and then it will be siphonically transferred in a roughly quantified manner (70 μL). It can be seen that through the optimized setting of the position of the mixed solution siphon channel 37 within the specific shape of the mixing chamber 35, the quantitative transfer of the plasma after successive purification and dilution to the chromatography strip 3 is realized to complete the IGRA detection.

[0058] For the same reason as the first capillary valve 30 is provided in the diluent transfer siphon channel 29, in addition to being connected through the mixed liquid siphon channel 37 between the mixing chamber 35 and the strip card slot 34, in order to prevent the mixed liquid of the diluted plasma from filling the mixed liquid siphon channel 37 due to capillary action and entering the strip card slot 34 in advance at the initial stage of detection, a second capillary valve 38 and a mixed liquid serpentine channel 39 are sequentially provided at the end of the mixed liquid siphon channel 37, that is, the second capillary valve 38 is provided at the end of the mixed liquid siphon channel 37, and one end of the mixed liquid serpentine channel 39 is connected to the end of the second capillary valve 38 and the other end is connected to the strip card slot 34. Therefore, the mixing chamber 35 and the strip card slot 34 are connected through the mixed liquid siphon channel 37, the second capillary valve 38 and the mixed liquid serpentine channel 39. Specifically, when the mixed liquid in the mixing chamber 35 fills the mixed liquid siphon channel 37, the liquid cannot continue to flow by siphon due to the resistance of the second capillary valve 38. However, when the servo motor rotates at a high speed, the mixed liquid filling the mixed liquid siphon channel 37 will break through the resistance of the second capillary valve 38 and continue to fill the mixed liquid serpentine channel 39 due to capillary action, and enter the sampling area on the chromatography strip 3 in the space where the strip card slot 34 is located, and then flow smoothly on the chromatography strip 3 and chromatographically diffuse to the detection T line and C line. Finally, the detection results of the mixed liquid at the T line and C line are observed and recorded from the sample detection unit 22.

[0059] Example 2 Assembly of the Centrifugal Microfluidic Detection Chip for Tuberculosis-Specific Cellular Immunity Since the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention mainly consists of three layers of structures: the chip bottom plate sealing plate 1, the chip substrate 2, and the chip top sealing plate 5 from bottom to top, the assembly is carried out sequentially from bottom to top, as Figures 1 - 2 shown in the exploded view. Since there are chip positioning structures 12 with the same shape and pin positioning holes 14 (preferably 3) at corresponding positions on the three layers of structures, the chip positioning structures 12 and pin positioning holes 14 on the three layers of structures are used for alignment and assembly in sequence. In order to ensure the accuracy of the IGRA detection, the assembly work needs to be completed in a clean environment. The specific assembly process is as follows: First step: Preparation work. Check whether the dimensional accuracy of the three layers of structures of the detection chip bottom plate sealing plate 1, the chip substrate 2, and the chip top sealing plate 5, the chip positioning structures 12, the pin positioning holes 14, etc. are matched, and check whether other accessories, reagents or materials to be installed are complete, clean and usable; Second step: Assembly of the chip bottom plate sealing plate 1. Preferably, a positioning fixing pin (the outer diameter of the pin part is 1 mm and the length is 8 mm) is used for locking and fixing. The positioning fixing pin is inserted from bottom to top through the pin positioning hole 14 on the chip bottom plate sealing plate 1, and it can be seen that the positioning fixing pin has a protruding positioning column (about 7 mm long); Step 3: Assembly of the chip substrate 2. After applying adhesive at spaced points on the chip base sealing plate 1, align the chip substrate 2 with the exposed positioning posts on the chip base sealing plate 1 and insert it through the pin positioning holes 14 on the chip substrate 2 and then lock it in place. Then, complete the encapsulation of the chip base sealing plate 1 and the chip substrate 2 by using adhesive glue. Fix the chromatographic paper strip 3 and the puncture valve 4 at the positions of the paper strip card slots 34 and the puncture holes 21 of each sample detection unit 22 of the chip substrate 2 respectively, and place the magnetic beads 53 into the mixing chamber 35. Load desiccant into the desiccant chamber 32. Pre-install specific liquids as negative control (N), positive control (P), and sample control (T) respectively in the sample enrichment preparation chamber 23 of the single-chamber centrifugal sedimentation structure 51. If only the matrix liquid is pre-installed in the sample enrichment preparation chamber 23, the corresponding sample detection unit is marked as negative control (N). If both the matrix liquid and the tuberculosis non-specific stimulating antigen (such as phytohemagglutinin PHA) are pre-installed in the sample enrichment preparation chamber 23, the corresponding sample detection unit is marked as positive control (P). If both the matrix liquid and the Mycobacterium tuberculosis specific antigen (such as ESAT-6 / CFP-10) are pre-installed in the sample enrichment preparation chamber 23, the corresponding sample detection unit is marked as sample control (T). Step 4: Assembly of the chip top sealing plate 5. After applying adhesive at a position near the pin positioning holes 14 on the chip substrate 2, align the chip top sealing plate 5 with the still-exposed positioning posts (about 1 mm long) on the chip substrate 2, insert it through the pin positioning holes 14 on the chip top sealing plate 5 and then lock it in place. Then, complete the encapsulation between the chip substrate 2 and the chip top sealing plate 5 by using adhesive glue. At the central axis position of the puncture hole 21, assemble the puncture airbag fixing plate 9 and the airbag 7 from bottom to top in sequence. At the central axis position of the air pressure buffer hole 20, assemble the buffer airbag fixing plate 8 and the airbag 7 from bottom to top in sequence. At the opening position of the paper strip detection window 18 on the chip top sealing plate 5, use a polypropylene film with a thickness of 0.1 mm as the pressure-sensitive encapsulation adhesive 6 for sealing.

[0060] So far, the entire assembly operation of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity is completed. Since the chip is pre-loaded with matrix liquid, tuberculosis non-specific stimulating antigen, Mycobacterium tuberculosis specific antigen, etc., the assembled and sealed centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity is stored at 2 - 8 °C for future use.

[0061] During the above assembly process, the three-layer structures of the chip base sealing plate 1, the chip substrate 2, and the chip top sealing plate 5 are not only locked and fixed through the positioning posts, but also sealed and assembled by using adhesive glue. In addition, in addition to the above-mentioned adhesive sealing, it can also be replaced with sealing methods such as thermal pressing sealing, laser welding, or ultrasonic welding to seal and connect the layers in sequence to further ensure the tightness of the chip.

[0062] Application of centrifugal microfluidic detection chip IGRA for tubercle-specific cellular immunity 1. Detection process of centrifugal microfluidic detection chip IGRA for tubercle-specific cellular immunity First step, detection preparation. Take Figures 1 - 2 the assembled centrifugal microfluidic detection chip for tubercle-specific cellular immunity with 3 detection units as shown. The 3 detection units are used as negative control (N), positive control (P) and sample control (T) respectively. The chip is equilibrated at room temperature for at least 20 minutes; Second step, sample addition and sealing. Take the sample to be tested and the diluent, and use a pipette to inject the sample to be tested in the form of whole blood and the diluent into the corresponding single-chamber centrifugal sedimentation structure 51 and diluent chamber 28 respectively from the sample addition hole 13 and diluent chamber injection hole 15 of each detection unit to complete the liquid addition operation. After liquid addition, preferably use polyester film as the seal to seal the 4 holes of the sample addition hole 13, exhaust hole 19, diluent chamber injection hole 15 and diluent chamber exhaust hole 16 respectively; Third step, incubation. Place the chip in a constant temperature environment of 37°C and incubate for 22 ± 4 hours. During the incubation process, the temperature will cause a change in the air pressure in the single-chamber centrifugal sedimentation structure 51. The single-chamber centrifugal sedimentation structure 51 is connected to the airbag 7 through the air pressure buffer chamber connection channel 50, and the elastic change of the airbag 7 is used to relieve the drastic change of air pressure; Fourth step, IGRA detection. Place the chip stably on the corresponding position of the upper flange block of the servo motor through the chip positioning structure 12, and then the subsequent operations of separation, reagent mixing and IGRA strip detection can be carried out, and the detection result can be observed through the strip detection window 18 on the top sealing plate 5 of the chip. Observe the fluorescence signals of the detection line and the quality control line on the chromatography strip 3 through the strip detection window 18. The fluorescence signals can be qualitatively determined by the naked eye, or quantitatively detected by a fluorescence scanner and the quantitative concentration of interferon-γ stimulated in the sample to be tested can be calculated. Specifically, after the operations of sample addition and incubation are completed on the chip, the specific process of specific IGAR detection is as follows: For the first centrifugation, after incubation stimulation, the sample to be tested undergoes preliminary centrifugal layering enrichment in the single-chamber centrifugal sedimentation structure 51 through the first centrifugation. The servo motor accelerates to a high-speed rotation state (2500 to 3000 rpm, preferably 3000 rpm) at an acceleration of 500 rpm / s and drives the chip to rotate for 2 to 6 minutes in the high-speed rotation state. When the servo motor reaches the set rotation time (preferably 3 minutes), it decelerates to a stationary state at a deceleration of 500 rpm / s; during this centrifugation process, the plasma and white blood cells in the whole blood are enriched in the sample enrichment preparation chamber 23 to be tested. Through the physical separation of the fence structure 25, the plasma layer containing interferon-γ is at the top layer of the sample enrichment preparation chamber 23 to be tested, the white blood cells are intercepted in the layer where the fence structure 25 is located, and the red blood cells settle to the red blood cell enrichment chamber 26; simultaneously with the first centrifugation, the diluent fills and wets the diluent transfer siphon channel 29.

[0063] Open the puncture valve 4. Apply a vertically downward force to the airbag 7 in the coaxial direction of the puncture hole 21 and the puncture valve 4 until the aluminum foil sacrificial layer 10 on the puncture valve 4 is punctured, so that the puncture valve 4 is in Figure 11 the open state shown.

[0064] Second centrifugation: The servo motor accelerates to a high-speed rotation state (2500 to 3000 rpm, preferably 2500 rpm) at an acceleration of 100 rpm / s and drives the chip to rotate for 2 to 6 minutes in the high-speed rotation state. When the servo motor reaches the set rotation time (preferably 3 minutes), it decelerates to a stationary state at a deceleration of 500 rpm / s; during this centrifugation process, the diluent in the diluent storage chamber 28 breaks through the resistance of the first capillary valve 30 and, under the combined action of siphon and centrifugal force, enters the position of the lower chamber 60 of the siphon valve inlet of the mixing chamber 35 along the diluent transfer siphon channel 29; during the second centrifugation, since the puncture valve 4 is in the open state, the plasma initially stratified and enriched in the plasma extraction inlet 24 of the single-chamber centrifugal sedimentation structure 51 from the sample to be tested in the enrichment preparation chamber 23 flows into the plasma transfer channel 48, then flows out through the puncture valve fixed stepped hole 47 to the annular flow channel 46, and then flows into the plasma quantitative chamber 45 and the red blood cell collection chamber 42 of the concentric collinear interconnection structure 52 through the annular flow channel 46. Under the synergistic action of centrifugal force and Euler force, the remaining red blood cells in the plasma that has been enriched for the first time from the sample to be tested in the enrichment preparation chamber 23 will enter the red blood cell collection chamber 42 through the three-way middle inlet stepped channel 41. The gas in the red blood cell collection chamber 42 will be discharged along the three-way right exhaust stepped channel 43. As the plasma in the sample to be tested in the enrichment preparation chamber 23 continues to flow in and undergoes centrifugal stratification, the remaining red blood cells are secondarily enriched to the lower layer of the red blood cell collection chamber 42. After the red blood cell collection chamber 42 is filled, relatively pure plasma continuously fills the plasma quantitative chamber 45. After the plasma quantitative chamber 45 completes the quantitative function of 100 μL, the excess plasma liquid enters the waste liquid collection chamber 44 along the annular flow channel 46; among them, although there are individual red blood cells that enter the siphon transfer channel 36 connected to the three-way left stepped channel 40 in advance during the process of flowing into the red blood cell collection chamber 42, however, since there is a certain elevation angle θ between the siphon transfer channel 36 and the perpendicular line of the three-way left stepped channel 40, this elevation angle θ enables the red blood cell particles 57 that enter the siphon transfer channel 36 in advance to move at a uniform speed under the action of the centrifugal force component 59 in the far-center direction of the centrifugal force 58 until finally all the red blood cells will settle in the red blood cell collection chamber 42. Therefore, the plasma preliminarily enriched from the sample to be tested in the enrichment preparation chamber 23 completes the filtration of the second-stage red blood cells and the enrichment of pure plasma in the concentric collinear interconnection structure 52, and the pure plasma enriched in the second stage is quantitatively stored in the plasma quantitative chamber 45. After the second centrifugation stops, the pure plasma in the plasma quantitative chamber 45 fills the siphon transfer channel 36, and when the siphon transfer channel 36 is filled with plasma, the third centrifugation is carried out.

[0065] For the third centrifugation, the servo motor accelerates to a high-speed rotation state (1000 to 2000 rpm, preferably 1500 rpm) at an acceleration of 300 rpm / s and drives the chip to rotate for 1 to 5 minutes in the high-speed rotation state. When the servo motor reaches the set rotation time (preferably 2 minutes), it decelerates to a stationary state at a deceleration of 100 rpm / s; under the action of the third centrifugation, 100 μL of pure plasma quantitatively enriched in the plasma quantification chamber 45 is transferred to the mixing chamber 35 through the siphon transfer channel 36 and is fully mixed with 100 μL of diluent flowing in under the action of the magnetic beads. When the mixed system of 100 μL of pure plasma and 100 μL of diluent coexists at the positions of the lower chamber 60 and the upper chamber 61 of the siphon valve inlet in the mixing chamber 35, the fourth centrifugation is carried out.

[0066] For the fourth centrifugation mixing, the acceleration and deceleration of the servo motor are both set to 100 rpm / s, the high-speed rotation speed is 1000 to 2000 rpm, preferably 1500 rpm; the high-speed rotation time is 0.5 to 3 minutes, preferably 1 minute; the fourth centrifugation is a repeated acceleration and deceleration operation, and each completion of an acceleration and deceleration cycle is regarded as a mixing process. In order to ensure sufficient and uniform mixing, 5 - 15 mixing processes need to be completed, preferably 10 mixing processes; this centrifugation mixing operation utilizes the Euler force generated during acceleration and deceleration in the centrifugation process to shorten the mixing time and improve the mixing efficiency; in addition, due to the presence of magnetic beads 53 in the mixing chamber 35, the magnetic beads 53 act as a dynamic "stirrer" and continuously move under the influence of centrifugal force and other inertial forces, and this movement enhances the contact and diffusion between liquids, improving the mixing efficiency and uniformity; it can be seen that the pure plasma and diluent can further effectively stir and mix the mixed liquid in the mixing chamber 35 through the synergistic effect of the repeated centrifugation mixing process and the dynamic stirring of the magnetic beads 53 in the mixing chamber 35. After the last centrifugation mixing process is completed, it decelerates to a stationary state at a deceleration of 300 rpm / s. After the centrifugation stops, the mixed liquid (i.e., diluted plasma) fills the mixed liquid siphon channel 37. When the mixed liquid siphon channel 37 is filled with the diluted plasma, the fifth centrifugation is carried out.

[0067] For the fifth centrifugation, the servo motor accelerates to a high-speed rotation state at an acceleration of 300 rpm / s (2500 to 3500 rpm, preferably 3000 rpm), and drives the chip to rotate for 1 to 4 minutes in the high-speed rotation state. When the servo motor reaches the set rotation time (preferably 2 minutes), it decelerates to a stationary state; 70 μL of diluted plasma at the position of the upper chamber 61 of the siphon valve inlet in the mixing chamber 35 is transferred to the sample application hole position of the sample pad on the chromatography strip 3 fixedly installed on the strip card slot 34. The absorbent pad at the other end of the sample pad on the chromatography strip 3 promotes the fluorescence signal shown after the diluted plasma flows on the chromatography strip 3 to be observed or quantitatively detected. Among them, the diluted plasma at the position of the lower chamber 60 of the siphon valve inlet will not be transferred because its liquid level is lower than the lower edge of the liquid outlet of the mixing chamber 35 (the inlet of the mixed liquid siphon channel 37).

[0068] 2. Data analysis of the centrifugal microfluidic detection chip IGRA for the detection of tuberculous specific cellular immunity The intensity values of the fluorescence signals corresponding to the test line (T line) and the quality control line (C line) on the chromatography strip 3 of the detection units of the negative control (N), positive control (P), and sample control (T) are quantitatively detected by a fluorescence scanner to obtain a double-peak or single-peak curve, and the following Figure 15 data preprocessing steps shown are carried out. The process is as follows: Figure 15 In [reference], 1 is the original data for obtaining the fluorescence signal. Corresponding to the distribution of the fluorescence intensity values on the chromatography strip 3, generally the curve presents a double-peak or single-peak curve diagram as shown in Figure 15 [reference]. The first peak curve corresponds to the IFN-γ test line T line on the chromatography strip 3, and contains the numerical curve of the original data of the test results of the negative control N, positive control P, and sample control T. The second peak curve corresponds to the quality control line C line. The numerical values of the fluorescence signal intensity gradually decrease on both sides of the peak of the curve, indicating that there are no non-specific binding sites.

[0069] Figure 15 In [reference], 2 is the filtering and normalization analysis. That is, the original data is subjected to filtering smoothing and normalization processing.

[0070] Figure 15 In [reference], 3 is the peak searching algorithm analysis. By using the first-order differential processing and monotonicity feature extraction, the starting point 1 A1, peak point 1 F1, and ending point 1 Z1 corresponding to the peak curve of the test line and the starting point 2A2, peak point 2 F2, and ending point 2 Z2 corresponding to the peak curve of the quality control line are respectively determined. Among them, whether it is the test line or the quality control line, its peak point (F1, F2) can be used as a characteristic value representing the positive correlation between the fluorescence signal intensity and the IFN-γ concentration, that is, the higher the numerical value of the peak point, the stronger its fluorescence signal intensity and the higher the IFN-γ concentration it represents.

[0071] Figure 15 In 4, the calculation of the baseline and the integral area is involved. For the calculation of the baseline of the detection line (left baseline and right baseline), the left baseline is calculated by first moving leftward from the peak point 1 F1 by a distance 1 (d1), where d1 = 2×d2 and d2 is the distance 2. Starting from the new position after moving d1, then move rightward by a distance 2 (d2), and calculate the average value of the fluorescence signal intensity values within the range from moving left by d1 to moving right by d2. This average value is used as the left baseline of the detection line; the right baseline of the detection line is obtained by a similar method; then the average value of the two baselines (left baseline and right baseline) of the detection line is used as the final baseline value corresponding to the peak curve of the detection line. The baseline final value of the quality control line is obtained by a similar method.

[0072] Calculate the integral area S: According to the above, the baseline final values of the detection line and the quality control line are calculated respectively. The area of the peak curve above the baseline final value is the integral area S. The integral area of the detection line is the area of the peak curve from the starting point 1 A1 to the ending point 1 Z1 above the baseline final value (S T ), and the integral area of the quality control line C is the area of the peak curve from the starting point 2 A2 to the ending point 2 Z2 above the baseline final value (S C ). Then, by calculating the ratio of the integral area of the detection line (S T ) to the integral area of the quality control line (S C ), the fluorescence signal intensity of the detection line is quantified. Among them, the ratio of the integral areas of the two is simply called the T / C value, that is, T / C value = S T ÷S C .

[0073] Under normal circumstances, the control line serves as a reference for comparing the fluorescence signal intensity of the test line, providing benchmark data of stable fluorescence signals to achieve the correction of fluorescence background signals, compensation for systematic errors, and filtering of non-specific fluorescence signals, thereby ensuring the stability, reliability, and effectiveness of the chromatographic strip detection system. Therefore, based on the detection data of the control line, semi-quantitative analysis and determination of the IGRA test results can be performed through the numerical range of the T / C value, that is, qualitative judgment and semi-quantitative analysis of the negative or positive IGRA test results are completed by comparing the T / C value with the thresholds (0.3, 0.5, and 1). Among them, the thresholds (0.3, 0.5, and 1) are obtained through statistical analysis based on existing experimental data and may be appropriately adjusted as the statistical sample size expands in the future. The specific determination method for semi-quantifying the test line is as follows: when the T / C value ≥ 1.0, it is determined as strongly positive (+++); when 0.5 ≤ T / C value < 1.0, it is determined as positive (++); when 0.3 ≤ T / C value < 0.5, it is determined as weakly positive (+); and when the T / C value < 0.3, it is determined as negative (-). In addition, for IGRA testing, the final result determination also needs to combine the test results of the negative control N, positive control P, and sample control T corresponding to the test sample. Among them, the semi-quantitative determination method for the test lines corresponding to N, P, and T is as described above, and the determination criteria for the combination of N, P, and T are as follows: taking the T / C value of the negative control (N T / C ) as 0 ≤ N T / C < 0.3 as the determination benchmark for the effectiveness of the IGRA test results. On the premise that the IGRA test results are effective, if the difference between the T / C value of the positive control (P T / C ) and the T / C value of the negative control (N T / C ) is (P T / C - N T / C ) ≥ 0.3, it is determined that the function of T cells in the test sample is normal. At this time, the result of the sample control T is valid (or the IGRA detection system of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity is effective), that is, when both 0 ≤ N T / C < 0.3 and (P T / C - N T / C ) ≥ 0.3 are satisfied, the test results of the test sample using the chip of the present invention are valid.

[0074] On the premise that the chip IGRA detection system is effective, if 0 ≤ (T T / C - N T / C ) < 0.3, it is judged that the IGRA test of the test sample is negative, that is, there is no Mycobacterium tuberculosis in the test sample; if (T T / C - N T / C ) ≥ 0.3, it is judged that the IGRA test of the test sample is positive; similarly, using a determination method similar to the semi-quantification of the test line, the semi-quantification of the chip IGRA positive test results can also be further determined, and the determination criteria are as follows: (TT / C -N T / C When (T-N) ≥ 1.0, it is determined as strongly positive for IGRA (+++); when 0.5 ≤ (T-N) T / C -N T / C < 1.0, it is determined as positive for IGRA (++); when 0.3 ≤ (T-N) T / C -N T / C < 0.5, it is determined as weakly positive for IGRA (+).

[0075] 3. Comparative verification of IGRA detection of centrifugal microfluidic detection chip for tuberculous specific cellular immunity Three negative samples 1, 2, and 3 of Mycobacterium tuberculosis with clear clinical backgrounds were detected in parallel by traditional IGRA and the chip IGRA of the present invention respectively. The detection results of these three negative samples of Mycobacterium tuberculosis by traditional IGRA and chip IGRA are compared as Figure 16 shown. First of all, whether it is traditional IGRA or chip IGRA, the T / C value of the negative control all meets the determination criterion of 0 ≤ N T / C < 0.3, and the difference between the T / C value of the positive control (P T / C -N T / C -N T / C -N T / C -N T / C -N T / C -N T / C -N T / C -N T / C -N T / C -N T / C -N T / C -N

[0076] In the IGRA comparative detection of negative samples of Mycobacterium tuberculosis, the detection values of the positive control of chip IGRA (P T / C were respectively: 1.220, 1.663 and 1.171) were generally higher than the detection values of the positive control of traditional IGRA (P T / CThe results are helpful to enhance the sensitivity of chip IGRA detection. The possible reasons for this result are as follows: First, multiple centrifugation operations in the chip IGRA detection process are helpful to enhance the cell enrichment efficiency, that is, multiple active centrifugation operations accelerate the sedimentation of T cells in whole blood samples, improve the stratification and enrichment efficiency of plasma and other cells and the purity of plasma, and through the flow and spatial enrichment of red blood cells in various chambers and channels of the chip, the distance between T cells is shortened, and the probability of physical contact between cells is increased, which can promote the exchange of cytokines and signal transduction efficiency, and finally form a synergistic amplification effect of immune response. Secondly, the three-dimensional structural design of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity adopted by the chip IGRA of the present invention creates a larger specific surface area, providing an efficient channel for the diffusion and transmission of intercellular signal molecules (such as IFN-γ). Therefore, intercellular signal molecules can efficiently diffuse and transmit on a larger surface area, promoting contact and interaction between cells, and further improving the efficiency of intercellular communication, thereby not only enhancing the intensity and accuracy of the immune response, but also helping to accurately detect the secretion level of IFN-γ and improve detection sensitivity. Third, the assembly and preparation method of the chip of the present invention ensures the reliability of IGRA detection. For example, the chip is injection molded with PMMA material, combined with computer numerical control (CNC) processing technology to ensure the high precision and consistency of the flow channel structure. Among them, CNC processing removes surface materials by milling to avoid exogenous pollution. At the same time, it is assembled in a clean environment. The raw materials and accessories used in the assembly have all undergone clean testing, which can effectively control the endotoxin level. Although there may be trace residual factors on the surface of the chip, it can be seen from the parallel comparison test with the traditional IGRA that the chip of the present invention has been experimentally confirmed to be unaffected by the nonspecific stimulation of IFN-γ, and the detection specificity symbol requirements, such as the negative control N tube value is stable at 0≤N T / C <0.3.

[0077] Due to safety issues, it is difficult to obtain a positive Mycobacterium tuberculosis sample with a clear clinical background, so a tuberculosis-positive simulated sample was prepared. The specific preparation method is: add 80 μL of recombinant IFN-γ standard (wherein the initial concentration of IFN-γ standard is 500 pg / mL) to 720 μL of fresh anticoagulated whole blood, and after sufficient mixing, the concentration of IFN-γ stimulated by the Mycobacterium tuberculosis-positive sample can be simulated (the final concentration is 50 pg / mL). The tuberculosis-positive simulated sample was tested three times in parallel using the traditional IGRA and the chip IGRA of the present invention: 1, 2, 3, and the test results were compared. Figure 17 As shown, both the traditional IGRA and the chip IGRA, not only the T / C value of the negative control satisfies 0≤N T / CJudgment criterion of <0.3, and the difference between the T / C value of the positive control (P T / C ), and the T / C value of the negative control (N T / C ) satisfies the effective judgment criterion of the IGRA detection system that (P T / C -N T / C ) ≥ 0.3. Figure 17 It can be seen that (P T / C -N T / C ) > 0.5; most importantly, the semi - quantitative judgment result value of the sample to be tested (T T / C -N T / C ) is all within the range of (T T / C -N T / C ) ≥ 0.3, that is, within the positive range of IGRA detection. Figure 17 It can be seen that for the IGRA detection results of the simulated samples positive for tuberculosis, whether it is traditional IGRA or chip IGRA, (T T / C -N T / C ) ≥ 1.0, that is, it is determined as strongly positive (+++). It can be seen that the positive coincidence rate of the chip IGRA and traditional IGRA of the present invention is 100%. Combining with the results of the negative coincidence rate above, the total coincidence rate of the chip IGRA and traditional IGRA of the present invention is 100%. In addition, in the IGRA comparative detection of the simulated samples positive for tuberculosis, traditional IGRA still relies on manual operations (such as high - speed centrifugation, pipette extraction), and there is a potential risk of introducing errors due to differences in operator skills. While using the chip IGRA of the present invention, the chip can integrate multiple detection units, and is expected to truly realize the parallel processing of batch samples to be tested for IGRA. Only 50 μL of whole blood sample is required to complete the IGRA detection (200 - 500 μL is required for the traditional method); in terms of detection time, excluding the incubation time, the whole process of the chip IGRA to complete the N / P / T three - tube control detection takes less than 30 minutes (the operation time for traditional IGRA to simultaneously complete the N / P / T three - tube chromatography strip control detection for one sample is at least 35 - 50 minutes).

[0078] According to the overall concept of the present invention, a centrifugal microfluidic detection chip for tuberculosis - specific cellular immunity is provided, and the construction and performance evaluation of the tuberculosis - specific cellular IGRA (immunomicrofluidic detection chip method) detection platform are completed, and the total coincidence rate with traditional IGRA is preliminarily verified to be consistent. In the future, on the one hand, it is necessary to further improve and enhance the preparation and assembly accuracy of the centrifugal microfluidic detection chip for tuberculosis - specific cellular immunity, such as improving the processing accuracy and hardware quality of the chip, and improving the stability of large - scale chip production; on the other hand, it is also necessary to verify the application of the chip of the present invention to the detection of a large number of clinical samples, and compare and analyze the correlation of the data with the traditional IGRA detection results, so as to promote the detection application of chip IGRA in a larger scale of samples to be tested.

[0079] The specific embodiments of the present invention have been described in detail, making it easy for those skilled in the art to understand. Moreover, it can be understood that all or part of the steps for implementing the above embodiments can be completed by hardware, or can be completed by instructing related hardware through a program. The said program can be stored in a computer-readable storage medium. The above-mentioned storage medium can be a read-only memory, a magnetic disk or an optical disc, etc. However, the above specific embodiments are only used to specifically illustrate the technical solutions or preferred solutions of the present invention in combination with the accompanying drawings, rather than an exhaustive limitation of the protection scope of the present invention. According to all the descriptions that have been made public, details can be appropriately modified or replaced without departing from the essence and scope of the technical solutions of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity, characterized in that: A sealed chip mainly assembled from bottom to top by three layers of structures: a chip bottom sealing plate (1), a chip substrate (2), and a chip top sealing plate (5). The chip is provided with detection units for multiple samples; among them, Each sample detection unit (22) on the chip substrate (2) includes a strip card slot (34) for fixing the chromatography strip (3), 9 chambers that can accommodate gas or liquid, and channels for gas-liquid communication between the strip card slot (34) and the 9-chamber structure. The 9 chambers are respectively 2 chambers, namely the sample enrichment preparation chamber (23) and the red blood cell enrichment chamber (26) in the single-chamber centrifugal sedimentation structure (51), a pressure buffer chamber (49) for buffering the air pressure in the single-chamber centrifugal sedimentation structure (51), 2 quantitative chambers, namely the plasma quantification chamber (45) and the red blood cell collection chamber (42) that form a concentric collinear interconnected structure (52), a waste liquid collection chamber (44) for collecting excess liquid, a diluent chamber (28) for diluting the sample to be tested, a desiccant chamber (32) for humidity adjustment, and a mixing chamber (35) for mixing the diluent and purified plasma; among them, the single-chamber centrifugal sedimentation structure (51) includes a sample enrichment preparation chamber (23), a plasma extraction inlet (24), a fence structure (25), and a red blood cell enrichment chamber (26); the concentric collinear interconnected structure (52) includes a plasma quantification chamber (45), a three-way left stepped channel (40), a three-way middle liquid inlet stepped channel (41), a red blood cell collection chamber (42), and a three-way right exhaust stepped channel (43); On a single detection unit (17) of the chip top sealing plate, there are a strip detection window (18) for observing the IGRA detection result of the chromatography strip (3) and 6 gas-liquid interaction hole positions.

2. The centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity according to claim 1, wherein: The three-layer structures of the chip bottom sealing plate (1), the chip substrate (2), and the chip top sealing plate (5) are all provided with chip positioning structures (12) with the same shape and pin positioning holes (14) at corresponding positions; the number, positions, and related structures of the sample detection units (22) on the chip substrate (2) and the single detection units (17) on the chip top sealing plate correspond to each other; the number of the detection units can be 3 - 10 to adapt to the detection of multiple samples to be tested, and at least can be used to simultaneously complete the IGRA detection of sample controls for negative control, positive control, and 1 - 8 samples to be tested; The channels for gas-liquid communication of the 9-chamber structure include a plasma transfer channel (48) that connects the plasma extraction inlet (24) and the puncture valve fixed stepped hole (47) and promotes the liquid to flow in from the plasma extraction inlet (24) and out from the puncture valve fixed stepped hole (47); a puncture valve (4) is fixedly installed on the puncture valve fixed stepped hole (47), and the puncture valve (4) is a three-layer structure composed of two upper and lower nylon gaskets (11) and a middle aluminum foil sacrificial layer (10), belonging to an active valve that does not require additional driving structure cooperation; The six gas-liquid interaction pore positions are respectively a sample injection hole (13) and an exhaust hole (19) for gas-liquid interaction with the single-chamber centrifugal sedimentation structure (51), a diluent chamber liquid injection hole (15) and a diluent chamber exhaust hole (16) for gas-liquid interaction with the diluent chamber (28), a gas pressure buffer hole (20) for buffering the gas pressure in the buffer gas pressure buffer chamber (49), and a puncture hole (21) located in the upper part of the axial direction of the puncture valve (4).

3. The centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity according to claim 1, characterized in that: The paper strip detection window (18) is sealed with a pressure-sensitive encapsulation adhesive (6) made of a transparent material.

4. The centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity according to claim 1, wherein: Magnetic beads (53) for promoting the stirring and mixing of the solution are pre-placed in the mixing chamber (35).

5. The centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity according to claim 1, characterized in that: When only the matrix solution is pre-loaded in the to-be-detected sample enrichment preparation chamber (23), the corresponding sample detection unit is marked as a negative control; if both the matrix solution and the tuberculosis non-specific stimulating antigen are pre-loaded in the to-be-detected sample enrichment preparation chamber (23), the corresponding sample detection unit is marked as a positive control; if both the matrix solution and the Mycobacterium tuberculosis specific antigen are pre-loaded in the to-be-detected sample enrichment preparation chamber (23), the corresponding sample detection unit is marked as a sample control.

6. The centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity according to claim 2, characterized in that: The coaxial positions where the puncture valve (4) is located from top to bottom are in sequence: an airbag (7), a puncture airbag fixing plate (9), a puncture hole (21), a nylon gasket (11), an aluminum foil sacrificial layer (10), and a nylon gasket (11); the buffer airbag fixing plate (8) is installed on the chip top sealing plate (5), and the airbag (7) is installed on the buffer airbag fixing plate (8). Therefore, in this coaxial position from top to bottom, they are in sequence: an airbag (7), a buffer airbag fixing plate (8), a gas pressure buffer hole (20), and a gas pressure buffer chamber (49). The airbags (7) installed on the puncture airbag fixing plate (9) and the airbag (7) installed on the buffer airbag fixing plate (8) are both selected from elastic materials with a high elastic modulus.

7. The assembly process of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity described in any one of claims 1-6 includes: The first step: Preparation work; The second step: Assembly of the chip bottom sealing plate (1), and installing pins in the pin positioning holes (14) to form positioning columns; The third step: Assembly of the chip substrate (2): Through the positioning columns formed by the installation of the chip bottom sealing plate (1), the chip substrate (2) is locked and fixed with the chip bottom sealing plate (1), and the exposed positioning columns are still visible after fixation; The fourth step: Assembly of the chip top sealing plate (5): Through the positioning columns exposed after the assembly of the chip substrate (2), the chip top sealing plate (5) is locked and fixed with the chip substrate (2); at the central axis position of the puncture hole (21), the puncture airbag fixing plate (9) and the airbag (7) are assembled from bottom to top in sequence; at the central axis position of the gas pressure buffer hole (20), the buffer airbag fixing plate (8) and the airbag (7) are assembled from bottom to top in sequence; at the opening position of the paper strip detection window (18) on the chip top sealing plate (5), it is sealed with a pressure-sensitive encapsulation adhesive (6).

8. The detection process of chip IGRA using the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity described in any one of claims 1-6 includes: Detection preparation: Equilibrate the chip at room temperature for at least 20 minutes; Sample addition and sealing: Inject the test sample and diluent into the corresponding single-chamber centrifugal sedimentation structure (51) and diluent chamber (28) respectively from the sample addition hole (13) and the diluent chamber injection hole (15). After liquid addition, seal the hole positions; Incubation: Place the chip in a constant temperature environment of 37°C and incubate for 22 ± 4 hours; Chip IGRA detection: Place the chip on the servo motor and complete the separation of plasma in the test sample, liquid mixing, and chip IGRA detection through 5 centrifugation operations. Observe the detection results of chip IGRA at the strip detection window (18); Determination of chip IGRA detection results: Make a negative / positive determination of the test line and control line on the chromatography strip (3) either by the naked eye; Or perform optimization processing and semi-quantitative analysis on the intensity values of the fluorescence signals obtained by quantitative detection with a fluorescence scanner.

9. The detection process of chip IGRA using the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity according to claim 8, characterized in that: In the 5 centrifugations in the chip IGRA detection, the chip is centrifuged by the servo motor after being fixed on the flange block of the servo motor. Among them, it is accelerated to a high-speed rotation state of no higher than 3000 rpm at an acceleration of no higher than 500 rpm / s, and the centrifugation time is no higher than 6 minutes.

10. Application of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity described in any one of claims 1-6 in chip IGRA detection.

Images