Enrichment device, enrichment method and microfluidic chip for interferon gamma detection

Abstract

The application provides an enrichment device, an enrichment method and a micro-fluidic chip for interferon gamma detection, which comprises a cell culture device, an enrichment strip and a micro-fluidic detection chip, the cell culture device comprises at least three cell culture pools and an upper cover, the enrichment strip is detachably connected with the upper cover, the enrichment strip comprises a substrate and a protruding block, the substrate is elastic, the protruding block is made of porous material and coated with anti-interferon gamma antibodies on the surface, and the protruding block is inserted into a corresponding culture pool through the upper cover, so that the interferon gamma in the culture supernatant is combined with the antibodies on the surface of the protruding block and enriched on the surface of the protruding block. After the enrichment is completed, the enrichment strip is transferred to the detection chip for interferon gamma determination. The amount of blood used is controlled to be less than 1ml, so that the problem of insufficient detection sensitivity under the condition of micro-culture is effectively solved; and the micro-fluidic technology is combined, so that the rapid and convenient detection of interferon gamma is realized.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostics and immunoassay technology, and specifically relates to an enrichment device, enrichment method and microfluidic chip for the detection of gamma interferon. Background Technology

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (MTB), with pulmonary tuberculosis being the most common form. MTB infection is often latent, and latent carriers are the primary source of infection. Therefore, timely detection and diagnosis of TB, especially in asymptomatic latent carriers, is crucial. With the deepening of TB research and the application of modern immunological techniques, immunological diagnostic methods for TB are constantly being developed, and their diagnostic value is increasingly recognized. Relevant immunological tests include the tuberculin skin test, tuberculosis antigen or antibody detection, and interferon-gamma release assay. A positive tuberculin skin test indicates TB infection or successful BCG vaccination and is an important indicator for assessing active TB. Mycobacterium tuberculosis antigen is an early indicator of TB infection; and Mycobacterium tuberculosis IgG antibody levels can assist in the diagnosis of active TB. Interferon release assays (IGRA) utilize Mycobacterium tuberculosis-specific antigens to stimulate effector T cells in peripheral blood to rapidly activate and proliferate, releasing interferon gamma. The results are then interpreted based on the percentage of interferon-positive cells.

[0003] After a person is initially infected with Mycobacterium tuberculosis, sensitized T lymphocytes are present in the body. When the body is exposed to the same Mycobacterium tuberculosis antigens again, such as early secretory antigen target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10), the sensitized lymphocytes are rapidly activated into effector T lymphocytes and release high levels of cytokines (such as interferon-gamma). By detecting the secretion of interferon-gamma, it is possible to determine whether an MTB-specific cellular immune response exists. All Bacille Calmette-Guérin (BCG) strains and the vast majority of nontuberculous mycobacteria (NTMs) do not contain the two proteins mentioned above. Therefore, IGRA does not cross-contaminate with BCG and most NTMs, avoiding false positives caused by BCG vaccination and NTM infection. Commonly used clinical detection methods include T-SPOT and QFT methods. The difference between the two is that the T-SPOT method detects the gamma-interferon response after effector T cell activation using an enzyme-linked immunospot assay, while the QFT method detects the concentration of gamma-interferon secreted by effector T cells after activation using an enzyme-linked immunosorbent assay. Both methods can be used for the auxiliary diagnosis of Mycobacterium tuberculosis infection, but they cannot differentiate between active and latent tuberculosis infection.

[0004] Currently, the QFT method commonly uses the detection of interferon-gamma (γ) content in cell culture supernatants for γ-interferon release assays. However, this method faces two pressing problems: First, conventional culture methods require large volumes, necessitating the collection of 4-6 ml of blood. Microculture methods, on the other hand, have a limited number of T cells per culture volume, resulting in low concentrations of γ-interferon secreted into the supernatant, making direct detection with conventional techniques insufficiently sensitive. Second, conventional methods using enzyme-linked immunosorbent assays (ELISA) or chemiluminescence immunoassays (CIA) to detect γ-interferon in cell culture supernatants require 50 or 100 μL of sample. ELISA is complex, requires manual operation, is difficult to standardize, and is time-consuming. More importantly, each assay requires calibration curves and parallel replicates, leading to wasted microplates. Chemiluminescence immunoassays require expensive instruments, have high detection costs, and are difficult to configure as automated analyzers.

[0005] Furthermore, while microfluidic chip technology has been widely applied in the field of biomedical testing, the conventional design of microfluidic chips typically involves coating antibodies within the detection area of ​​the chip, suitable for the combined detection of multiple indicators in a single sample. However, the IGRA assay requires three control tubes simultaneously: a blank control tube (to measure background levels), a positive control tube (to verify T cell activity), and a tuberculosis antigen tube (to measure specific reactions). Diagnostic conclusions are derived through logical comparison of the results from these three control tubes. Conventional microfluidic chips struggle to simultaneously handle the detection requirements of multiple different samples (such as three control tubes) for the same indicator. Forcibly integrating these tubes often requires complex flow path switching designs or multiple independent chip channels, leading to complex chip structures, increased costs, and difficulty in ensuring consistency in the detection conditions of the three control tubes. Therefore, current technology lacks a microfluidic chip and supporting device that can easily and efficiently process three control tube samples in the IGRA assay simultaneously and achieve parallel detection of the same indicator. Summary of the Invention

[0006] The present invention aims to provide an enrichment device, enrichment method and microfluidic chip for gamma interferon detection, and solves the problems existing in the prior art by optimizing the enrichment method of gamma interferon.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: an enrichment device for detecting interferon gamma includes a cell culture device and an enrichment strip. The cell culture device includes a cell culture pool and a top cover. The top cover is fitted onto the cell culture pool, and the cell culture pool contains at least three culture chambers. The enrichment strip is detachably connected to the top cover. The enrichment strip includes a substrate and protrusions. The substrate is elastic, and there are multiple protrusions. The multiple protrusions are fixed along the length direction of the substrate on its lower surface. The protrusions are made of a porous material capable of binding proteins, and the surface of the protrusions is coated with anti-interferon gamma antibodies. The protrusions are inserted into their respective culture chambers through the top cover. Interferon gamma in the culture supernatant in the cell culture pool binds to the anti-interferon gamma antibodies on the surface of the protrusions and is enriched on the surface of the protrusions.

[0008] In one embodiment of the present invention, the porous material is at least one of nitrocellulose membrane and polyvinylidene fluoride membrane.

[0009] In one embodiment of the present invention, the protrusion is a cylindrical structure with a diameter of 0.5-1 mm and a height of 0.5-2 mm.

[0010] In one embodiment of the present invention, the substrate is made of PET material.

[0011] In one embodiment of the present invention, at least three culture chambers are respectively used to provide blank control, positive control and tuberculosis antigen stimulation, and each culture chamber is provided with two of the bumps.

[0012] On the other hand, the present invention also provides a method for enriching gamma interferon using the above-described enrichment device, comprising at least the following steps: S1. Add the sample to be tested into the at least three culture chambers respectively, and add blank control, positive control stimulus and tuberculosis-specific antigen to the corresponding culture chambers, and incubate for cell culture; S2. After the culture is completed, install the enrichment strip on the top cover so that the protrusion is inserted into the corresponding culture chamber; S3. Rotate or flip the cell culture tank so that the bump is submerged in the culture supernatant to capture gamma interferon.

[0013] In one embodiment of the present invention, in step S3, after the culture is completed, the cell culture pool is flipped so that the bumps are immersed in the culture supernatant and shaken. After the shaking is completed, the cell culture pool is restored to an upright state, and then the enrichment strip is removed from the top cover.

[0014] On the other hand, the present invention also provides a microfluidic chip for gamma interferon detection, including a substrate chip, a structural chip, and the enrichment strips mentioned above. The substrate chip and the structural chip are enclosed to form a microchannel. The structural chip is provided with a marking area and a detection area in sequence along the liquid flow direction. The structural chip is provided with an insertion hole corresponding to the bump at the position of the detection area. The bump is inserted into the insertion hole so that the lower surface of the bump is flush with the lower surface of the structural chip. The marking area is pre-positioned with a marker-coupled anti-gamma interferon detection antibody.

[0015] In one embodiment of the present invention, the marker is a fluorescent microsphere.

[0016] In one embodiment of the present invention, the number of the insertion holes is multiple, and each hole corresponds to a protrusion of the enrichment strip.

[0017] The enrichment device, enrichment method, and microfluidic chip for gamma interferon detection obtained through the above technical solution have the following advantages: 1. The enrichment device provided by this invention detachably mounts a porous material with protrusions coated with anti-gamma interferon antibodies onto the top cover of a cell culture chamber, and inserts the protrusions into the corresponding culture chambers to contact the culture supernatant, specifically capturing and enriching gamma interferon. Compared to the traditional QFT method, which only uses 50 μL of culture supernatant (approximately 1 / 10 of the total culture supernatant) for detection on an ELISA platform, this invention can enrich gamma interferon in the entire culture supernatant. Therefore, when using the same initial blood sample, the detection signal is stronger; or, under the premise of achieving the same detection sensitivity, the initial blood volume can be significantly reduced, making it particularly suitable for scenarios where blood collection is limited, such as with infants and young children.

[0018] 2. This invention integrates enrichment strips directly into the top cover of the culture tank. After enrichment, the enrichment strips are transferred to the detection area of ​​the microfluidic chip, and a lateral flow microfluidic chip is used for signal reading. This eliminates steps such as centrifugation, multiple pipetting, long-term incubation, and washing. After transferring the enrichment strips to the microfluidic chip, only buffer solution needs to be injected, and the detection antibody located in the labeled area of ​​the structured chip dissolves rapidly. Under the laminar flow of the microchannel, the dissolved detection antibody flows closely to the surface of the structured chip and flows through the detection area where the bumps immobilized with gamma interferon are located in a high-concentration, short-path manner. Because laminar flow reduces the longitudinal diffusion of the detection antibody, a high concentration of detection antibody can be maintained locally in the detection area, so that the antigen-antibody binding reaction can reach stability within minutes. The entire process can be controlled within 10 minutes, realizing rapid and simple detection of gamma interferon and significantly improving detection efficiency.

[0019] 3. This invention integrates antibody coating and interferon capture and enrichment functions into a single enrichment strip. Through a substrate and multiple bumps fixed on the lower surface of the substrate, the bumps are inserted into corresponding culture chambers (blank control, positive control, tuberculosis antigen), simultaneously capturing and enriching gamma interferon under different treatment conditions. After enrichment, the entire enrichment strip is transferred to a microfluidic chip. The microfluidic chip itself does not need to be coated with any antibody; it only serves as a signal reading platform. Multiple insertion holes on the chip receive the corresponding bumps, thus enabling parallel detection of multiple samples of the same indicator.

[0020] 4. The microfluidic chip of the present invention does not require the coating of antibodies inside the chip. It only requires the pre-placement of the marker-coupled detection antibody in the labeling area. The chip fabrication process is simplified and the cost is reduced. At the same time, the combination of the enrichment strip and the microfluidic chip is simple and does not require complex fluid control equipment. The signal can be read by a portable fluorescence reader, which reduces the investment cost of detection equipment. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the enrichment device for gamma interferon detection described in this invention; Figure 2 This is an exploded view of the enrichment device for gamma interferon detection described in this invention; Figure 3 This is a flowchart of enriching gamma interferon using the enrichment device for gamma interferon detection described in this invention. Figure 4 This is a schematic diagram of the microfluidic chip (transparent) for gamma interferon detection described in this invention; Figure 5 This is an exploded view of the microfluidic chip for gamma interferon detection described in this invention; Figure 6 This is a bottom view of the chip structure described in this invention; Figure 7 This is a cross-sectional view of the microfluidic chip for gamma interferon detection described in this invention.

[0022] In the figure, 1 is the cell culture device; 2 is the enrichment strip; 3 is the substrate chip; 4 is the structure chip; 5 is the microchannel; 11 is the cell culture pool; 11a is the culture chamber; 12 is the top cover; 21 is the substrate; 22 is the bump; 41 is the insertion hole; 51 is the marking area; and 52 is the detection area. Detailed Implementation

[0023] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0024] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0025] The present invention will be further explained and described below with reference to the embodiments and accompanying drawings. It should be understood that the present invention is not limited to the specific embodiments described.

[0026] like Figures 1-2 As shown, an enrichment device for detecting gamma interferon includes a cell culture device 1 and an enrichment strip 2. The cell culture device 1 includes a cell culture pool 11 and a top cover 12. The top cover 12 covers the cell culture pool 11. The cell culture pool 11 contains at least three culture chambers 11a. The enrichment strip 2 is detachably connected to the top cover 12. The enrichment strip 2 includes a substrate 21 and protrusions 22. The substrate 21 is elastic. There are multiple protrusions 22, which are fixed along the length of the substrate 21 on its lower surface. The protrusions 22 are made of a porous material capable of binding proteins. The surface of the protrusions 22 is coated with anti-gamma interferon antibodies and is conventionally sealed to prevent non-specific adsorption. The protrusions 22 are inserted into the corresponding culture chambers 11a through the top cover 12. Gamma interferon in the culture supernatant in the cell culture pool 11 binds to the anti-gamma interferon antibodies on the surface of the protrusions 22 and is enriched on the surface of the protrusions 22.

[0027] The substrate 21 is made of an elastic material, which allows the substrate 21 to fit tightly against the surface of the top cover 12 after the protrusion 22 is inserted into it. The elastic deformation fills the tiny gaps and prevents the culture supernatant from leaking out of the mounting hole when the cell culture pool is flipped or tilted. At the same time, during the process of installing the enrichment strip 2 with the protrusion 22 onto the top cover 12, the elasticity of the substrate 21 can absorb the tiny positional deviations caused by processing errors or human operation, so that the protrusion 22 can be smoothly inserted into the top cover 12.

[0028] Under normal circumstances, after peripheral blood cells are cultured and stimulated, the cell culture supernatant is collected by centrifugation, and only a small portion (about 1 / 10) of the supernatant solution is used for detection. During the detection process, the solid-phase antibody binds to the interferon in the culture supernatant, completing the specific binding of the interferon in the culture supernatant. However, due to the limitations of the detection method, not all of the culture supernatant can be used as the detection sample; only a small portion is taken. Taking a commercially available kit (such as the Mycobacterium tuberculosis-specific cellular immunoreaction detection kit - enzyme-linked immunosorbent assay) as an example, the routine operation is as follows: peripheral blood is collected using lithium heparin anticoagulant tubes, with a blood volume of approximately 0.8-1.2 ml (usually 1 ml) per tube. The blood is aliquoted into mitogen positive control tubes, blank control tubes, and tuberculosis antigen tubes, respectively. After incubation for 16-24 hours, the supernatant is collected by centrifugation, and 50 μL of the supernatant is taken. Then, the level of gamma interferon is detected using an enzyme-linked immunosorbent assay (ELISA) kit. Each tube uses approximately 1 ml of whole blood for culture, and the total blood volume usually requires 4-6 ml (including three control tubes). This large blood volume is unfriendly to infants, critically ill patients, or those who require repeated blood draws. If microculture (reducing blood volume) is attempted, the number of T cells in the culture system is limited, resulting in a low concentration of interferon-gamma secreted into the culture supernatant. When directly detecting these cells using conventional detection techniques, the sensitivity is insufficient.

[0029] The enrichment device provided by this invention detachably mounts a porous material protrusion 22 coated with anti-gamma interferon antibody onto the top cover 12 of the cell culture tank 11, and inserts the protrusion 22 into the corresponding culture chamber 11a to contact the culture supernatant, specifically capturing and enriching gamma interferon in the supernatant. Compared to the traditional QFT method, which only uses 50 μL of culture supernatant (approximately 1 / 10 of the total culture supernatant) for detection on an ELISA platform, this invention can enrich gamma interferon in the entire culture supernatant, thus providing a stronger detection signal when using the same initial blood sample; or, under the premise of achieving the same detection sensitivity, it can significantly reduce the initial blood volume.

[0030] The porous material is at least one of nitrocellulose membrane and polyvinylidene fluoride membrane. Nitrocellulose membrane (NC membrane) and polyvinylidene fluoride membrane (PVDF membrane) have high physical adsorption capacity for proteins, enabling antibodies to be stably coated and effectively capture gamma interferon.

[0031] The protrusion 22 is a cylindrical structure with a diameter of 0.5-1mm and a height of 0.5-2mm.

[0032] The substrate 21 is made of PET material. The moderate elasticity of PET material ensures the seal when the bump 22 is inserted into the top cover. At the same time, PET film has excellent chemical stability and biocompatibility, does not affect the accuracy of cell culture and gamma interferon detection, and is inexpensive, making it suitable for single use.

[0033] At least three culture chambers 11a are used to provide blank control, positive control and tuberculosis antigen stimulation, respectively. Each culture chamber 11a is provided with two bumps 22. The two bumps 22 can be used to evaluate the consistency of enrichment and detection, and the mean of two points can be calculated and used in subsequent logical calculations.

[0034] Among them, the blank control corresponding to the three culture chambers 11a contains no stimulants (only blood and culture medium), and its main function is to measure the non-specific background level of interferon-gamma to correct the test results; the positive control contains non-specific stimulants (such as mitogens), which can strongly activate all T cells without relying on antigen specificity, causing them to release interferon-gamma, and is used to verify whether the T lymphocytes in the tested blood sample have normal activity and function; the nuclear antigen stimulation contains Mycobacterium tuberculosis specific antigen to measure specific reactions.

[0035] Taking a case where there are 3 culture chambers 11a, the 3 culture chambers 11a are arranged in a row, with a total length of 20mm, width of 6mm, and height of 10mm. Each culture chamber 11a is circular with an inner diameter of 5mm. Each top cover 12 has two holes, and two protrusions 22 are embedded in it. The two protrusions have the same diameter and fit together to prevent liquid from overflowing. This allows each culture chamber 11a to correspond to 2 γ-interferon enrichment sites, and the three culture chambers 11a carry 6 interferon enrichment sites.

[0036] like Figure 3 As shown, the present invention also provides a method for enriching gamma interferon using the above-described enrichment device, comprising at least the following steps: S1. The sample to be tested is added to the at least three culture chambers 11a respectively, and blank control, positive control stimulus and tuberculosis-specific antigen are added to the corresponding culture chambers 11a, and cell culture is carried out by incubation; wherein, the cell culture medium, blank control, positive control stimulus and tuberculosis-specific antigen involved in the cell culture of the present invention are all set according to conventional methods.

[0037] S2. After the culture is completed, the enrichment strip 2 is installed on the upper cover 12, so that the protrusion 22 is inserted into the corresponding culture chamber 11a; S3. Rotate or flip the cell culture pool 11 so that the bump 22 is immersed in the culture supernatant to capture γ interferon.

[0038] In step S3, after the culture is completed, the cell culture tank 11 is flipped so that the protrusion 22 is immersed in the culture supernatant and shaken. After the shaking is completed, the cell culture tank 11 is restored to an upright state, and then the enrichment strip 2 is removed from the top cover 12.

[0039] likeFigures 4-7 As shown, the present invention also provides a microfluidic chip for gamma interferon detection, including a substrate chip 3, a structural chip 4, and the enrichment strip 2 mentioned above. The substrate chip 3 and the structural chip 4 enclose a microchannel 5. The microchannel 5 is provided with a marking area 51 and a detection area 52 in sequence along the liquid flow direction. The structural chip 4 is provided with an insertion hole 41 corresponding to the bump 22 at the position of the detection area 51. The bump 22 is inserted into the insertion hole 41, so that the lower surface of the bump 22 is flush with the lower surface of the structural chip 4, so that the bump 22 is in contact with the liquid in the microchannel 5. If the bump 22 of the enrichment strip 2 is inserted into the microchannel 3, it will disrupt the dynamics of the liquid flow in the microchannel 5. Therefore, it is required that after the bump 22 is inserted, it can fill the hole position of the insertion hole 41 of the structural chip 4 and form a plane. Therefore, it is required that the lower surface of the bump 22 is flush with the lower surface of the structural chip 4. The marking area 51 is pre-filled with a marker-coupled anti-gamma interferon detection antibody.

[0040] The typical design approach for microfluidic chips is to simultaneously detect multiple different indicators (e.g., multiple cytokines such as IL-6, TNF-α, and IFN-γ) on a single sample using a single chip. These chips typically have antibodies targeting different indicators coated in different detection zones within the chip.

[0041] The requirements of the IGRA test are exactly the opposite: the core of the IGRA test is to determine the presence of tuberculosis infection by detecting the secretion level of gamma interferon. Therefore, the IGRA test requires testing the same indicator (gamma interferon) in three different samples (blank, positive, and antigen) and drawing a diagnostic conclusion through logical comparison. If this is to be achieved using conventional microfluidic chips, either three independent chips are needed (high cost and complex operation), or three independent flow paths need to be set up on the same chip (complex structure and difficulty in ensuring consistency). In this case, the inherent differences between chips will destroy the comparability between the three test results, making the logical comparison based on the signals of blank, positive, and antigen tubes lose a reliable benchmark and making it difficult to reasonably interpret the test results.

[0042] This invention separates the enrichment strip 2, which has been enriched with gamma interferon, from the cell culture device 1, and then assembles this enrichment strip 2 with a lateral flow chip, so that the position of the capture point matches the position of the detection point of the microfluidic chip. Since multiple bumps 22 are fixed on one enrichment strip 2 (e.g., 6 bumps, 2 corresponding to each culture chamber), each bump 22 is inserted into the corresponding culture chamber 11a (blank, positive, antigen), capturing gamma interferon in that chamber respectively. Since multiple insertion holes 41 on the chip receive the corresponding bumps 22 respectively, by using a detector adapted to the microfluidic chip to simultaneously read the signals on multiple bumps, parallel detection of the same indicator (gamma interferon) in multiple samples (three control tubes) can be completed, perfectly adapting to the three-tube control logic of IGRA.

[0043] The marker is a fluorescent microsphere.

[0044] There are multiple insertion holes 41, and each hole corresponds to a protrusion 22 of the enrichment strip 2.

[0045] The microchannel is formed by the grooves on the lower surfaces of the substrate chip 3 and the structural chip 4, and the two ends of the microchannel 5 are respectively connected to the buffer injection hole and the waste liquid area.

[0046] Example 1: Fabrication of the enrichment device and detection chip 1. Fabrication of cell culture apparatus The cell culture tanks are made of polystyrene and are manufactured using 3D printing equipment according to pre-set three-dimensional drawings, resulting in a one-piece, interconnected culture chamber structure. Perforations are drilled in the top cover of each tank, with the number and location of the perforations matching the cylindrical structures in the enrichment strips. Subsequently, the cell culture tanks undergo a hydrophilic treatment to facilitate uniform cell adhesion and growth.

[0047] 2. Preparation of interferon-gamma enrichment strips (1) Fabrication of PET film Using biaxial stretching and cutting processes, PET resin is processed into PET films with a length of 20 mm, a width of 4 mm, and a thickness of 1 mm.

[0048] (2) Fabrication of the shielding mold PMMA material was selected and processed into a structure with the same size as PET film using laser cutting technology. Then, double wells were made at the positions corresponding to the blank control well (NC), positive control well (PC), and antigen stimulation well (A-TB) of the cell culture pool. The center-to-center distance between the double wells was 3 mm and the diameter was 1 mm.

[0049] (3) Assembly, spraying and demolding First, the PET film surface is corona treated, and then it is laid flat and fixed on a flat table. Second, the masking mold is placed on the PET film to ensure a tight fit. Third, nitrocellulose slurry is evenly sprayed onto the openings until each opening is filled. Finally, after the nitrocellulose slurry has completely dried, the mold is removed. At this point, six cylindrical structures with a diameter of 1 mm and a thickness of 1 mm are formed on the surface of the PET film.

[0050] (4) Coating of biomolecules First, spot 3 μL of 10 μg / mL interferon antibody onto the lower surface of each cylinder and dry for 30 min; second, wash three times with TBST solution; third, block with 5% skim milk for 30 min; finally, wash three times with TBST solution.

[0051] 3. Fabrication of the detection chip The detection chip consists of a structural chip and a substrate chip, both manufactured from PMMA material using a laser etching machine. The structural chip includes a buffer injection well, a labeling region, an enrichment strip insertion well, and a waste liquid region. The labeling region contains pre-dried fluorescent microspheres conjugated with anti-gamma interferon antibodies. The structural chip and the substrate chip are tightly bonded together to form the main chip structure.

[0052] Example 2: Verification of the enrichment effect of the enrichment device 1. Whole blood sample collection Following standard operating procedures, venous blood from the subject was collected into a blood collection tube containing lithium heparin anticoagulant and immediately inverted to mix.

[0053] 2. Blood incubation Four blood volume gradients were set up: 100 μL, 300 μL, 500 μL, and 1 mL. For each gradient, whole blood was added to three culture chambers of the cell culture apparatus: blank control chamber (NC), positive control chamber (PC), and antigen stimulation chamber (A-TB). At the same time, 1 mL of whole blood was added to each QFT blood culture tube (containing NiL, Mitogen, and TB antigen tubes) as a control.

[0054] Add equal amounts of stimulants to each culture tank and QFT tube: PHA was added to the PC tank and Mitogen tube at a final concentration of 5 μg / mL; ESAT-6 and CFP-10 were added to the A-TB tank and QFT TB antigen tube at a final concentration of 0.5 μg / mL; no stimulants were added to the NC tank and QFT NiL tube. The cell culture tanks and QFT tubes were placed in a 37°C incubator and incubated for 16 hours.

[0055] 3. Enrichment and Detection of Gamma Interferon After incubation, the enrichment strip coated with anti-interferon-gamma antibody was inserted into the top cover of the cell culture tank, and the cell culture apparatus was inverted and gently shaken at 50 rpm for 1.5 hours on a shaker to ensure full contact between the enrichment strip bumps and the culture supernatant, specifically capturing interferon-gamma. After the reaction, the cell culture apparatus was uprighted, the enrichment strip was removed, and inserted into the matching microfluidic detection chip. 40 μL of buffer was added to the sample buffer injection well to dissolve the fluorescent microspheres in the labeled area conjugated with anti-interferon-gamma antibody. Under laminar flow, the antibody dissolved and flowed through the detection area, binding with the interferon-gamma captured on the bumps. The remaining liquid was collected in the waste liquid area. After 5 minutes, the chip was placed in a fluorescence reader to detect the signal value.

[0056] After incubation in QFT culture tubes, centrifuge at 2000-3000 RCF for 15 minutes and collect plasma. Transfer 50 μL of plasma to an ELISA microplate and perform quantitative detection of interferon-gamma according to the kit instructions.

[0057] 4. Test data Table 1 Detection Data

[0058] As can be seen from the above data, under the same blood volume (1 mL), the signal values ​​detected by the present invention are significantly higher than those of the traditional QFT, indicating that enrichment can effectively improve the detection sensitivity.

[0059] When the blood volume is reduced to 300 μL, the signal value detected by this invention is still comparable to the detection value when QFT uses 1 mL of whole blood.

[0060] When the blood volume was reduced to 100 μL, the A-TB signal value was close to the NC background value, indicating that when the blood volume was too small, the number of cells was too low to achieve effective detection.

[0061] In summary, using the enrichment device and detection method provided by this invention, only 300 μL of blood is needed per tube to obtain detection results similar to those of standard QFT. Based on the calculation that each subject needs to be tested with three tubes (NC, PC, and A-TB), the total blood volume can be reduced to 900 μL (<1 mL), significantly reducing the blood collection volume compared to traditional QFT (which requires 3-4 mL of whole blood), making it particularly suitable for infants or patients with difficult blood collection.

[0062] Example 3: Evaluation of the accuracy of microfluidic chip detection 1. Sample Collection Five groups (numbered #1, #2, #3, #4, and #5) of stimulated plasma samples were collected from the clinic. Each group contained plasma samples aliquoted from a blank control tube, a positive control tube, and an antigen stimulation tube. Sample #1 was clinically confirmed as positive, while the others were negative.

[0063] 2. Enrichment of interferon-gamma First, the cell culture device, consisting of three cell culture chambers (NC, PC, and A-TB), is taken out. Each chamber has two 1mm diameter orifices at the top, which match the cylindrical protrusions of the enrichment strip. Second, the sample is injected into the corresponding cell culture chamber, with a sample volume of 50μL added to each chamber. Third, the enrichment strip is inserted into the orifice at the top of the culture medium. Finally, the enrichment device is inverted so that the sample in the culture chamber comes into contact with the anti-gamma interferon site on the enrichment strip, and incubated for 1.5 h to achieve the enrichment of gamma interferon in the sample.

[0064] 3. Detection and comparison of gamma-interferon results First, place the cell culture apparatus upright and remove the enrichment strip. Second, insert the enrichment strip into the corresponding well of the structured chip, ensuring the lower surface of the enrichment strip bump is flush with the lower surface of the structured chip. Third, add 40 μL of buffer solution to the buffer injection well of the chip to dissolve the anti-gamma interferon antibody conjugated to the fluorescent microspheres in the labeled area. The antibody flows through the detection area with the buffer solution and reacts with the gamma interferon on the lower surface of the bump. The remaining buffer solution is collected in the waste liquid area. After 5 minutes, place the chip in a fluorescence analyzer for detection and compare the results with those of an ELISA test.

[0065] 4. Test data Table 2 Comparison Data

[0066] In the above comparative experiments, cell stimulation and culture were not involved; only the supernatant after culture was collected. 50 μL was used for conventional ELISA to detect interferon-gamma, simulating the detection mode of traditional QFT. Another 50 μL was processed using the device of this invention (including enrichment strips) and detected on a microfluidic chip to verify the accuracy of the microfluidic detection platform. The data show that the results obtained by the microfluidic platform are consistent with those of the ELISA platform, indicating that the microfluidic detection platform used in this invention has good detection accuracy.

[0067] The above technical solutions only embody the preferred technical solutions of the present invention. Any modifications that may be made by those skilled in the art to certain parts thereof embody the principles of the present invention and fall within the protection scope of the present invention.

[0068] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0069] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicating orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. Furthermore, the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined with "first," "second," etc., may explicitly or implicitly include one or more of that feature. In the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0070] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "setting" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal communication between two components. Those skilled in the art will understand the specific meaning of the above terms in this invention based on the specific circumstances.

Claims

1. An enrichment device for detecting gamma interferon, characterized in that, The device includes a cell culture apparatus (1) and an enrichment strip (2). The cell culture apparatus (1) includes a cell culture pool (11) and a cover (12). The cover (12) is fitted onto the cell culture pool (11). The cell culture pool (11) contains at least three culture chambers (11a). The enrichment strip (2) is detachably connected to the cover (12). The enrichment strip (2) includes a base plate (21) and protrusions (22). The base plate (21) is elastic, and the number of protrusions (22) is multiple. Multiple bumps (22) are fixed along the length of the substrate (21) on the lower surface of the substrate (21). The bumps (22) are made of a porous material that can bind proteins. The surface of the bumps (22) is coated with anti-gamma interferon antibodies. The bumps (22) are inserted into the corresponding culture chambers (11a) through the top cover (12). The gamma interferon in the culture supernatant in the cell culture pool (11) binds to the anti-gamma interferon antibodies on the surface of the bumps (22) and accumulates on the surface of the bumps (22).

2. The enrichment device for gamma interferon detection according to claim 1, characterized in that, The porous material is at least one of nitrocellulose membrane and polyvinylidene fluoride membrane.

3. The enrichment device for gamma interferon detection according to claim 1, characterized in that, The protrusion (22) is a cylindrical structure with a diameter of 0.5-1mm and a height of 0.5-2mm.

4. The enrichment device for gamma interferon detection according to claim 1, characterized in that, The substrate is made of PET material.

5. The enrichment device for gamma interferon detection according to claim 1, characterized in that, At least three culture chambers (11a) are used to provide blank control, positive control and tuberculosis antigen stimulation, respectively, and each culture chamber (11a) is provided with two of the bumps (22).

6. An enrichment method for the detection of gamma interferon, characterized in that, The enrichment apparatus according to any one of claims 1-5 comprises at least the following steps: S1. The sample to be tested is added to the at least three culture chambers (11a) respectively, and blank control, positive control stimulus and tuberculosis-specific antigen are added to the corresponding culture chambers (11a) and incubated for cell culture; S2. After the culture is completed, the enrichment strip (2) is installed on the upper cover (12) so that the protrusion (22) is inserted into the corresponding culture chamber (11a); S3. Rotate or flip the cell culture pool (11) so that the bump (22) is immersed in the culture supernatant to capture γ interferon.

7. The enrichment method for gamma interferon detection according to claim 6, characterized in that, In step S3, after the culture is completed, the cell culture pool (11) is flipped so that the bump (22) is immersed in the culture supernatant and shaken. After the shaking is completed, the cell culture pool (11) is restored to an upright state and the enrichment strip (2) is removed from the top cover (12).

8. A microfluidic chip for gamma interferon detection, characterized in that, The device includes a substrate chip (3), a structural chip (4), and an enrichment strip (2) enriched with γ-interferon using the enrichment method described in claim 6 or 7. The substrate chip (3) and the structural chip (4) enclose a microchannel (5). The structural chip (4) is provided with a marking area (51) and a detection area (52) in sequence along the liquid flow direction. The structural chip (4) is provided with an insertion hole (41) corresponding to the bump (22) at the position of the detection area (52). The bump (22) is inserted into the insertion hole (41) so that the lower surface of the bump (22) is flush with the lower surface of the structural chip (4). The marking area (51) is pre-filled with a marker-coupled anti-γ-interferon detection antibody.

9. The microfluidic chip for gamma interferon detection according to claim 8, characterized in that, The marker is a fluorescent microsphere.

10. The microfluidic chip for gamma interferon detection according to claim 8, characterized in that, The number of the insertion holes (41) is multiple, and each hole corresponds to a protrusion (22) of the enrichment strip (2).

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