A microfluidic detection chip for tuberculosis-specific cellular immunity

Abstract

The present invention provides a microfluidic detection chip for tuberculosis-specific cellular immunity, belonging to the technical field of Mycobacterium tuberculosis detection. The chip is primarily composed of a three-layer structure, assembled sequentially from bottom to top: a chip bottom sealing plate, a chip substrate, and a chip top sealing plate. The chip is equipped with detection units for multiple samples, capable of simultaneously performing IGRA detection of negative controls, positive controls, and a sample control of 1-8 test samples. Furthermore, the present invention provides an assembly process for a centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity, as well as a detection method and application of chip-based IGRA using the chip. The chip-based IGRA demonstrated a 100% overall concordance rate between test results and those of conventional IGRA. Furthermore, the chip-based IGRA enables detection of very small numbers of test samples, while improving the throughput and speed of IGRA detection, thus showing significant application prospects for IGRA detection of large numbers of clinical samples.

Description

Technical Field

[0001] The present invention relates to the technical field of Mycobacterium tuberculosis detection, and in particular to a tuberculosis-specific cellular immunity microfluidic detection chip established by combining Mycobacterium tuberculosis-specific cellular immune response detection with centrifugal microfluidic detection. Background Art

[0002] Tuberculosis (TB) is caused by infection with a single pathogen, Mycobacterium tuberculosis (MBT). Since 2007, TB infection has been a serious global public health problem. Currently, the interferon-gamma release assay (IGRA) measures IFN-γ levels in peripheral blood stimulated with M. tuberculosis antigens. IGRAs are frequently used for the auxiliary diagnosis of latent tuberculosis infection (LTBI) and active TB, and are recommended by the WHO for auxiliary diagnosis of TB infection, due to their high specificity, lack of interference from BCG vaccination and most NTMs, and rapid detection compared to the traditional tuberculin skin test (TST). Furthermore, IGRAs have a single threshold, facilitating standardization of test results. However, IGRA testing includes multiple steps such as sample collection, centrifugation, plasma separation, antigen stimulation, cell culture, and cytokine detection. These steps rely on a variety of bulky professional equipment (including centrifuges, constant temperature cell incubators, and fluorescence analyzers), and 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, he needs to use a pipette to slowly aspirate the centrifuged plasma supernatant to complete plasma separation. This process easily absorbs impurities such as red blood cells; after that, the plasma needs to be manually mixed with the diluent and cultured, and a quantitative volume of diluted plasma needs to be aspirated for testing. These manual processing steps of blood samples are not only time-consuming, but also prone to introduce human errors, thereby affecting the accuracy of the final IGRA test results.

[0003] Microfluidic technology can be divided into three main types based on the principle of liquid manipulation: self-powered, cartridge and centrifugal. 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, cultivation, washing, analysis or preparation on a small disc, and uses centrifugal force to drive the flow of liquid to process related liquids. It can avoid the problems of self-powered and cartridge types such as the introduction of bubbles, uneven mixing, and low parallel processing capabilities. It has the advantages of miniaturization, high throughput, integration, automation, fast detection, small sample volume requirement, closed type to reduce pollution, and portable to achieve instant detection. Among them, valve operation is the key to achieving precise liquid control in centrifugal microfluidics, which mainly relies on non-contact valves, including passive valves (also known as self-propelled valves) that do not require external energy input and active valves that require exogenous forces such as pressure, magnetism, and electricity to drive; passive valves such as capillary valves, steam traps, 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 require additional driving structures to be embedded in the microfluidic chip, which will increase the number of layers of the chip. Summary of the Invention

[0004] The purpose of the present invention is to overcome the defects of the existing interferon-γ release assay (IGRA) and provide a simple and easy-to-operate centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity, which can be used for IGRA detection.

[0005] The present invention also aims to provide a centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity, which can complete the simultaneous detection of three tubes: negative control (N), positive control (P), and sample control (T) at one time.

[0006] The present invention also aims to combine centrifugal microfluidics with Mycobacterium tuberculosis-specific cellular immunity to prepare a new centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity, thereby avoiding the human errors that may be introduced by traditional IGRA and improving the precision and accuracy of detection.

[0007] In order to achieve the above purpose, the present invention provides the following technical solutions:

[0008] In the first aspect, the present invention provides a centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity, which is mainly composed of a three-layer structure of a chip bottom plate sealing plate 1, a chip substrate 2 and a chip top sealing plate 5, which are assembled from bottom to top to form a closed chip. The chip is provided with a plurality of sample detection units, preferably 3-10, to adapt to the detection of multiple samples to be tested, and can at least be used to simultaneously complete the IGRA detection of negative controls, positive controls and 1-8 sample controls to be tested. The number of sample detection units 22 on the chip substrate 2 and the number of single detection units 17 on the chip top sealing plate is as follows: , position and related structures; wherein, each sample detection unit 22 on the chip substrate 2 includes a paper strip card slot 34 for fixing the chromatography paper strip 3, 9 chambers that can accommodate gas or liquid, and a channel for gas-liquid communication between the paper strip card slot 34 and the 9 chamber structures, wherein the 9 chambers are respectively the two chambers of the sample enrichment preparation chamber 23 and the red blood cell enrichment chamber 26 in the single-chamber centrifugal sedimentation structure 51, the air pressure buffer chamber 49 for buffering the air pressure in the single-chamber centrifugal sedimentation structure 51, the plasma quantitative chamber 45 and the red blood cell quantitative chamber 46 that constitute the center-collinear interconnection structure 52. The collection chamber 42 comprises two quantitative chambers, 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 regulation, and a mixing chamber 35 for mixing the diluent and purified plasma; wherein, the single-chamber centrifugal sedimentation structure 51 comprises a sample enrichment preparation chamber 23, a plasma extraction inlet 24, a fence structure 25 and a red blood cell enrichment chamber 26; the center-colinear interconnected structure 52 comprises a plasma quantitative chamber 45, a three-branch left stepped channel 40, a three-branch middle liquid inlet stepped channel 41, and a red blood cell collection chamber 42. , a three-branch right exhaust stepped channel 43; the channel includes 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 into the plasma extraction inlet 24 and out of the puncture valve fixed stepped hole 47; the puncture valve 4 is fixedly installed on the puncture valve fixed stepped hole 47, and the puncture valve 4 is a three-layer structure consisting of two layers of nylon gaskets 11 and a middle layer of aluminum foil sacrificial layer 10, which is an active valve that does not require additional drive structure cooperation; the single detection unit 17 on the sealing plate at the top of the chip includes a device for observing the chromatography paper strip 3 The IGRA test result strip detection window 18 and 6 gas-liquid interaction holes are respectively the sample addition hole 13 and the exhaust hole 19 for gas-liquid interaction with the single-chamber centrifugal sedimentation structure 51, the diluent chamber injection hole 15 and the diluent chamber exhaust hole 16 for gas-liquid interaction with the diluent chamber 28, the air pressure buffer hole 20 for buffering the air pressure in the air pressure buffer chamber 49, and the puncture hole 21 located at the upper axial direction of the puncture valve 4.

[0009] According to a preferred embodiment of the present invention, in the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity, the three-layer structure 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 of the same shape and pin positioning holes 14 at corresponding positions.

[0010] 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 using a pressure-sensitive packaging adhesive 6 made of a transparent material.

[0011] According to a preferred embodiment of the present invention, in the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity, the coaxial position of the puncture valve 4 is, from top to bottom,: 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 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, so at this coaxial position, from top to bottom, they are: the airbag 7, the buffer airbag fixing plate 8, the air pressure buffer hole 20 and the air pressure buffer chamber 49, and the airbag 7 installed on the puncture airbag fixing plate 9 and the airbag 7 installed on the buffer airbag fixing plate 8 are preferably made of elastic material with a high elastic modulus.

[0012] 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 stirring and mixing of the solution are pre-placed in the mixing chamber 35 .

[0013] According to a preferred embodiment of the present invention, in the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity, when only matrix fluid is pre-loaded in the sample enrichment preparation chamber 23 to be tested, the corresponding sample detection unit is marked as a negative control; if both matrix fluid and tuberculosis non-specific stimulation antigen are pre-loaded in the sample enrichment preparation chamber 23 to be tested, the corresponding sample detection unit is marked as a positive control; if both matrix fluid and Mycobacterium tuberculosis-specific antigen are pre-loaded in the sample enrichment preparation chamber 23 to be tested, the corresponding sample detection unit is marked as a sample control.

[0014] In the second aspect, the present invention provides an assembly process of a centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity, including: the first step: preparatory work; the second step: assembling the chip bottom plate sealing plate 1, installing pins in the pin positioning holes 14 to form positioning columns; the third step: assembling the chip base 2: through the positioning columns formed by the chip bottom plate sealing plate 1, the chip base 2 and the chip bottom plate sealing plate 1 are locked and fixed, and the exposed positioning columns are still visible after fixation; the fourth step: assembling the chip top sealing plate 5: through the positioning columns exposed after the chip base 2 is assembled, the chip top sealing plate 5 and the chip base 2 are locked and fixed; at the central axis position of the puncture hole 21, the puncture airbag fixing plate 9 and the airbag 7 are assembled in sequence from bottom to top; at the central axis position of the air pressure buffer hole 20, the buffer airbag fixing plate 8 and the airbag 7 are assembled in sequence from bottom to top; the opening position of the paper strip detection window 18 on the chip top sealing plate 5 is sealed with pressure-sensitive packaging glue 6.

[0015] In a third aspect, the present invention provides a chip IGRA detection process using the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity, comprising: detection preparation: equilibrating the chip at room temperature for at least 20 minutes; sample addition and sealing: injecting the sample to be tested and the diluent into the corresponding single-chamber centrifugal sedimentation structure 51 and the diluent chamber 28 from the sample addition hole 13 and the diluent chamber injection hole 15, respectively, and completing the sealing of the hole position after adding liquid; incubation: placing the chip in a constant temperature environment of 37°C for incubation for 22±4 hours; chip IGRA detection: the chip is centrifuged on a servo motor for 5 times to complete the separation of plasma in the sample to be tested, liquid mixing and chip IGRA detection, and the chip IGRA detection results are observed in the paper strip detection window 18; determination of the chip IGRA detection results: the detection line and the quality control line on the chromatography paper strip 3 are judged to be positive or negative by the naked eye; or the intensity value of the fluorescence signal obtained by quantitative detection with a fluorescence scanner is optimized and semi-quantitatively analyzed.

[0016] According to a preferred embodiment of the present invention, during chip-IGRA testing using a centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity, the five centrifugations in the chip-IGRA test are performed by fixing the chip on a flange block of a servo motor and then allowing the servo motor to drive the chip for centrifugation, wherein the centrifugation is performed at an acceleration of no more than 500 rpm / s to a high-speed rotation state of no more than 3000 rpm, and the centrifugation lasts no more than 6 minutes.

[0017] In a fourth aspect, the present invention provides an application of chip IGRA detection using a centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity.

[0018] According to a preferred embodiment of the present invention, in chip IGRA detection applications, the intensity value of the fluorescence signal obtained by quantitative detection with a fluorescence scanner can be optimized and then the baseline and integrated area are calculated to achieve semi-quantitative analysis of the IGRA detection results.

[0019] Beneficial effects of the present invention:

[0020] The present invention provides a microfluidic detection chip for tuberculosis-specific cellular immunity. The chip mainly forms a three-layer structure by a chip bottom plate sealing plate, a chip substrate and a chip top sealing plate. The chip substrate is provided with a chromatography paper strip, a puncture valve and other structures. The chip top sealing plate is provided with a pressure-sensitive packaging glue, an airbag, a buffer airbag fixing plate and a puncture airbag fixing plate and other structures. The chip body is designed with innovative structures such as a single-chamber centrifugal sedimentation structure, a center-collinear interconnection structure, a mixing chamber and a paper strip detection chamber, which can automatically complete key steps such as whole blood incubation, red blood cell separation, reagent mixing and paper strip detection. The single-chamber centrifugal sedimentation structure of the chip uses centrifugal force to separate plasma and red blood cells. The plasma enters the center-collinear 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 paper strip card slot by siphoning to complete the paper strip chromatography detection. The chip is also designed with an active normally closed valve, which controls the opening and closing of the valve by applying pressure through an airbag, ensuring precise control of the fluid and release on demand. In order to improve the mixing efficiency, magnetic balls can be placed in the mixing chamber, and the reagents can be stirred by using the Euler force generated by the acceleration and deceleration of the servo motor, so that the components of the reagents in the chamber are in full contact, thereby improving the detection sensitivity. In addition, obstacles can be integrated into 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 design of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention focuses on the simplicity and automation of operation, and has a high-throughput detection function. It can complete the simultaneous detection of three tubes, namely the negative control N, the positive control P, and the sample control T, at one time, realizing "sample in - result out" on-site rapid instant detection (POCT). By using the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention for IGRA testing, the entire testing process can be completed using a single intelligent servo motor (e.g., a centrifugal microfluidic system) that combines centrifugation and constant temperature control functions, eliminating the need for multiple independent devices such as a centrifuge, constant temperature cell incubator, and fluorescence analyzer. This significantly improves detection throughput and operational convenience, and enables fully automated, high-throughput, "sample-in, result-out" IGRA testing. Furthermore, the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention can improve the accuracy and reliability of IGRA testing and is suitable for auxiliary diagnosis of latent tuberculosis infection (LTBI) and active tuberculosis.

[0021] The present invention also provides a centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity. The chip can perform high-throughput parallel testing of multiple test samples. Specifically, the chip can distribute at least one test sample into three independent detection units, respectively performing IGRA (Integrated Gene Rat Assay) test on the negative control, positive control, and sample control. Therefore, three independent control tubes—negative control (N), positive control (P), and sample control (T)—can be tested simultaneously on a single chip. Three tuberculosis-negative samples and a tuberculosis-positive simulated sample were then compared and tested in parallel using both conventional IGRA and the chip IGRA of the present invention. Preliminary test results confirmed that the negative, positive, and overall coincidence rates can reach 100%.

[0022] 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 state of equilibrium in the closed space. Among them, the puncture valve is an active valve that does not require an additional driving structure. The flow of liquid after active opening is achieved by squeezing the airbag. This operation method will not destroy the sealing of the chip. The air pressure expansion buffer in the chip is achieved 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 paper strip detection. This "sample in-result out" design has a higher degree of functional integration, making on-site rapid and instant detection possible, and can complete detection tasks more efficiently, reduce manual operation links, reduce human errors, and improve the accuracy and reliability of detection.

[0023] Regarding valves, active valves typically increase the number of chip layers due to their associated drive structure. However, this invention utilizes an active, normally closed valve—a puncture valve—to achieve precise control and on-demand release of liquids. The puncture valve's design cleverly exploits differences in material physical properties. Through its modular structure, the puncture valve achieves efficient integration of diverse functions without increasing chip fabrication complexity or the number of chip layers. This modular design allows the puncture valve to be flexibly combined with other functional modules, enhancing the chip's scalability and adaptability.

[0024] The IGRA tuberculosis detection centrifugal microfluidic chip of the present invention features 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 center-colinear interconnected structure. The first stage is completed within the single-chamber centrifugal sedimentation structure. By adding a fence structure to the single-chamber centrifugal sedimentation structure, the fence structure can accumulate clumped red blood cells to the lower layer, physically separating red blood cells and plasma, thereby reducing the impact of clumped red blood cells on plasma transfer. The outlet is positioned at the point where the red blood cell and plasma separation is most obvious in the single-chamber centrifugal sedimentation structure, thereby minimizing the "fluid-solid coupling effect" between plasma and red blood cells. The second stage is completed in the center-collinear interconnected structure. By controlling the centrifugal force generated by the direction and magnitude of the servo motor's rotational angular velocity and the density difference of different components in the blood sample, as well as microfluidic technology, the direction of the inertial force acting on the liquid in the center-collinear interconnected structure is controlled, so that the liquid containing more red blood cells is prompted to flow into the red blood cell collection chamber through the three-way liquid inlet stepped channel, while the gas in the red blood cell collection chamber is discharged through the three-way right exhaust stepped channel. Therefore, the red blood cell collection chamber of the blind hole structure is preferentially filled with liquid containing more red blood cells, and the liquid containing relatively less red blood cells (equivalent to pure plasma) is subsequently quantitatively filled into the plasma quantitative chamber, further reducing the possibility of red blood cells indirectly flowing into the chromatography paper strip through the plasma siphon channel. In addition, there is a potential third-stage red blood cell filtration structure, which is mainly completed in the mixing chamber. Through the optimization of the shape of the mixing chamber, the lower chamber of the siphon valve inlet, and the position of the liquid outlet, after the above-mentioned two-stage red blood cell filtration structure, the plasma is basically pure. Red blood cells are no longer observable to the naked eye in the mixing chamber. However, it is not ruled out that in extremely rare cases, a few red blood cells have entered the mixing chamber. Under the action of centrifugal force, a few red blood cells will sink into the lower chamber of the siphon valve inlet. The only liquid outlet in the mixing chamber can only discharge the liquid in the "siphon valve inlet upper chamber" space above the liquid level of the siphon valve inlet to the chromatography paper strip. Therefore, the optimized design of the mixing chamber structure and the position of the liquid outlet achieves a third enrichment of red blood cells and can further prevent the few red blood cells that have entered the mixing chamber from flowing onto the chromatography paper strip and affecting the detection accuracy. In summary, it can be seen that the chip can achieve multi-stage red blood cell enrichment and separation, purification, and quantitative extraction of plasma without the use of red blood cell filters and cell separation fluids.

[0025] The chip of the present invention is used for chip-IGRA testing. By adding a diluent and pre-installing magnetic beads in the mixing chamber, the problem of plasma viscosity hindering flow on the chromatography paper strip can be solved, thereby avoiding the poor detection effect of the mixed liquid on the chromatography paper strip due to the influence of liquid viscosity, thereby improving the accuracy of the chip-IGRA test results. In the chip-IGRA test, the inertial force generated by five centrifugations is used to improve the efficiency of enrichment, directional flow, and liquid mixing, effectively avoiding the interference of red blood cells in the whole blood sample to be tested, or the poor chromatography effect of the paper strip caused by uneven mixing of plasma and diluent, thereby improving the reliability of the test results.

[0026] The centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity described in the present invention has a flat, short cylindrical shape with a thickness of about 1 cm and a diameter of no more than 10 cm. It is small and compact overall and adopts a modular design, making IGRA operation simple and convenient, and can be used for auxiliary detection of Mycobacterium tuberculosis infection. The modular design of the chip described in the present invention, in particular, the single-chamber centrifugal sedimentation structure, the center-of-circle colinear interconnection structure, the nine-chamber structure including the mixing chamber and the paper strip detection chamber, the puncture valve structure of the normally closed active valve, etc., cooperate with each other to automatically perform key steps such as whole blood incubation, red blood cell separation, reagent mixing, and paper strip detection with the assistance of centrifugal force, integrating whole blood incubation-blood separation-reagent mixing-paper 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 through the sample loading hole and the diluent chamber injection hole of the chip respectively. After that, no additional manual operation is required. The chip automatically completes gas-liquid exchange in the closed space according to the optimized path, and realizes the in vitro detection experiment of the level of gamma interferon release after tuberculosis-specific antigen stimulation. The test results are displayed by immunofluorescence paper strips, and the test results can also be quantitatively analyzed, so that "sample in-result out" instant detection can be quickly and automatically realized on site. The test results are divided into three types: positive, negative, and uncertain. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is an exploded view of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention;

[0028] Figure 2 An exploded view of a portion of the structure on the top sealing plate of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention;

[0029] Figure 3 A top view of the sealing plate on the top of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention;

[0030] Figure 4 The centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention is in the state where the puncture valve is closed. Figure 3 Longitudinal cross-sectional view at angle aa;

[0031] Figure 5 This is an enlarged view of a single detection unit on the top sealing plate of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention;

[0032] Figure 6 A top view of the chip substrate of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention;

[0033] Figure 7 A single detection unit of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention, with a top view of the various well positions of the chip base structure arranged at corresponding positions on the sealing plate at the top of the chip;

[0034] Figure 8 This 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, viewed from above;

[0035] Figure 9 This is a schematic diagram of the design principle of a radial stack formed by centrifuging a blood sample in a 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;

[0036] Figure 10 This 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;

[0037] Figure 11 The axis line of the single-chamber centrifugal sedimentation structure in the single sample detection unit on the chip substrate in the state where the puncture valve of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention is open Figure 10 Longitudinal cross-section of the middle bb;

[0038] Figure 12 This is a partial enlarged view of the center-collinear interconnected 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, viewed from above;

[0039] Figure 13 This is a schematic diagram of the design principle of the mixing chamber for transferring diluted plasma 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;

[0040] Figure 14 This 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;

[0041] Figure 15The invention provides four steps for data analysis of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity IGRA detection: step 1 is to obtain the raw data of the fluorescence signal, step 2 is filtering and normalization analysis, step 3 is peak search algorithm analysis, and step 4 is the calculation of the baseline and integrated area.

[0042] Figure 16 The results of IGRA detection of three Mycobacterium tuberculosis-negative samples were compared using the traditional IGRA and the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention, respectively. 1-3 represent three Mycobacterium tuberculosis-negative samples, A represents the traditional IGRA, and B represents the chip IGRA of the present invention.

[0043] Figure 17 Comparison of the results of three repeated IGRA tests on tuberculosis-positive simulated samples using the traditional IGRA and the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention, where 1-3 represent three repeated tests, A represents the traditional IGRA, and B represents the chip IGRA of the present invention;

[0044] Figures 1-14Middle: 1. Chip bottom sealing plate, 2. Chip substrate, 3. Chromatography paper strip, 4. Puncture valve, 5. Chip top sealing plate, 6. Pressure-sensitive packaging 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. Sample addition hole, 14. Pin positioning hole, 15. Dilution chamber injection hole, 16. Dilution chamber exhaust hole, 17. Chip top sealing plate Single detection unit, 18, paper strip detection window, 19, exhaust hole, 20, air pressure buffer hole, 21, puncture hole, 22, sample detection unit, 23, sample enrichment preparation room, 24, plasma extraction inlet, 25, fence structure, 26, red blood cell enrichment chamber, 27, internal gas circulation channel, 28, diluent chamber, 29, diluent transfer siphon channel, 30, first capillary valve, 31, desiccant chamber first connecting channel, 32, dry Desiccant chamber, 33, desiccant chamber second connecting channel, 34, paper strip slot, 35, mixing chamber, 36, plasma siphon channel, 37, mixed liquid siphon channel, 38, second capillary valve, 39, mixed liquid serpentine channel, 40, three-branch left stepped channel, 41, three-branch middle liquid inlet stepped channel, 42, red blood cell collection chamber, 43, three-branch right exhaust stepped channel, 44, waste liquid collection chamber, 45, plasma quantitative chamber, 46, annular flow channel Channel, 47. Piercing valve fixed stepped hole, 48. Plasma transfer channel, 49. Air pressure buffer chamber, 50. Air pressure buffer chamber connecting channel, 51. Single-chamber centrifugal sedimentation structure, 52. Center-collinear interconnected 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 distal direction, 60. Siphon valve inlet lower chamber, 61. Siphon valve inlet upper chamber. DETAILED DESCRIPTION

[0045] The embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In order 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 fully improve and understand the overall inventive concept of the present invention. However, in other cases, one or more well-known embodiments may also be implemented without these specific details.

[0046] The present invention claims protection for 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 of a sample to be tested on a chromatography paper strip on the chip, belonging to the field of in vitro diagnostic reagents. The paper strip card slot of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention is installed with an IGRA immunofluorescence chromatography paper strip, which can be used to perform IGRA detection on the sample to be tested. During the chip detection process, preferably, a centrifugal function that can 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 a 37°C constant temperature function are used. The servo motor is a small device independently developed by the laboratory of the applicant of the present invention.

[0047] The following is combined with Figure 1-17 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 IGRA detection using the chip are described in detail.

[0048] Example 1 Structure of a Centrifugal Microfluidic Detection Chip for Tuberculosis-Specific Cellular Immunity

[0049] The centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention is disc-shaped, and multiple sample detection units are evenly arranged on the chip along the circumference, preferably 3-10, to accommodate the detection of multiple samples to be tested. At least one detection unit on the chip needs to be set up for a negative control (N) and one for a positive control (P), and the other detection units are used to detect different sample controls (T). Therefore, the number of detection units for a chip that can simultaneously detect 1-8 samples to be tested (n≦8) can be set to 3, 4, 5, 6, 7, 8, 9, or 10; or at least one detection unit is set up to be shared as a negative control (N), and each sample to be tested is set up in parallel for separate verification as a positive control (Pn), and one is set up for sample control (Tn). Therefore, the number of detection units for a chip that can simultaneously detect 1-4 samples to be tested (n≦4) can be set to 3, 5, 7, or 9; or each sample to be tested is set up with a complete set of control tubes (Nn, Pn, Tn). Therefore, the number of detection units for a chip 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 set to 3, 6, or 9. It can be seen that the number of detection units is at least 3. In order to clearly illustrate the structure of each detection unit, the drawings of the present invention are mainly drawn by setting up 3 detection units as an example. The 3 detection units can simultaneously complete the three-tube control experiment including the negative control (N), the positive control (P) and the sample control (T) when IGRA detects one sample to be tested. The sample control (T) is sometimes also called the test control (T). The following description is all exemplified by setting up 3 detection units.

[0050] The present invention provides a centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity. Figure 1 This is an exploded schematic diagram of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention. Figure 2 This is an exploded schematic diagram of part of the structure on the chip top sealing plate 5. Figure 1 and Figure 2 As can be seen from the above, the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention comprises a three-layer structure, which comprises, from bottom to top, a chip bottom plate sealing plate 1, a chip base 2, and a chip top sealing plate 5. A chromatography paper strip 3 and a puncture valve 4 are arranged on the chip base 2. The upper and lower parts of the chip base 2 are sealed and protected by the chip bottom plate sealing plate 1 and the chip top sealing plate 5, respectively. The number of chromatography paper strips 3 and puncture valves 4 is set to the same as the number of chip detection units. Figure 1 and Figure 2 In the figure, three detection units are set, so there are three chromatography paper strips 3 and three puncture valves 4; Figure 1 and Figure 2 It can also be seen that the upper part of the chip top sealing plate 5 is also provided with a pressure-sensitive packaging glue 6, a buffer airbag fixing plate 8 and a puncture airbag fixing plate 9, which are consistent with the number of detection units. The upper part of the buffer airbag fixing plate 8 and the puncture airbag fixing plate 9 are respectively provided with airbags 7, so the number of airbags 7 is twice the number of detection units.

[0051] Specifically, the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention is a short flat cylinder with a thickness of about 1 cm, wherein the preparation materials of the chip bottom plate sealing plate 1, the chip substrate 2 and the chip top sealing plate 5 can all be polymer materials with good biocompatibility, preferably polymethyl methacrylate (PMMA). In addition to good biocompatibility, PMMA is also a polymer with excellent light transmittance (90%-92%); the chip bottom plate 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 preferably formed by micro-milling technology. Figure 1 As shown, if the number of detection units is 3, the chip bottom plate sealing plate 1 is in the shape of 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 chip substrate 2 is in the shape of 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 1The chip substrate 2 shown is arranged with three sample detection units 22 arranged at a 120° angle around the center of the circle. Therefore, the three sample detection units 22 can perform IGRA testing and simultaneously complete control testing for at least the three types of detection tubes required for the negative control (N), positive control (P), and sample control (T). When PMMA is used to form the chip top sealing plate 5, the chip top sealing plate 5 is in the shape of 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, a cylinder with a diameter of 120 mm and a thickness of 1 mm.

[0052] In addition, when preparing the chip top sealing plate 5, although the preferred PMMA has excellent light transmittance, if the detection results of the chromatography paper strip 3 on the chip substrate 2 are directly observed through the 1 mm thick PMMA, the PMMA will still have a certain impact on the fluorescence signal on the chromatography paper strip 3: first, the fluorescence signal is weakened, because PMMA will absorb and scatter part of the fluorescence signal, resulting in a decrease in the detected effective signal intensity; second, the absorption characteristics of PMMA under certain specific wavelengths of light will hinder the transmission of the fluorescence signal, that is, the light transmittance of PMMA is limited and is not completely non-absorbent, so 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 chip top sealing plate 5, it is necessary to place the chromatography paper strip 3 on the chip substrate 2 above the paper strip slot 34 at the position corresponding to the chip top sealing plate 5, and to process a paper strip detection window 18 by hollowing out and grooving, thereby forming an opening for observing the detection results on the chromatography paper strip 3 without passing through PMMA; further, in order to ensure the sealing of the chip, it is also necessary to use a pressure-sensitive packaging glue 6 with a thickness of 0.05-0.2 mm to seal the opening position of the paper strip detection window 18 on the chip top sealing plate 5. The preferred material of the pressure-sensitive packaging glue 6 is polypropylene film. Compared with PMMA, the polypropylene film will neither weaken the fluorescence signal nor hinder the collection of the fluorescence signal due to absorption of fluorescence.

[0053] 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 a pin positioning hole 14 is set on the sealing plate 5 at the top of the chip. Figure 6This 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 a pin positioning hole 14. In fact, a pin positioning hole 14 is also provided on the chip bottom plate sealing plate 1. The chip is fixed by the pin positioning holes 14 at corresponding positions on the two-layer structure of the chip top sealing plate 5 and the chip substrate 2. It is preferably fixed by a combination of fixing accessories such as positioning fixing pins, interference fit pins, cotter pins with pins, retaining rings with pins, threaded pins with nuts, etc. After fixing, the corresponding position forms a positioning column, which can effectively prevent the chip from being displaced or rotated during the preparation and assembly process, thereby improving the assembly accuracy and reliability of the chip. In addition, a longitudinal cross-sectional view of the puncture valve in the closed state is shown from the aa angle of the puncture airbag fixing plate 9. Figure 4 shown. Figure 4 The puncture valve 4 is in a complete state, that is, in a closed state. Figure 4 Combine Figure 1 、 Figure 5 As can be seen, the airbag 7 is fixedly mounted on the puncture airbag fixing plate 9, which is in turn fixedly mounted on the chip top sealing plate 5. The chip top sealing plate 5 is provided with a puncture hole 21. Below the puncture hole 21 is the puncture valve 4, shown in dashed lines. The puncture valve 4 consists of two layers of nylon gaskets 11, upper and lower, and an aluminum foil sacrificial layer 10 in between. The cross-sectional structure of the coaxial position where the puncture valve 4 is located, from top to bottom, is: airbag 7, puncture airbag fixing plate 9, puncture hole 21, nylon gasket 11, aluminum foil sacrificial layer 10, and nylon gasket 11. The puncture hole 21 is coaxially aligned with the puncture valve 4, ensuring that the force applied through the puncture hole 21 on the chip top sealing plate 5 can be accurately transmitted to the aluminum foil sacrificial layer 10 of the puncture valve 4 below.

[0054] Regarding the relevant 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 in the vertical direction along 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, consisting of two layers of nylon gaskets 11 and a middle layer of 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 with two layers of polytetrafluoroethylene gaskets and a middle layer of stainless steel sacrificial layer to improve durability. Whether it is nylon with aluminum foil or polytetrafluoroethylene with stainless steel, the puncture valve 4 uses the physical properties of the material to control the opening and closing of the puncture valve by actively applying an external mechanical force. It is an active valve that does not require an additional drive structure. The puncture valve 4 is usually Figure 4The closed state shown effectively prevents unintended fluid flow. To open the channel, simply squeeze the airbag 7 above the puncture hole 21. The airbag 7 and the aluminum foil sacrificial layer 10 deform to varying degrees under pressure. The aluminum foil sacrificial layer 10, due to its poor elasticity, is preferentially damaged, while the airbag 7, due to its elasticity, remains intact. This material combination ensures that the active valve not only opens upon application of force, but also maintains the overall sealing of the microfluidic chip.

[0055] Figure 5 This 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. Figure 1 It can be seen that the chip positioning structures 12 of the same shape are arranged at the centers of the three layers of the chip bottom sealing plate 1, the chip base 2 and the chip top sealing plate 5. Figure 5 It can be seen that the chip positioning structure 12 is a combination of a circular and a flat keyway shape, and the flat keyway is preferably round-headed 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, and ensuring that not only the chip of the present invention can rotate coaxially with the servo motor, but also the chip will not shift during the rotation process, so as to ensure the stability and accuracy of the rotation.

[0056] Figure 5 The dotted line specifically shows a partial enlarged view of a single detection unit 17 on the chip top sealing plate. It can be seen that: on the chip top sealing plate 5, with the center of the circle as the center, three identical single detection units 17 on the chip top sealing plate are distributed at an angle of 120°, and the single detection unit 17 on the chip top sealing plate includes a pin positioning hole 14, a paper strip detection window 18 and 6 holes for gas-liquid interaction. The pin positioning hole 14 is used to prevent the chip from being displaced or rotated during the bonding process, thereby improving the assembly accuracy and reliability of the chip; the paper strip detection window 18 is located above the paper strip slot 34 on the chip substrate 2, and the chromatography paper strip 3 is positioned and placed in the paper strip slot 34, so the detection line and quality control line of the lower chromatography paper strip 3 can be observed from the paper strip detection window 18; the paper strip detection window 18 can be sealed with a transparent pressure-sensitive packaging glue 6 to ensure the sealing of the entire chip; the pressure-sensitive packaging glue 6 is preferably a transparent material with a thickness of 0.1 mm.

[0057] Figure 7 This is a top view of the various hole positions of the chip substrate 2 on the corresponding positions of the chip top sealing plate 5 of the present invention, specifically showing the hole 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 used for sample addition and exhaust of the sample enrichment preparation chamber 23 to be tested, respectively; 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. After the sample to be tested is added from the sample addition hole 13, it can flow to the single-chamber centrifugal sedimentation structure 51, 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 tape or a polyester film (PET film); the diluent chamber injection hole 15 and the diluent chamber exhaust hole 16 are gas-liquid interaction holes of the diluent chamber 28, which are respectively used to add diluent to the diluent chamber 28 and exhaust gas; in terms of position, the diluent chamber injection hole 15 and the diluent chamber exhaust hole 16 are symmetrically distributed at both 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 that of the sample addition hole 13 and the exhaust hole 19 is adopted: preferably, a single-sided tape or a polyester film is used to seal the two holes of the diluent chamber injection hole 15 and the diluent chamber exhaust hole 16. 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 buffer and adjust the air pressure in the single-chamber centrifugal sedimentation structure 51 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 transferred to the aluminum foil sacrificial layer 10 of the puncture valve 4, thereby opening the active valve like the puncture valve 4.

[0058] 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. Each sample detection unit 22 includes a paper strip holder 34 and nine chambers and channels directly or indirectly connected thereto. The chromatography paper strip 3 is positioned and fixed in the paper strip holder 34. The liquid addition and detection operations are identical for each sample detection unit 22. Figure 9 This is a radial stack image formed by centrifuging a blood sample by the 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 10It can be seen from the combination that the sample enrichment preparation chamber 23, the fence structure 25, the red blood cell enrichment chamber 26 and the plasma extraction inlet 24 located on the sample enrichment preparation chamber 23 together form Figure 9 、 Figure 10 The single-chamber centrifugal sedimentation structure 51 shown by the dotted line can be designed in the shape of a fan, an ellipse, a square, a circle, etc. to achieve uniform distribution of centrifugal force. Figure 9-10 The inverted sector ring shown can achieve uniform centrifugal force distribution. 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.

[0059] Since whole blood is mainly composed of plasma and red blood cells, of which plasma accounts for (50%-55%) of the whole blood volume, while red blood cells account for only 45% of the whole blood volume, the IGRA detection marker is gamma-interferon (IFN-γ) secreted by immune cells. The molecular weight of IFN-γ is relatively small, usually 17100 Daltons (Da). After the whole blood sample is centrifuged, cells and larger particles will settle to the bottom, while IFN-γ with a smaller molecular weight will remain in the supernatant where the plasma layer is located. Testing the supernatant collected by centrifugation can effectively avoid the interference of red blood cells in the whole blood sample and improve the accuracy of detecting the gamma-interferon content. Specifically, if the sample to be tested is whole blood, the sample to be tested is first incubated in a single-chamber centrifugal sedimentation structure 51 for 22±4 hours, and then centrifuged. The mass density of each component in the whole blood is different. Under the action of the rotating centrifugal field, as the rotation time increases, along the direction of the radius of the circle, the density increases from low to high, namely plasma, white blood cells and red blood cells. Therefore, a radial stack structure consisting of pure plasma supernatant and red blood cell particles at the bottom of the container will be formed in the chamber of the single-chamber centrifugal sedimentation structure 51. Figure 9 shown.

[0060] Specifically, under the centrifugal force brought by the servo motor, the whole blood sample is sequentially separated into radial stacks in the single-chamber centrifugal sedimentation structure 51 from the proximal end to the distal end. The upper layer is the supernatant of pure plasma, and the lower layer is particles with larger molecular weight such as white blood cells and red blood cells. The layers from the proximal end to the distal end are as follows: Figure 9Shown in order are: plasma layer 54, white blood cell layer 55 and red blood cell layer 56. Since when the servo motor stops rotating, the stratified state between the plasma layer 54, the white blood cell layer 55 and the red blood cell layer 56 will no longer be maintained, and the stack-like structure formed will no longer exist. Therefore, the present invention uses a physical separation method to ensure the continued maintenance of the stratified state by adding a filter mesh-like structure in the single-chamber centrifugal sedimentation structure 51. The present invention preferably provides a filter mesh-like structure in the single-chamber centrifugal sedimentation structure as a fence structure 25. The mesh 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 fence structure 25 is equidistantly arranged around the center of the circle in a stepped filter mesh channel, which provides sufficient interaction space for the red blood cells to settle downward into the red blood cell enrichment chamber 26, allowing the red blood cells to smoothly enter the red blood cell enrichment chamber 26.

[0061] After the sample is loaded and incubated on the chip, it enters the IGAR test. The stimulated sample is first preliminarily enriched in the single-chamber centrifugal sedimentation structure 51. That is, under the centrifugal force generated by the high-speed rotation of the servo motor driving the chip, red blood cells are gradually collected in the red blood cell enrichment chamber 26, and the plasma and white blood cells in the whole blood are enriched in the sample enrichment preparation chamber 23. Through the physical separation of the fence structure 25, the different components in the whole blood sample are preliminarily enriched in layers. That is, red blood cells are sedimented in the red blood cell enrichment chamber 26, and white blood cells are trapped in the layer where the fence structure 25 is located. The top layer is the plasma layer containing gamma interferon. At this time, the puncture valve 4 is in Figure 4 In the closed state shown, the sample in the single-chamber centrifugal sedimentation structure 51 will not be released into the next-stage chamber through the plasma extraction inlet 24 .

[0062] Among them, the plasma extraction inlet 24 is set at the center position of the intersection of the sample enrichment preparation chamber 23 and the fence structure 25, which can also be said to be located at the impact interface between the preliminary stratified enriched plasma layer and the red blood cells, that is, at the liquid surface junction of the upper purified plasma and the lower red blood cells, and ensure that when the liquid level of the liquid in the single-chamber centrifugal sedimentation structure 51 is lower than the plasma extraction inlet 24, the liquid will not continue to transfer to 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 no less than 1 mL, wherein the main accommodating chambers in the single-chamber centrifugal sedimentation structure 51 include the sample enrichment preparation chamber 23 and the red blood cell enrichment chamber 26. The volume of the sample 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 enrichment preparation chamber 23 is 613 μL, and the volume of the red blood cell enrichment chamber 26 is 450 μL.

[0063] Figure 10 It is also shown that the center points of the plasma extraction inlet 24 on the single-chamber centrifugal sedimentation structure 51 and the puncture valve fixed stepped hole 47 below are located on the same axis bb of the chip. Figure 11 Specifically, a longitudinal cross-sectional view of the axis line bb is shown. It can be seen that when the puncture valve 4 is open, the flow direction of the liquid in the single sample detection unit 22 on the chip substrate 2 and in the plasma transfer channel 48 is as follows: Figure 10 Indicated by the arrow. Figure 10 and Figure 11 It can be seen that the plasma transfer channel 48 is located at the bottom of the chip substrate 2, and the plasma extraction inlet 24 and the puncture valve fixed stepped hole 47 are connected through the plasma transfer channel 48. After the liquid flows into the plasma extraction inlet 24, it flows through the plasma transfer channel 48 and then flows out of the puncture valve fixed stepped hole 47. Specifically, the puncture valve fixed 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 middle puncture valve fixed stepped hole 47 as a fixed 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 an open state, the first stepped large hole is communicated with the annular flow channel 46, and the liquid flows out of the first stepped large hole of the puncture valve fixed 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 an aluminum hole for the puncture valve 4 After the foil sacrificial layer 10 is punctured, the reserved space for the residual aluminum foil prevents the residual aluminum foil from clogging the liquid flow channel, ensuring the smooth flow of the liquid. The second step hole is smaller in diameter and shallower in depth than the first step large hole, and is drilled downward from the step surface of the first step large hole. The depth of the second step hole is 0.5-1.5mm, preferably 1mm; the third step small hole formed within the innermost circle serves as the main outflow hole for the liquid, extending deeply downward to connect with the plasma transfer channel 48. The plasma transfer channel 48 is 1-3 mm long, and the inner diameter of the plasma transfer channel 48 is 15-200 microns. 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. Due to Figure 11 The diagram shows the state where the puncture valve 4 is open, i.e., the aluminum foil sacrificial layer 10 in the middle of the puncture valve 4 has been destroyed and is connected to the puncture hole 21 on the sealing plate 5 at the top of the chip. Although the puncture valve 4 can be connected to the puncture hole 21 when opened, there is still sealing protection provided by the airbag 7 on the sealing plate 5 at the top of the chip, which is coaxially installed with the puncture hole 21 and the puncture valve 4. Therefore, when the puncture valve 4 is open, the chip is still in a sealed environment.

[0064] Figure 8 Each sample detection unit 22 includes 9 chambers that can accommodate gas or liquid, in addition to the above Figure 9-10The single-chamber centrifugal sedimentation structure 51 includes two chambers, namely the sample enrichment preparation chamber 23 and the red blood cell enrichment chamber 26, and further includes an air pressure 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 red blood cell collection chamber 42, which form a circular center 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. Figure 8 It can be seen that the liquid of the sample to be tested flows through different chambers and various channels for gas-liquid interaction between different chambers. Finally, the diluted plasma is fully mixed in the mixing chamber 36 and then transferred to the chromatography paper strip 3 at the position of the paper strip card slot 34 by siphoning for IGRA detection.

[0065] Among them, the air pressure buffer chamber 49 is connected to the single-chamber centrifugal sedimentation structure 51 through the air pressure buffer chamber connecting channel 50. The air pressure buffer chamber 49 of the chip substrate 2 is opposite to the air pressure buffer hole 20 of the upper chip top sealing plate 5. At the position of the air pressure buffer hole 20, the buffer airbag fixing plate 8 is installed on the chip top sealing plate 5, and the airbag 7 is further installed on the buffer airbag fixing plate 8. Therefore, from top to bottom in this vertical direction, they are: airbag 7, buffer airbag fixing plate 8, air pressure buffer hole 20, and air pressure buffer chamber 49. Therefore, the air pressure changes in the single-chamber centrifugal sedimentation structure 51 can be transmitted to the air pressure buffer chamber 49 through the air pressure buffer chamber connecting channel 50, and the air pressure received in the air pressure buffer chamber 49 can also be transmitted to the airbag 7 on the upper layer of the chip, and finally buffered by the airbag 7 made of 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 to the internal gas circulation channel 27 from left to right include the diluent chamber 28, the mixing chamber 35, the two quantitative chambers of the plasma quantitative chamber 45 and the red blood cell collection chamber 42 in the center-colinear interconnection structure 52, and the waste liquid collection chamber 44; since the flow of liquid between the chambers is not in a vacuum state, it is necessary to have gas in the constructed closed space to circulate continuously to assist in driving the flow of the liquid, so the internal gas circulation channel 27 ensures the gas-liquid replacement in the closed space where the chip is located.

[0066] Figure 12 This is a partial enlarged view of the center-collinear interconnection structure 52 in a single sample detection unit 22 on the chip substrate 2, combined with Figure 8 、 Figure 11 and Figure 12 Visible: Due to Figure 12The center-collinear interconnected structure 52 shown by the middle dotted line includes two chambers, a plasma quantitative chamber 45 and a red blood cell collection chamber 42, and three stepped channels connecting the two chambers: a three-way left stepped channel 40, a three-way middle liquid inlet stepped channel 41, and a three-way right exhaust stepped channel 43. Since the center of the circle between the two chambers is located on the same axis, and the three stepped channels are located in the middle of the two chambers and connect the two, the structure consisting of the two chambers and the three stepped channels is called a center-collinear interconnected structure 52, wherein the three stepped channels are also called "three-way stepped channels" or "center-collinear interconnected stepped channels". The synergistic effect of the three stepped channels can provide sufficient interaction space for the formation of the stratified interface and stratified transfer between plasma and red blood cells. The center-collinear interconnection structure 52 is connected to the mixing chamber 35 through the plasma siphon channel 36, wherein the entrance of the plasma siphon channel 36 is connected to the three-way left stepped channel 40 in the center-collinear interconnection structure 52, which is conducive to extracting the pure plasma sample quantitatively collected in the plasma quantitative chamber 45 into the mixing chamber 35 through the siphon action of the plasma siphon channel 36. Since the entrance of the plasma siphon channel 36 is located below the plasma quantitative chamber 45, the siphon action only collects and transfers the plasma in the upper plasma quantitative chamber 45, and can also avoid collecting the blood sample containing red blood cells in the lower red blood cell collection chamber 42.

[0067] Regarding the center-colinear interconnected structure 52, specifically: of 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 volumes 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 differences in size between the three stepped channels. The diameters of the two side channels of the three-branch left stepped channel 40 and the three-branch right exhaust stepped channel 43 are 0.2-1 mm. The diameter of the liquid inlet stepped channel 41 in the middle of the three-branch is slightly larger than the diameters of the two side channels. The optional channel diameter 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 the diameter of the three-branch middle liquid inlet stepped channel 41. The adjacent three-branch middle liquid inlet stepped channel 41 and the three-branch right exhaust stepped channel 43 form a channel aperture with such a significant difference in diameter, which can reduce the resistance of the liquid to flow through the three-branch middle liquid inlet stepped channel 41. For example, the agglomerated red blood cells increase the viscosity of the liquid. The wider diameter of the three-branch middle liquid inlet stepped channel 41 can ensure that liquids containing red blood cells of different viscosities preferentially flow through the three-branch middle liquid inlet stepped channel 41 into the red blood cell collection chamber 42.

[0068] The specific channel diameters can be customized based on the specific requirements of the IGRA reaction system. The diameters of the three-branch left stepped channel 40 and the three-branch right exhaust stepped channel are not significantly different, preferably being the same at 0.4 mm. The preferred diameter of the three-branch middle liquid inlet stepped channel 41 is 1 mm. Since the plasma siphon channel 36 communicates with the three-branch left stepped channel 40, the diameter difference between the two channels is also not significant. The diameter of the plasma siphon channel 36 is 0.2-1 mm, preferably 0.4 mm. In particular, the entrance of the plasma siphon channel 36 forms a certain angle with the three-way left stepped channel 40 after being connected, that is, the elevation angle θ formed by the plasma siphon channel 36 and the vertical line of the three-way left stepped channel 40 ranges from 10° to 45°. Within this angle range, combined with the length adjustment of the plasma siphon channel 36, it is ensured that when the servo motor drives the chip to rotate, the centrifugal force generated on the transfer siphon channel 36 and the plasma quantitative chamber 45 at the same centrifugal radius is consistent, that is, the liquid levels of plasma in the transfer siphon channel 36 and the plasma quantitative chamber 45 are maintained at 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 still receive the centrifugal force 58 along the distal direction of the centrifugal force component 59. Under the action of the centrifugal force component 59, the red blood cell particles 57 therein can be caused to settle into the red blood cell collection chamber 42, thereby not only avoiding the accumulation of red blood cell particles 57 in the plasma siphon channel 36 to cause blockage, but also avoiding the red blood cell particles 57 from being transferred through the plasma siphon channel 36 into the mixing chamber 35.

[0069] Therefore, the design of the three-branch middle liquid inlet stepped channel 41 with a larger diameter and lower resistance, in conjunction with the design of maintaining a certain elevation angle θ between the plasma siphon channel 36 and the three-branch left stepped channel 40, not only ensures that the red blood cell liquid preferentially flows smoothly through the three-branch middle liquid inlet stepped channel 41 into the red blood cell collection chamber 42, but also ensures that red blood cell particles 57 that accidentally enter the plasma siphon channel 36 prematurely flow smoothly into the red blood cell collection chamber 42 under the action of the external centrifugal force component 59. In addition, it allows the gas generated during the liquid flow process to be smoothly discharged through the three-branch right exhaust stepped channel 43 with a smaller channel diameter, ensuring that the liquid and gas can freely exchange gas and liquid in the centrally collinear interconnected structure 52. The centrally collinear interconnected structure 52 can also control the direction and magnitude of the angular velocity of the servo motor, thereby controlling the flow direction of the blood samples of different layers based on their respective mass characteristics and the inertial force they are subjected to. It can be seen that the mutual cooperation between the three stepped channels in the center-collinear interconnected structure 52 not only ensures the smooth communication of the liquid between the two chambers, but also the difference in diameters between the three stepped channels and the angle design of the elevation angle θ between the plasma siphon channel 36 and the three-branch left stepped channel 40 make the three stepped channels each assume a specific function.

[0070] Regarding the waste liquid collection chamber 44. Since the single-chamber centrifugal sedimentation structure 51 and the center-of-circle colinear 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, the plasma extraction inlet 24 of the single-chamber centrifugal sedimentation structure 51 and the puncture valve fixed stepped hole 47 are connected through the plasma transfer channel 48, and since the puncture valve fixed stepped hole 47 above the plasma transfer channel 48 can be connected to the annular flow channel 46, there is an arc-shaped drop channel away from the center of the circle at the connection between the puncture valve fixed stepped hole 47 and the annular flow channel 46, so that the distance between the annular body of the annular flow channel 46 and the center of the circle (that is, the radius of the annular flow channel 46) is greater than the distance between the puncture valve fixed stepped hole 47 and the center of the circle. 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 puncture valve fixed stepped hole 47. Figure 12As shown, when the puncture valve 4 is destroyed by external force and is in an open state, the liquid flows out of the puncture valve fixed stepped hole 47 through the plasma transfer channel 48, and can further start from the puncture valve fixed stepped hole 47, through the annular flow channel 46 connected to the puncture valve fixed stepped hole 47 to flow into the plasma quantitative chamber 45 and the red blood cell collection chamber 42 of the circle center collinear interconnection structure 52, wherein the red blood cell collection chamber 42 is a blind hole structure. In the preliminary experiment, only two stepped channels were designed (equivalent to the three-way liquid inlet stepped channel 41 and the three-way right exhaust stepped channel 43, and the inlet of the plasma siphon channel 36 was directly connected to the three-way liquid inlet stepped channel 41). 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 for the present invention Figure 12 The three stepped gas-liquid replacement channel structures shown can avoid the phenomenon of liquid flowing into and blocking the exhaust channel, thereby improving the gas-liquid replacement efficiency of the red blood cell collection chamber 42 and ensuring smooth inflow of liquid and free discharge of gas.

[0071] Annular flow channel 46 is positioned above plasma quantification chamber 45 and waste liquid collection chamber 44, connecting these two chambers from above. Since both plasma quantification chamber 45 and red blood cell collection chamber 42 have fixed capacities (e.g., preferably 100 μL each), when liquid flowing from single-chamber centrifugal sedimentation structure 51 into centrally collinear interconnected structure 52 exceeds a certain volume of the two chambers, and the excess liquid level rises above annular flow channel 46, it can flow through annular flow channel 46 into waste liquid collection chamber 44, thereby ensuring that plasma quantification chamber 45 is filled with the required fixed volume (e.g., preferably 100 μL) of pure plasma. The waste liquid collection chamber 44 is thus primarily due to the specific configuration of annular flow channel 46 and the fixed capacity of the two chambers in centrally collinear interconnected structure 52. From another perspective, the provision of waste liquid collection chamber 44 and annular flow channel 46 ensures that the plasma quantification chamber 45 in the single-chamber centrifugal sedimentation structure 51 can retain a fixed amount of pure plasma solution.

[0072] Regarding the diluent chamber 28, since the plasma quantification chamber 45 contains pure plasma, which generally has a high viscosity, if directly transferred to the chromatography paper strip 3, not only will it not flow smoothly, but the high concentration may also lead to a false negative test result due to the "hook effect," making it difficult to ensure the accuracy of the IGRA test performed on the chromatography paper strip 3. Therefore, the high-viscosity plasma can be diluted with a diluent to reduce the viscosity of the plasma to be tested and ensure smooth flow and migration on the chromatography paper strip 3. Generally, the concentration of the plasma can be diluted in a multiple ratio, that is, diluted twice. If the quantitative volume of the plasma quantification chamber 45 is preferably 100 μL, the volume of the diluent chamber 28 can be set to no less than 100 μL. The diluent chamber 28 is connected to the mixing chamber 35 via the diluent transfer siphon channel 29, and the diluent can be guided from the diluent chamber 28 to the mixing chamber 35 by siphoning. 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 detection, a first capillary valve 30 is set in a certain part of the diluent transfer siphon channel 29. The function of the first capillary valve 30 is to provide a certain resistance. Since the total energy of the liquid will increase when it expands at the solid-liquid interface, the energy of the flowing liquid that slowly increases during the expansion process can gradually overcome the resistance obstacle of the first capillary valve 30. 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 described in the present invention to rotate, the diluent can be transferred to the mixing chamber only when a certain critical speed is reached. For example, when the sample to be tested is centrifuged and preliminarily enriched, under the action of the centrifuge, the diluent flowing in the diluent transfer siphon channel 29 can break through the resistance of the first capillary valve 30, and thus gradually transferred to the mixing chamber 35 under the action of the siphon.

[0073] Desiccant chamber 32 contains a solid desiccant that absorbs and removes gaseous or liquid water molecules, thereby reducing the humidity in the chamber connected to desiccant chamber 32. One end of desiccant chamber 32 is connected to mixing chamber 35 via a first desiccant chamber connection channel 31, while the other end is connected to the chamber containing the paper strip slot 34 via a second desiccant chamber connection channel 33. Therefore, desiccant chamber 32 is capable of maintaining the humidity in two enclosed chambers: mixing chamber 35 and paper strip slot 34, where the chromatography paper strip 3 is located. In particular, since the whole blood sample to be tested undergoes an incubation process at 37°C in the single-chamber centrifugal sedimentation structure 51 of the chip of the present invention, the incubation process will form water mist in the entire closed chamber. 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 paper strip slot 34 is located. If there are too many water droplets, they will drip onto the interference chromatography paper strip 3, thereby affecting the intensity of the fluorescence signal on the test line and the quality control line, and thus affecting the accuracy of the test. Therefore, by providing a desiccant chamber 32 connected to the mixing chamber 35 and the paper strip slot 34 in the last two steps of liquid flow, the moisture in these two closed chambers is absorbed to ensure the optimal reaction humidity conditions in the final IGRA detection environment, thereby improving the accuracy of IGRA detection.

[0074] Regarding the mixing chamber 35, the mixing chamber 35 is used as a chamber for mixing the plasma of the sample to be tested and the diluent to obtain the diluted plasma. For plasma with higher viscosity, magnetic beads 53 can be added to the mixing chamber 35 as a dynamic "agitator", which will be affected not only by the centrifugal force and the Euler force generated by the acceleration and deceleration of the servo motor, but also by the Coriolis force generated by the movement under the action of the centrifugal force; therefore, the plasma and diluent in the mixing chamber 35 are preferably fully contacted and mixed under the combined action of the magnetic beads and the inertial forces such as the centrifugal force, the Euler force, and the Coriolis force; wherein the centrifugal force is the inertial force introduced in the rotating reference frame, "simulating" the object trying to fly in the tangential direction due to inertia. The Euler force is an inertial force generated in a non-uniformly rotating reference frame (where the angular acceleration of the rotation is non-zero). Its direction is perpendicular to the plane defined by the angular acceleration and the object's position vector (i.e., a deflection force perpendicular to the radial direction during uniform acceleration). Its magnitude depends on the angular acceleration, the object's mass, and its distance from the rotation axis. The Coriolis force is an inertial force exerted on an object due to linear motion in a rotating reference frame. Its direction is perpendicular to the plane defined by the angular velocity vector and the object's relative velocity vector. Its magnitude depends on the angular velocity, the object's mass, and its relative velocity. Furthermore, other mixing methods that are beneficial for mixing the plasma and diluent may be employed in the mixing chamber 35, such as ultrasonic mixing, mechanical stirring, vortex oscillation, and the like.

[0075] Figure 13 This is a schematic diagram of the design of the mixing chamber 35 for transferring the diluted plasma obtained after mixing. 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, and they gradually narrow asymmetrically from wide to narrow. Therefore, under the action of centrifugal force, the liquid flowing into the mixing chamber 35 will theoretically form a radial stacking layer similar to that 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 stacked layers formed by the inflowing liquid are concentrated at the bottom of the mixing chamber 35, which is biased towards the wider side, that is, gradually filling the mixing chamber 35. Figure 12 As shown in the figure, the siphon valve inlet is at the lower cavity; 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 into the mixed liquid siphon channel 37. As the liquid flows into 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 into 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 at Figure 13 At the position of the siphon valve inlet upper chamber 61 shown, liquid will continue to flow through the liquid outlet until all the liquid in the siphon valve inlet upper chamber 61 is completely transferred out through the siphon principle. When the liquid in the mixing chamber 35 only remains to the volume of the siphon valve inlet lower chamber 60, that is, when the liquid level drops below the liquid outlet position of the mixing chamber 35, the siphon phenomenon is broken and the siphon flow of liquid will cease. The arc between the stacked layers of liquid in the mixing chamber 35 is the theoretical stratification line of the two virtual chambers: the siphon valve inlet lower chamber 60 and the siphon valve inlet upper chamber 61.

[0076] 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: an inlet for transferring diluent from the diluent chamber 28 into the mixing chamber 35 through the diluent transfer siphon channel 29; an inlet for transferring a predetermined amount of plasma from the plasma quantitative chamber 45 of the circular collinear interconnection structure 52 into the mixing chamber 35 through the plasma siphon channel 36; an inlet for communicating with the desiccant chamber 32 through the desiccant chamber second connecting channel 33 and capable of maintaining the humidity of the mixing chamber 35; and an inlet for the internal air circulation channel 27 for sealing the free circulation of air within the chip. Figure 8 It can be seen that the inlets located at the upper end of the mixing chamber 35 are arranged from left to right in sequence as the inlets of the desiccant chamber second connecting channel 33, the diluent transfer siphon channel 29, the internal gas circulation channel 27 and the plasma siphon channel 36.

[0077] As can be seen, after mixing the plasma and diluent flowing into the mixing chamber 35, the mixing liquid is then quantitatively transferred to the chromatography paper strip 3 for IGRA analysis using the siphon principle. Regarding the volume of the mixing chamber 35, if the custom volume of the plasma quantification chamber 45 is 100 μL, the corresponding volume of the diluent chamber 28 is generally custom-set to 100 μL. The theoretical total volume of diluted plasma ultimately flowing into the mixing chamber 35 and obtained after mixing is 200 μL. Since only the liquid forming the fan-shaped ring in the upper chamber 61 of the siphon valve inlet is above the arc line (or delamination line), it can flow out through the mixed liquid siphon channel 37. For example, if the custom volume of the lower chamber 60 of the siphon valve inlet is preferably 130 μL, then approximately 70 μL of liquid will remain in the upper chamber 61 of the siphon valve inlet, allowing a roughly quantitative amount (70 μL) of liquid to be siphoned away. It can be seen that by optimizing the position of the mixed liquid siphon channel 37 within the specific shape of the mixing chamber 35, the quantitative transfer of the sequentially purified and diluted plasma to the chromatography paper strip 3 is achieved to complete the IGRA detection.

[0078] For the same reason as the first capillary valve 30 is provided in the diluent transfer siphon channel 29, in addition to being connected to the mixing chamber 35 and the paper strip slot 34 through the mixed liquid siphon channel 37, in order to prevent the diluted plasma mixture from filling the mixed liquid siphon channel 37 and entering the paper strip slot 34 prematurely due to capillary action at the initial stage of detection, a second capillary valve 38 and a mixed liquid serpentine channel 39 are provided in sequence 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 paper strip slot 34. Therefore, the mixing chamber 35 and the paper strip slot 34 are connected through the mixed liquid siphon channel 37 and the mixed liquid serpentine channel 39. The second capillary valve 38 is connected to 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 through the siphon due to the resistance of the second capillary valve 38. However, when the servo motor rotates at high speed, the mixed liquid filled in 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. It enters the sample loading area on the chromatography paper strip 3 in the space where the paper strip slot 34 is located along the mixed liquid serpentine channel 39, and then flows smoothly on the chromatography paper strip 3 and diffuses chromatographically to the detection T line and C line. Finally, the detection results of the mixed liquid on the T line and C line are observed and recorded by the sample detection unit 22.

[0079] Example 2 Assembly of a Centrifugal Microfluidic Detection Chip for Tuberculosis-Specific Cellular Immunity

[0080] Since the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity of the present invention mainly consists of three layers from bottom to top: the chip bottom sealing plate 1, the chip substrate 2 and the chip top sealing plate 5, the assembly is sequentially assembled from bottom to top, such as Figure 1-Figure 2 As shown in the exploded view. Since all three layers have chip positioning structures 12 of the same shape and corresponding pin positioning holes 14 (preferably three), the chip positioning structures 12 and pin positioning holes 14 on the three layers are sequentially aligned for assembly. To ensure the accuracy of IGRA testing, assembly work needs to be completed in a clean environment. The specific assembly process is as follows:

[0081] Step 1: Preparation. Check the dimensional accuracy of the three-layer structure of the chip bottom plate sealing plate 1, chip substrate 2, and chip top sealing plate 5, and check whether the chip positioning structure 12 and the pin positioning hole 14 match each other. Check whether other accessories, reagents, or materials to be installed are complete, clean, and usable.

[0082] Step 2: Assemble the chip base plate sealing plate 1. Preferably, a positioning pin (1 mm outer diameter, 8 mm long) is used for locking. Insert the positioning pin from bottom to top through the pin positioning hole 14 on the chip base plate sealing plate 1. The positioning pin has an exposed positioning column (approximately 7 mm long).

[0083] Step 3: Assembly of chip substrate 2. After the chip bottom plate sealing plate 1 is glued at intervals, the chip substrate 2 is aligned with the exposed positioning pins on the chip bottom plate sealing plate 1 and inserted into the pin positioning holes 14 on the chip substrate 2 to lock and fix them. The chip bottom plate sealing plate 1 and the chip substrate 2 are packaged by gluing. The chromatography paper strip 3 and the puncture valve 4 are respectively fixed to the paper strip slot 34 position and the puncture hole 21 of each sample detection unit 22 of the chip substrate 2, and the magnetic beads 53 are placed in the mixing chamber 35. The desiccant chamber 32 is filled with desiccant. The specific liquid is pre-loaded in the sample enrichment preparation chamber 23 of the single-chamber centrifugal sedimentation structure 51 to make the sample enrichment preparation chamber 23. The negative control (N), positive control (P) and sample control (T); if only matrix liquid is pre-installed in the sample enrichment preparation chamber 23 to be tested, the corresponding sample detection unit is marked as negative control (N); if both matrix liquid and non-specific stimulating antigen of tuberculosis (such as phytohemagglutinin PHA) are pre-installed in the sample enrichment preparation chamber 23 to be tested, the corresponding sample detection unit is marked as positive control (P); if both matrix liquid and Mycobacterium tuberculosis specific antigen (such as ESAT-6 / CFP-10) are pre-installed in the sample enrichment preparation chamber 23 to be tested, the corresponding sample detection unit is marked as sample control (T);

[0084] Step 4: Assemble the chip top sealing plate 5. After applying glue near the pin positioning hole 14 on the chip substrate 2, align the chip top sealing plate 5 with the positioning post (approximately 1 mm long) that is still exposed on the chip substrate 2. Insert the pin positioning hole 14 on the chip top sealing plate 5 and lock it in place. The chip substrate 2 and the chip top sealing plate 5 are packaged together using glue. At the center axis of the puncture hole 21, the puncture airbag fixing plate 9 and the airbag 7 are assembled from bottom to top. At the center axis of the air pressure buffer hole 20, the buffer airbag fixing plate 8 and the airbag 7 are assembled from bottom to top. At the opening of the paper strip detection window 18 on the chip top sealing plate 5, a 0.1 mm thick polypropylene film is used as a pressure-sensitive encapsulating adhesive 6 to seal the opening.

[0085] At this point, the entire assembly process for the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity is complete. Since the chip is pre-loaded with matrix fluid, nonspecific TB stimulating antigens, and Mycobacterium tuberculosis-specific antigens, the assembled, sealed centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity is stored at 2-8°C until use.

[0086] During the assembly process, the three-layer structure—chip bottom plate sealing plate 1, chip substrate 2, and chip top sealing plate 5—is not only locked in place by positioning pins but also sealed by adhesive dispensing. Furthermore, in addition to the aforementioned adhesive sealing, other sealing methods such as heat press sealing, laser welding, or ultrasonic welding can also be used to sequentially seal each layer to further ensure the chip's sealing properties.

[0087] Example 3 Application of the Centrifugal Microfluidic Detection Chip IGRA for Tuberculosis-Specific Cellular Immunity

[0088] 1. IGRA detection process of tuberculosis-specific cellular immunity using a centrifugal microfluidic chip

[0089] The first step is to prepare for the test. Figure 1-Figure 2 The assembled tuberculosis-specific cellular immunity centrifugal microfluidic detection chip with three detection units is shown. The three detection units serve as negative control (N), positive control (P), and sample control (T), respectively. The chip is equilibrated at room temperature for at least 20 minutes.

[0090] The second step is sample addition and sealing. The sample and diluent to be tested are taken and pipetted into the corresponding single-chamber centrifugal sedimentation structure 51 and diluent chamber 28 through the sample addition hole 13 and diluent chamber injection hole 15 of each detection unit, respectively, to complete the liquid addition operation. After the liquid is added, polyester film is preferably used as a sealant to seal the sample addition hole 13, vent 19, diluent chamber injection hole 15, and diluent chamber vent 16.

[0091] Step 3: Incubation. Place the chip in a constant temperature environment at 37°C for 22±4 hours. During the incubation process, the temperature will cause changes 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 via the air pressure buffer chamber connecting channel 50. The elastic changes of the airbag 7 mitigate the drastic changes in air pressure.

[0092] Step 4: IGRA detection. The chip is placed steadily in the corresponding position of the flange block on the servo motor using the chip positioning structure 12. Subsequent separation, reagent mixing, and IGRA strip detection operations can then be performed, and the test results can be observed through the strip detection window 18 on the sealing plate 5 at the top of the chip. The fluorescence signals of the test line and quality control line on the chromatography strip 3 are observed through the strip detection window 18. The fluorescence signals can be qualitatively determined by the naked eye or quantitatively detected and calculated using a fluorescence scanner to determine the quantitative concentration of the stimulated γ-interferon produced in the test sample. Specifically, after the sample loading and incubation operations are completed on the chip, the specific process of IGRA detection begins as follows:

[0093] During the first centrifugation, after incubation and stimulation, the sample undergoes initial centrifugal stratification and enrichment in the single-chamber centrifugal sedimentation structure 51. The servo motor accelerates to a high-speed rotation state (2500 to 3000 rpm, preferably 3000 rpm) at an acceleration of 500 rpm / s, driving the chip to rotate at high speed for 2 to 6 minutes. After the servo motor reaches the set rotation time (preferably 3 minutes), it decelerates to a stationary state at a deceleration rate of 500 rpm / s. During this centrifugation, plasma and white blood cells in the whole blood are enriched in the sample enrichment preparation chamber 23. Physically separated by the fence structure 25, the top layer of the sample enrichment preparation chamber 23 contains the plasma layer containing gamma interferon, white blood cells are trapped in the layer containing the fence structure 25, and red blood cells settle into the red blood cell enrichment chamber 26. Simultaneously with the first centrifugation, the diluent fills the diluent transfer siphon channel 29.

[0094] Open the puncture valve 4. Apply a vertical downward force to the airbag 7 on 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 a Figure 11 Shown in open state.

[0095] During the second centrifugation, the servo motor is accelerated 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 at a high speed for 2 to 6 minutes. When the servo motor reaches the set rotation time (preferably 3 minutes), it is decelerated to a stationary state at a deceleration rate 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 siphoning and centrifugal force, enters 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, the diluent in the diluent storage chamber 28 breaks through the resistance of the first capillary valve 30 and, under the combined action of siphoning and centrifugal force, enters the lower chamber 60 of the siphon valve inlet of the mixing chamber 35 along the diluent transfer siphon channel 29. When the puncture valve 4 is in the open state, the plasma that has been initially stratified and enriched in the sample enrichment preparation chamber 23 in the single-chamber centrifugal sedimentation structure 51 flows from the plasma extraction inlet 24 to the plasma transfer channel 48, and then flows out from 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 center-collinear interconnected structure 52 through the annular flow channel 46. Under the synergistic effect of centrifugal force and Euler force, the residual red blood cells in the plasma that has been enriched for the first time from the sample enrichment preparation chamber 23 will enter the red blood cell collection chamber 42 through the three-way inlet stepped channel 41, and the red blood cell collection chamber 42 will be collected. The gas in the collection chamber 42 will be discharged along the three-branch right exhaust stepped channel 43. As the plasma in the sample enrichment preparation chamber 23 continues to flow in and centrifuge, the remaining red blood cells are enriched for the second time in the lower layer of the red blood cell collection chamber 42. After the red blood cell collection chamber 42 is filled, the relatively pure plasma continues to fill 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 three-branch left stepped channel 40 connected to the red blood cell collection chamber 42 in advance during the process of flowing into the red blood cell collection chamber 42. However, due to the certain elevation angle θ between the siphon transfer channel 36 and the perpendicular to the three-branch left stepped channel 40, this elevation angle θ causes the red blood cell particles 57 that have entered the siphon transfer channel 36 to move at a uniform speed under the influence of the centrifugal force 58 and the centrifugal force component 59 in the distal direction, until all the red blood cells are finally settled in the red blood cell collection chamber 42. Therefore, the plasma that has undergone preliminary enrichment from the sample enrichment preparation chamber 23 undergoes secondary red blood cell filtration and pure plasma enrichment in the centrally aligned interconnected structure 52. The purified plasma from the second enrichment is then quantitatively stored in the plasma quantitative chamber 45. After the second centrifugation ends, the purified plasma in the plasma quantitative chamber 45 fills the siphon transfer channel 36. Once the siphon transfer channel 36 is completely filled with plasma, a third centrifugation is performed.

[0096] During the third centrifugation, the servo motor accelerates at 300 rpm / s to a high-speed rotation state (1000 to 2000 rpm, preferably 1500 rpm), driving the chip to rotate at high speed for 1 to 5 minutes. After the servo motor reaches the set rotation time (preferably 2 minutes), it decelerates at 100 rpm / s to a stationary state. During the third centrifugation, 100 μL of pure plasma quantitatively enriched in the plasma quantification chamber 45 is transferred through the siphon transfer channel 36 to the mixing chamber 35, where it is thoroughly mixed with the 100 μL of diluent introduced during the first centrifugation by the magnetic beads. When the mixture of 100 μL of pure plasma and 100 μL of diluent is present in both the siphon valve inlet lower chamber 60 and the siphon valve inlet upper chamber 61 of the mixing chamber 35, a fourth centrifugation is performed.

[0097] For the fourth centrifugal 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, each completed acceleration and deceleration cycle is regarded as a mixing process, in order to ensure sufficient and uniform mixing, it is necessary to complete 5-15 mixing processes, preferably 10 mixing processes; the centrifugal mixing operation utilizes the Euler force generated by the acceleration and deceleration during the centrifugal process to shorten the mixing time and improve the mixing efficiency; in addition, since there are magnetic beads 53 in the mixing chamber 35, the magnetic beads 53 act as It is a dynamic "stirrer" that moves continuously under the influence of centrifugal force and other inertial forces. This movement enhances the contact and diffusion between the liquids, thereby improving the mixing efficiency and uniformity. It can be seen that the pure plasma and the diluent are mixed in the mixing chamber 35 through repeated centrifugal mixing and the synergistic effect of the dynamic stirring of the magnetic beads 53, which can further effectively stir and mix the mixed liquid in the mixing chamber 35. After completing the last centrifugal mixing process, it is decelerated to a stationary state at a deceleration rate 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 diluted plasma, the fifth centrifugation is performed.

[0098] During the fifth centrifugation, the servo motor accelerates at 300 rpm / s to a high-speed rotation state (2500 to 3500 rpm, preferably 3000 rpm), driving the chip to rotate at high speed for 1 to 4 minutes. After the servo motor reaches the set rotation time (preferably 2 minutes), it decelerates to a stationary state. The 70 μL of diluted plasma in the upper chamber 61 at the siphon valve inlet of the mixing chamber 35 is transferred to the sample well of the sample pad on the chromatography paper strip 3 fixed to the paper strip holder 34. The absorbent pad at the other end of the sample pad on the chromatography paper strip 3 facilitates the observation or quantitative detection of the fluorescent signal displayed by the diluted plasma flowing on the chromatography paper strip 3. The diluted plasma in the lower chamber 60 at the siphon valve inlet is not transferred because its liquid level is below the lower edge of the liquid outlet of the mixing chamber 35 (the inlet of the mixed liquid siphon channel 37).

[0099] 2. Data Analysis of IGRA Detection of Tuberculosis-Specific Cellular Immunity Using a Centrifugal Microfluidic Detection Chip

[0100] The intensity values ​​of the fluorescence signals corresponding to the detection line (T line) and the quality control line (C line) on the chromatographic paper strip 3 of the detection unit where the negative control (N), positive control (P) and sample control (T) are located are quantitatively detected by a fluorescence scanner to obtain a double-peak or single-peak curve, and the following is performed: Figure 15 The data preprocessing steps shown are as follows:

[0101] Figure 15 1 is the raw data of the fluorescence signal. The distribution of the fluorescence intensity value on the corresponding chromatography paper strip 3 generally shows the following curve: Figure 15 In the double-peak or single-peak curve graph shown in FIG, the first peak curve corresponds to the IFN-γ detection line T on the chromatography paper strip 3, and includes a numerical curve of the raw data of the negative control N, positive control P, and sample control T test results. The second peak curve corresponds to the quality control line C. The fluorescence signal intensity values ​​on both sides of the peak of the curve gradually decrease, indicating the absence of nonspecific binding sites.

[0102] Figure 15 2 is the filtering and normalization analysis, that is, filtering, smoothing and normalization processing are performed on the original data.

[0103] Figure 15 Figure 3 shows the peak-finding algorithm analysis. Using first-order differential processing and monotonic feature extraction, we determined the starting point 1A1, peak point 1F1, and end point 1Z1 of the peak curve for the test line, and the starting point 2A2, peak point 2F2, and end point 2Z2 of the peak curve for the quality control line. Peak points F1 and F2 (for both the test and quality control lines) can be used as characteristic values ​​to indicate a positive correlation between fluorescence signal intensity and IFN-γ concentration. Specifically, a higher peak value indicates a stronger fluorescence signal and a higher IFN-γ concentration.

[0104] Figure 15 Figure 4 shows the calculation of the baseline and integrated area. The left baseline of the test line (left and right baselines) is calculated by first moving the peak point F1 to the left by a distance of 1 (d1), where d1 = 2 × d2, and d2 is distance 2. Starting from the new position at d1, the fluorescence signal intensity is then moved to the right by a distance of 2 (d2). The average value of the fluorescence signal intensity between the left shift d1 and the right shift d2 is calculated, and this average value is used as the left baseline of the test line. A similar method is used to calculate the right baseline of the test line. The average of these two baselines (left and right) is then used as the final baseline value corresponding to the peak curve of the test line. The final baseline value of the quality control line is calculated in a similar manner.

[0105] Calculate the integral area S: Obtain the final baseline values ​​of the test line and the quality control line according to the above calculations. The area of ​​the peak curve above the final baseline value is the integral area S. The integral area of ​​the test line is the area of ​​the peak curve from the starting point 1 A1 to the ending point 1 Z1 above the final baseline value (S T ), 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 ), and then calculate the integral area of ​​the detection line (S T ) and the integral area of ​​the quality control line (S C ) to quantify the fluorescence signal intensity of the detection line, where the ratio of the integrated areas of the two is referred to as T / C value, that is, T / C value = S T ÷S C .

[0106] Generally, a quality control line serves as a reference for comparing fluorescence signal intensity to the test line. It provides a stable baseline for fluorescence signal intensity, enabling correction of fluorescence background signals, compensation for systematic errors, and filtering of nonspecific fluorescence signals, thereby ensuring the stability, reliability, and effectiveness of the chromatographic strip test system. Therefore, based on the test data from the quality control line, a semi-quantitative analysis of IGRA test results can be performed using the T / C value range. Specifically, the T / C value is compared with thresholds (0.3, 0.5, and 1) to determine the negative or positive IGRA test result qualitatively and semi-quantitatively. The thresholds (0.3, 0.5, and 1) are determined through statistical analysis of existing experimental data and may be adjusted as the sample size increases. The specific semi-quantitative determination method for the test line is: a T / C value ≥ 1.0 is considered strongly positive (+++); 0.5 ≤ T / C < 1.0 is considered positive (++); 0.3 ≤ T / C < 0.5 is considered weakly positive (+); and a T / C value < 0.3 is considered negative (-). In addition, IGRA testing also requires the combination of the negative control N, positive control P and sample control T test results corresponding to the sample to be tested to make the final result judgment. Among them, the semi-quantitative judgment method of the corresponding test lines of N, P and T is as described above, and the judgment standard of the combination of N, P and T is: the T / C value of the negative control (N T / C ) is 0≤N T / C <0.3 is used as the criterion for the validity of IGRA test results. Under the premise that the IGRA test results are valid, if the positive control T / C value (P T / C ) and the T / C value of the negative control (N T / C ) is (P T / C -N T / C ) ≥ 0.3, the T cell function in the sample to be tested is determined to be normal. At this time, the result of the sample control T is valid (or the centrifugal microfluidic detection chip IGRA detection system of tuberculosis-specific cellular immunity is valid), that is, 0≤N T / C <0.3 and (P T / C -N T / C ) ≥ 0.3, the detection result of the sample to be tested using the chip of the present invention is valid.

[0107] Under the premise that the chip IGRA detection system is effective, if 0≤(T T / C -N T / C ) < 0.3, the IGRA test of the sample to be tested is judged to be negative, that is, Mycobacterium tuberculosis does not exist in the sample to be tested; if (T T / C -N T / C ) ≥ 0.3, the IGRA test of the sample to be tested is judged to be positive; similarly, a semi-quantitative judgment method similar to the detection line can also be used to further semi-quantitatively judge the positive IGRA test results of the chip. The judgment standard is: (TT / C -N T / C )≥1.0 was considered as IGRA strong positive (+++); 0.5≤(T T / C -N T / C ) <1.0 was considered as IGRA positive (++); 0.3≤(T T / C -N T / C ) < 0.5 was determined as IGRA weakly positive (+).

[0108] 3. Comparative Validation of IGRA Detection Using a Centrifugal Microfluidic Detection Chip for Tuberculosis-Specific Cellular Immunity

[0109] Three negative samples of Mycobacterium tuberculosis with clear clinical backgrounds were tested in parallel using conventional IGRA and the chip IGRA of the present invention. The test results of these three negative samples of Mycobacterium tuberculosis were compared using conventional IGRA and chip IGRA. Figure 16 As shown, first, whether it is traditional IGRA or chip IGRA, the T / C value of the negative control satisfies 0≤N T / C The judgment standard is <0.3, and the positive control T / C value (P T / C ) and the T / C value of the negative control (N T / C ) difference (P T / C -N T / C ) also satisfy (P T / C -N T / C ) ≥ 0.3 is the effective judgment standard of the IGRA detection system, especially the detection results of traditional IGRA and chip IGRA reach (P T / C -N T / C )>0.5, indicating that the T cells in the three Mycobacterium tuberculosis negative samples are functioning normally, that is, they have normal response capacity to stimulate the production of IFN-γ; the most important thing is the semi-quantitative determination result value of the sample to be tested (T T / C -N T / C ) are all within 0≤(T T / C -N T / C )<0.3, it can be judged that the IGRA detection results of the three Mycobacterium tuberculosis negative samples by both the traditional IGRA and the chip IGRA are negative. It can be seen that the preliminary verification shows that the negative coincidence rate of the chip IGRA of the present invention and the traditional IGRA is 100%.

[0110] In the IGRA comparison test of Mycobacterium tuberculosis negative samples, the detection value of the chip IGRA positive control (P T / C were 1.220, 1.663 and 1.171 respectively) which were generally higher than the detection values ​​of traditional IGRA positive control (P T / CThe results are as follows: 0.812, 1.508, and 0.721, respectively. This result is conducive to enhancing the sensitivity of chip-IGRA detection. The reasons for this result may be: First, the multiple centrifugation operations during the chip-IGRA detection process are conducive to enhancing 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 improve the purity of plasma. In addition, the flow and spatial enrichment of red blood cells in various chambers and channels of the chip shorten the distance between T cells and increase the probability of physical contact between cells, thereby promoting the exchange of cytokines and signal transduction efficiency, and ultimately forming a synergistic amplification effect of the 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 signaling molecules (such as IFN-γ). Therefore, intercellular signaling molecules can efficiently diffuse and transmit over 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 methods of the chip of the present invention ensure the reliability of IGRA detection. For example, the chip is injection-molded using PMMA material and combined with computer numerical control (CNC) processing technology to ensure high precision and consistency of the flow channel structure. CNC processing removes surface material by milling to avoid exogenous contamination. At the same time, it is assembled in a clean environment, and the raw materials and accessories used in the assembly have all undergone cleanliness testing, which can effectively control the level of endotoxin. Although there may be trace amounts of residual factors on the chip surface, parallel comparison with traditional IGRA tests shows 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.

[0111] Due to safety concerns, it is difficult to obtain a tuberculosis-positive sample with a clear clinical background. Therefore, a tuberculosis-positive simulated sample was prepared. The specific preparation method is: 80 μL of recombinant IFN-γ standard (wherein the initial concentration of IFN-γ standard is 500 pg / mL) is added to 720 μL of fresh anticoagulated whole blood. After thorough mixing, the concentration of IFN-γ produced by the tuberculosis-positive sample can be simulated (final concentration is 50 pg / mL). This tuberculosis-positive simulated sample was repeatedly tested in parallel three times using traditional IGRA and the chip IGRA of the present invention: 1, 2, and 3. The test results are compared. Figure 17 As shown, both the traditional IGRA and chip IGRA, not only the T / C value of the negative control satisfies 0≤N T / CThe judgment standard is <0.3, and the positive control T / C value (P T / C ) and the T / C value of the negative control (N T / C ) satisfies the difference (P T / C -N T / C ) ≥0.3 is the effective criterion for the IGRA detection system. Figure 17 Visible (P T / C -N T / C )>0.5; the most important thing is the semi-quantitative determination result value of the sample to be tested (T T / C -N T / C ) are located at (T T / C -N T / C ) ≥0.3 means the IGRA test is within the positive range, Figure 17 It can be seen that the IGRA test results of both traditional IGRA and chip IGRA for tuberculosis-positive simulated samples (T T / C -N T / C ) ≥ 1.0, it is considered strongly positive (+++). This indicates that the positive concordance rate between the chip-based IGRA of the present invention and the traditional IGRA is 100%. Combined with the negative concordance rate results above, the overall concordance rate between the chip-based IGRA of the present invention and the traditional IGRA is 100%. Furthermore, in the IGRA comparison of tuberculosis-positive simulated samples, the traditional IGRA still relies on manual operations (such as high-speed centrifugation and pipette extraction), which are prone to the potential risk of error due to operator skill differences. However, the chip-based IGRA of the present invention can integrate multiple detection units, potentially enabling parallel IGRA processing of batches of test samples. Only 50 μL of whole blood is required for IGRA testing (conventional methods require 200-500 μL). Excluding incubation time, the chip-based IGRA completes the entire N / P / T three-tube control test in under 30 minutes (conventional IGRAs require at least 35-50 minutes to complete the N / P / T three-tube control test on a single sample).

[0112] According to the overall concept of the present invention, a centrifugal microfluidic chip for detecting tuberculosis-specific cellular immunity was provided. The construction and performance evaluation of a tuberculosis-specific cellular IGRA (immunoassay chip-based microfluidic assay) detection platform were completed, with preliminary verification that the overall agreement rate was consistent with that of traditional IGRA. Future work is needed to further refine and improve the fabrication and assembly precision of the centrifugal microfluidic chip for detecting tuberculosis-specific cellular immunity, such as by improving chip processing accuracy and hardware quality, and enhancing the stability of large-scale chip production. Furthermore, the present chip should be applied to a large number of clinical samples for testing and validation, and the correlation between the results and those of traditional IGRA should be compared and analyzed, thereby promoting the application of chip-based IGRA in testing larger sample sizes.

[0113] The specific embodiments of the present invention have been described in detail so that those skilled in the art can easily understand them. Moreover, it can be understood that all or part of the steps of implementing the above embodiments can be completed by hardware, or can be completed by instructing relevant hardware through a program, and the program can be stored in a computer-readable storage medium, and the above-mentioned storage medium can be a read-only memory, a disk or an optical disk, etc. However, the above specific embodiments are only used to specifically illustrate the technical solution or preferred solution of the present invention in conjunction with the accompanying drawings, and are not an exhaustive limitation on the scope of protection of the present invention. According to all the descriptions that have been disclosed, the details can be appropriately modified or replaced without departing from the essence and scope of the technical solution of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity, characterized by: A sealed chip is mainly formed by assembling three layers of structures, namely, a chip bottom plate sealing plate (1), a chip substrate (2) and a chip top sealing plate (5), from bottom to top. The three layers of structures, namely, the chip bottom plate sealing plate (1), the chip substrate (2) and the chip top sealing plate (5), are provided with chip positioning structures (12) of the same shape and pin positioning holes (14) at corresponding positions; the chip is provided with a plurality of sample detection units; wherein, Each sample detection unit (22) on the chip substrate (2) includes a paper strip card slot (34) for fixing the chromatography paper strip (3), 9 chambers that can accommodate gas or liquid, and a channel for gas-liquid communication between the paper strip card slot (34) and the 9 chamber structures, wherein the 9 chambers are respectively the two chambers of the sample enrichment preparation chamber (23) and the red blood cell enrichment chamber (26) in the single-chamber centrifugal sedimentation structure (51), the air pressure buffer chamber (49) for buffering the air pressure in the single-chamber centrifugal sedimentation structure (51), the two quantitative chambers of the plasma quantitative chamber (45) and the red blood cell collection chamber (42) that constitute the center-colinear interconnected structure (52), the waste liquid collection chamber (44) for collecting excess liquid, the diluent chamber (28) for diluting the sample to be tested, and the desiccant for humidity regulation. The chamber (32) and the mixing chamber (35) for mixing the diluent and the purified plasma; wherein 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 center-colinear interconnected structure (52) includes a plasma quantitative chamber (45), a three-branch left stepped channel (40), a three-branch middle liquid inlet stepped channel (41), a red blood cell collection chamber (42), and a three-branch right exhaust stepped channel (43); the channel for gas-liquid communication of the nine chamber structures includes a plasma transfer channel (48) that connects the plasma extraction inlet (24) and the puncture valve fixed stepped hole (47) and promotes liquid to flow into the plasma extraction inlet (24) and out of the puncture valve fixed stepped hole (47); The upper part of the chip top sealing plate (5) is provided with a pressure-sensitive packaging glue (6), a buffer airbag fixing plate (8) and a puncture airbag fixing plate (9), and the upper parts of the buffer airbag fixing plate (8) and the puncture airbag fixing plate (9) are respectively provided with airbags (7); the single detection unit (17) on the chip top sealing plate includes a paper strip detection window (18) for observing the IGRA detection result of the chromatography paper strip (3) and 6 gas-liquid interaction holes; the 6 gas-liquid interaction holes 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 injection hole (15) and a diluent chamber exhaust hole (16) for gas-liquid interaction with the diluent chamber (28), an air pressure buffer hole (20) for air pressure buffering adjustment in the buffer air pressure buffer chamber (49), and a puncture hole (21) located at the upper part of the axial center direction of the puncture valve (4).

2. The centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity according to claim 1, characterized in that: The number, position and related structure of the sample detection unit (22) on the chip substrate (2) correspond to the number of the single detection unit (17) on the sealing plate at the top of the chip; the number of the detection units can be 3-10 to accommodate the detection of multiple samples to be tested, and can at least be used to simultaneously complete the IGRA detection of negative controls, positive controls and sample controls of 1-8 samples to be tested; The puncture valve (4) is fixedly mounted on the puncture valve fixed stepped hole (47). The puncture valve (4) is a three-layer structure consisting of two upper and lower nylon gaskets (11) and a middle aluminum foil sacrificial layer (10), and is an active valve that does not require an additional driving structure.

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 using a transparent pressure-sensitive encapsulating adhesive (6).

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

5. The centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity according to claim 1, characterized in that: When only matrix liquid is pre-installed in the sample enrichment preparation chamber (23), the corresponding sample detection unit is marked as a negative control; if matrix liquid and tuberculosis non-specific stimulating antigen are pre-installed in the sample enrichment preparation chamber (23), the corresponding sample detection unit is marked as a positive control; if matrix liquid and Mycobacterium tuberculosis specific antigen are pre-installed in the 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 position of the puncture valve (4) is sequentially arranged from top to bottom as follows: 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 buffer airbag fixing plate (8) is mounted on the chip top sealing plate (5), and the airbag (7) is mounted on the buffer airbag fixing plate (8), so the coaxial position is sequentially arranged from top to bottom as follows: the airbag (7), the buffer airbag fixing plate (8), the air pressure buffer hole (20) and the air pressure buffer chamber (49), and the airbag (7) mounted on the puncture airbag fixing plate (9) and the airbag (7) mounted on the buffer airbag fixing plate (8) are both selected from elastic materials with a high elastic modulus.

7. The method for assembling the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity according to any one of claims 1 to 6, characterized in that: The assembly process includes: Step 1: Preparation; Step 2: Assembling the chip bottom plate sealing plate (1), installing pins in the pin positioning holes (14) to form positioning columns; Step 3: Assembling the chip substrate (2): The chip substrate (2) and the chip bottom plate sealing plate (1) are locked and fixed by the positioning columns formed by the chip bottom plate sealing plate (1). After fixing, the exposed positioning columns are still visible; Step 4: Assembling the chip top sealing plate (5): The chip top sealing plate (5) is locked and fixed to the chip base (2) through the positioning column exposed after the chip base (2) is assembled; the puncture airbag fixing plate (9) and the airbag (7) are assembled in sequence from bottom to top at the central axis position of the puncture hole (21); the buffer airbag fixing plate (8) and the airbag (7) are assembled in sequence from bottom to top at the central axis position of the air pressure buffer hole (20); the opening position of the paper strip detection window (18) on the chip top sealing plate (5) is sealed with pressure-sensitive packaging glue (6).

8. A method for performing chip IGRA detection using the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity according to any one of claims 1 to 6, characterized in that: The testing process includes: Assay preparation: Equilibrate the chip at room temperature for at least 20 minutes; Sample addition 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) from the sample addition hole (13) and the diluent chamber injection hole (15), and complete the sealing of the hole position after adding liquid; Incubation: Place the chip in a constant temperature environment at 37°C for 22±4 hours; Chip IGRA detection: The chip is centrifuged on a servo motor for 5 times to complete the separation of plasma in the sample to be tested, liquid mixing and chip IGRA detection, and the chip IGRA detection results are observed on the paper strip detection window (18); Determination of chip IGRA test results: the detection line and the quality control line on the chromatography paper strip (3) are judged by the naked eye as positive or negative; or the intensity value of the fluorescence signal obtained by quantitative detection with a fluorescence scanner is optimized and semi-quantitatively analyzed; The chip IGRA detection process does not include disease diagnosis and treatment methods.

9. The centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity according to claim 8 is used for chip IGRA detection, characterized in that: The five centrifugations in the chip IGRA test were performed by fixing the chip on the flange block of the servo motor and then driving the chip for centrifugation by the servo motor, wherein the acceleration was increased from no more than 500 rpm / s to a high-speed rotation state of no more than 3000 rpm, and the centrifugation lasted no more than 6 minutes.

10. Use of the centrifugal microfluidic detection chip for tuberculosis-specific cellular immunity according to any one of claims 1 to 6 in the preparation of IGRA detection products.

Images