Micro-fluidic chip for TSHFT3FT4 joint quantitative analysis and application of micro-fluidic chip

Abstract

The invention provides a TSHamp (TSHamp; fT3amp, FT3amp; the invention discloses a microfluidic chip for FT4 joint quantitative analysis and application thereof, the microfluidic chip comprises a substrate and a cover plate pressed on the substrate, the substrate and the cover plate enclose to form a microchannel, the left end of the microchannel is communicated with a buffer solution injection hole formed in the cover plate, the microchannel is sequentially provided with a marking area and a detection area from left to right, and the marking area is communicated with the detection area. The marking area comprises a first marking area arranged on the lower surface of the cover plate and a second marking area arranged on the upper surface of the substrate, the detection area comprises an FT4 detection area arranged on the lower surface of the cover plate, an FT3 detection area arranged on the upper surface of the substrate and a TSH detection area, the first marking area is coated with a mark T3, and the second marking area is coated with a mark T4. The first marking area is coated with a marker T2 and a marked TSH antibody, the second marking area is coated with a marker T2 and a marked TSH antibody, the FT4 detection area is coated with an anti-FT4 antibody, the FT3 detection area is coated with an anti-FT3 antibody, and the TSH detection area is coated with an anti-TSH antibody. The problem of mutual interference during FT3 and FT4 detection can be effectively avoided, and the detection sensitivity is improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of in vitro diagnosis and immunodetection, and relates to a microfluidic chip for combined quantitative analysis of TSH, FT3 and FT4 and application thereof, in particular, combined quantitative analysis of TSH, FT3 and FT4. BACKGROUND

[0002] As the largest endocrine gland in the human body, the thyroid gland regulates whole-body metabolism and organ function through the secretion of thyroid hormones. Its dysfunction can cause various thyroid diseases, including hyperthyroidism, hypothyroidism and autoimmune thyroiditis. According to statistics, the prevalence of thyroid disease has been increasing year by year in recent years. Hyperthyroidism is thyrotoxicosis caused by thyroid disease, and hypothyroidism is a group of systemic low-metabolic syndrome caused by reduced synthesis and secretion of thyroid hormone or tissue utilization disorders. Thyroid dysfunction is an important cause of the above diseases. The thyroid gland is a key organ of the endocrine system, which synthesizes and releases thyroid hormones to regulate normal growth and development and metabolism in the human body.

[0003] Among them, thyroid-stimulating hormone (TSH) is a hormone secreted by the anterior pituitary, which mainly regulates the synthesis and secretion of thyroid hormones. TSH promotes the synthesis and release of triiodothyronine (T3) and thyroxine (T4) in thyroid cells by binding to TSH receptors on the cell surface. The secretion of TSH is negatively regulated by feedback, that is, when the concentration of T3 and T4 in the blood increases, the secretion of TSH decreases, and vice versa. In clinical detection, TSH is often used as the preferred indicator for thyroid function screening to assess the functional status of the thyroid gland.

[0004] T3 is an iodinated amino acid generated by direct secretion of the thyroid gland (20%) and peripheral tissue T4 deiodination (80%), containing 3 iodine atoms (3, 5, 3' positions), with a molecular weight of 651 Da. Although total T3 can reflect the total amount of hormones in circulation, its concentration is significantly affected by the levels of binding proteins such as thyroid-binding globulin and albumin, and cannot truly represent biological activity; free T3 (FT3) as the free form not bound to proteins, directly participates in nuclear receptor binding and regulates metabolic rate, and is a sensitive indicator of hyperthyroidism. In clinical detection, FT3 can more accurately reflect the immediate metabolic activity of the thyroid gland by avoiding the interference of binding proteins.

[0005] T4 is a hormone synthesized and secreted by thyroid follicular epithelial cells, containing 4 iodine atoms (3, 5, 3', 5' positions), with a molecular weight of 777 Da. About 99.6% of T4 in the blood is combined with binding protein, and its total amount is easily affected by drugs such as estrogen, glucocorticoid, and pathological conditions (such as nephrotic syndrome), resulting in false elevation or reduction; while free T4 (FT4) is not affected by the fluctuation of binding protein concentration, and is a key indicator of hypothyroidism.

[0006] In summary, TSH, FT4, and FT3 have become three important indicators for thyroid function tests. When patients have abnormal thyroid function, these three indicators will show abnormal changes. Therefore, accurate and high-sensitivity detection of TSH, FT4, and FT3 levels is beneficial to assist clinicians in the diagnosis and treatment of thyroid diseases.

[0007] TSH is a large molecule protein, and is usually detected by a double antibody sandwich mode, that is, a pair of matched antibodies are selected, one is used as a coating antibody (capture antibody), and the other is used as a labeled antibody (detection antibody). The capture antibody and the labeled antibody can bind to different antigen sites on the surface of TSH to form a double antibody sandwich complex.

[0008] FT3 and FT4 are both small molecule haptens, and their detection usually uses competitive immunoassay. In FT3 detection, an analog with high structural matching to natural FT3 (such as T3-carboxymethyl derivative or 3-maleimide propionic acid-T3) is selected as a competitive antigen; in FT4 detection, a T4 structural analog (such as T4-succinimidyl ester) is selected as a competitive antigen. The amount of antibody used needs to be strictly limited during detection, so that the antigen to be detected (FT3 / FT4 in the sample) and the labeled competitive antigen compete for the binding of a limited amount of specific antibody. After removing the free label, the signal intensity of the bound label is detected, which is inversely proportional to the concentration of the antigen to be detected. To improve the detection sensitivity, the following strategies can be used to optimize the competition system: ① Select a competitive substance with slightly lower affinity than the antigen to be detected to enhance the signal suppression of low concentration samples, such as selecting T2 as a competitive antigen for FT3 detection and T3 as a competitive antigen for FT4 detection; ② Use sequential competition mode, the antigen to be detected is preferentially combined with the antibody, and then the competitive antigen is combined with the antibody; ③ Use high-affinity antibodies to improve antigen recognition efficiency; ④ Optimize the molar ratio of competitive substance to antibody to avoid excessive competitive substance leading to signal saturation.

[0009] Currently, domestic hospitals mainly rely on enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassay (CLIA) for thyroid function testing. ELISA has been gradually phased out due to its cumbersome operation, long time consumption, and inferior sensitivity and detection range compared to CLIA. While CLIA has high sensitivity and accuracy, it is limited by the mandatory equipment binding of imported closed systems, high instrument costs, and premiums for dedicated reagents. In addition, the current technology requires separate detection of three indicators, TSH, FT3, and FT4, resulting in repeated reagent consumption and low operational efficiency.

[0010] In recent years, the incidence of thyroid diseases has been rising, seriously endangering human health. Even after a clear diagnosis, patients still need regular monitoring of their thyroid hormone levels to provide clinicians with a basis for adjusting medication concentrations. Therefore, the market urgently needs an integrated, low-cost, timely, and highly sensitive point-of-care testing (POCT) product for thyroid function testing to improve testing efficiency and reduce patients' waiting time.

[0011] Microfluidics is a technology that manipulates microfluidics through channels at the micrometer scale (tens to hundreds of micrometers). It integrates complex processes such as sample pretreatment, loading, reaction, separation, and detection required for detection and analysis into a chip at the square centimeter scale, providing the closed environment necessary for in vitro diagnostics, and achieving low sample consumption, rapid, and efficient analysis. Its greatest advantage lies in the flexible combination of various functional units, which not only significantly reduces reagent consumption and supports multi-indicator joint detection, but also has significant advantages such as high integration and strong device compatibility, creating new technical pathways for the joint detection of TSH, FT3, and FT4.

[0012] To improve the sensitivity of small molecule detection, the FT3 / FT4 competitive assay can use a competitor with a slightly lower affinity than the analyte antigen (e.g., T2 is used as the competing antigen for FT3 detection, and T3 for FT4 detection) to enhance signal suppression in low-concentration samples. In traditional lateral flow assays, the detection zone needs to be coated with anti-FT3 antibody, anti-FT4 antibody, and anti-TSH antibody, respectively, while the labeling zone is pre-dried with fluorescent microspheres labeled with T2 (FT3 competitor), T3 (FT4 competitor), and anti-TSH antibody. During sample migration: ① TSH preferentially binds to the labeled anti-TSH antibody in the liquid phase, forming a complex that migrates to the detection zone and is captured by the solid-phase antibody (double antibody sandwich assay); ② FT3 competes with the labeled T2 for binding to the solid-phase anti-FT3 antibody; ③ FT4 competes with the labeled T3 for binding to the solid-phase anti-FT4 antibody. This design presents two major problems: ① Low TSH sensitivity: During TSH detection, the rates of the two binding reactions differ significantly. TSH is less efficiently captured by the immobilized antibody as it flows through the detection zone, and there is no enrichment process. This often makes accurate quantification difficult for patients with severe hyperthyroidism. ② Cross-interference from competing antigens: Labeled T3 (used for FT4 detection) can competitively bind to the solid-phase anti-FT3 antibody, leading to falsely low FT3 detection results. Therefore, in traditional lateral flow microfluidic chips, since the reactions occur at the same level, it is impossible to integrate the detection of FT3 and FT4 into the same channel. Summary of the Invention

[0013] This invention aims to provide a microfluidic chip for combined quantitative analysis of TSH, FT3, and FT4 and its applications. By optimizing the chip design and detection methods, it solves the problems existing in the prior art and proposes a laminar flow-based microfluidic chip. Through a vertically layered design, FT4 competitive detection is integrated on the top layer of the chip, while FT3 competitive detection and TSH sandwich detection are integrated on the bottom layer. The height of the microchannels (20-50 μm) is precisely controlled to maintain a stable laminar flow state. After a single sample is injected, FT3, FT4, and TSH can be detected synchronously and without interference.

[0014] To achieve the above objectives, the technical solution of the present invention is as follows: a microfluidic chip for combined quantitative analysis of TSH, FT3, and FT4 includes a substrate and a cover plate pressed onto the substrate. The substrate and the cover plate enclose a microchannel with a height of 20-50 μm. The left end of the microchannel communicates with a buffer injection hole on the cover plate, and the right end of the microchannel is provided with a flow control valve. The flow control valve controls the opening and closing of the flow path of liquid in the microchannel to the flow control valve. From left to right, the microchannel is provided with a marking area and a detection area. The marking area includes a first marking area on the lower surface of the cover plate and a second marking area on the upper surface of the substrate. The detection area includes an FT4 detection area on the lower surface of the cover plate, an FT3 detection area and a TSH detection area on the upper surface of the substrate. The H detection areas do not overlap on the vertical plane. The cover plate is also provided with a sample injection hole, which is located between the detection area and the flow control valve. A magnetically controlled intercept valve is provided between the labeling area and the detection area of ​​the microchannel. The magnetically controlled intercept valve attracts and controls the opening and closing of the flow path between the labeling area and the detection area in the microchannel by a magnet. The first labeling area is coated with T3 label, the second labeling area is coated with T2 label and TSH antibody label, the FT4 detection area is coated with anti-FT4 antibody, the FT3 detection area is coated with anti-FT3 antibody, and the TSH detection area is coated with anti-TSH antibody.

[0015] In one embodiment of the present invention, the FT3 detection area, the TSH detection area, and the FT4 detection area are arranged sequentially from left to right along the microchannel.

[0016] In one embodiment of the present invention, a groove is provided on the lower surface of the cover sheet along its length direction, and the cover sheet forms a microchannel by means of the groove and the upper surface of the substrate, wherein the width of the microchannel is 2-3mm.

[0017] In one embodiment of the present invention, the labeled T3, labeled T2, and labeled TSH antibodies are fluorescent microspheres.

[0018] In one embodiment of the present invention, the cover plate is provided with a flow guide hole, the bottom of the magnetically controlled intercept valve passes through the flow guide hole and contacts the upper surface of the substrate, the top of the magnetically controlled intercept valve is provided with an iron material that can be attracted by a magnet, the bottom of the magnetically controlled intercept valve detaches from the flow guide hole after being attracted by magnetic force, and the bottom of the magnetically controlled intercept valve is provided with a water-absorbing material.

[0019] In one embodiment of the present invention, the flow control valve is a movable absorbent material, which moves to contact or move away from the microchannel.

[0020] On the other hand, the present invention also provides an application for joint detection of TSH, FT3 and FT4 using a microfluidic chip in any of the above technical solutions, comprising at least the following steps: 1) Move the flow control valve to the far right so that it is not connected to the microchannel. After adding the sample to be tested into the sample injection hole, the sample flows to the left and right. The liquid flowing to the right stays at the end of the microchannel, while the liquid flowing to the left can continue to flow. When it reaches the detection area, the upper liquid flows into contact with the lower surface of the cover plate. When it reaches the FT4 detection area, the FT4 contained in the sample is captured by the anti-human FT4 antibody solidified here, and the remaining substances are collected into the magnetically controlled intercept valve. The lower liquid flows into contact with the upper surface of the substrate. The TSH contained in the sample binds to the anti-TSH antibody solidified in the TSH detection area, and the FT3 in the sample is captured by the anti-FT3 antibody solidified in the FT3 detection area. After all the liquid in the channel has been collected into the magnetically controlled intercept valve, remove the intercept valve. At this time, the microchannel is restored to an unobstructed state. 2) Move the flow control valve to the far left so that it is embedded in the microchannel. Add buffer solution to the buffer injection well. Under the action of capillary driving force, the buffer solution enters the microchannel and flows forward in a laminar flow state. The upper liquid flow contacts the lower surface of the cover plate. When it reaches the first labeling area, the label T3 here is dissolved. When the label T3 reaches the FT4 detection area with the liquid flow, it binds to the remaining antibody sites. The excess liquid is collected into the control valve with the liquid flow. The lower liquid flow contacts the upper surface of the substrate. When it reaches the second labeling area, the label T2 and the labeled anti-TSH antibody here are dissolved. When the label T2 reaches the FT3 detection area, it binds to the remaining antibody sites. When the labeled anti-TSH antibody reaches the TSH detection area, it binds to the TSH that has been captured here. The remaining liquid flow continuously washes the microchannel and is collected into the flow control valve. 3) Read the signal values ​​of the FT3 detection area, TSH detection area, and FT4 detection area using conventional microfluidics.

[0021] The microfluidic chip for combined quantitative analysis of TSH, FT3, and FT4 obtained through the above technical solution and its application have the following beneficial effects: 1. Layered design effectively avoids mutual interference during FT3 and FT4 detection: In the dual-layer microfluidic chip, FT4 detection is set on the top layer of the chip, while FT3 and TSH detection are set on the bottom layer. At the same time, by strictly controlling the height of the microchannel (20-50μm) to maintain a stable laminar flow, the reactions of the upper and lower layers of the chip can proceed independently, avoiding the cross-interference problem of competing antigens, and realizing the joint detection of TSH, FT3 and FT4 in the same microchannel.

[0022] 2. Sandwich immunoassay is performed in steps to improve sensitivity: When detecting TSH, the sample preferentially binds to the capture antibody, and enrichment can be achieved by continuously adding samples. Then, the labeled anti-TSH antibody is dissolved, which binds to the TSH already captured in the detection area, which can enhance the capture of TSH in the sample, thereby effectively improving sensitivity.

[0023] 3. Enhance the sensitivity of competitive immunoassays: The sensitivity of competitive immunoassays can be enhanced in two ways: ① Select a competitor with a slightly lower affinity than the target antigen to enhance signal suppression in low-concentration samples. For FT3 detection, T2 is selected as the competitive antigen, and for FT4 detection, T3 is selected as the competitive antigen; ② Adopt a sequential competition mode, where the target antigen binds to the antibody first, and then the competitive antigen binds to the antibody. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the microfluidic chip (transparent) for combined quantitative analysis of TSH, FT3, and FT4 as described in this invention; Figure 2 This is a schematic diagram of the cover plate (transparent) of the present invention; Figure 3 This is a schematic diagram of the structure of the substrate described in this invention; Figure 4 This is a cross-sectional view of the microfluidic chip for combined quantitative analysis of TSH, FT3, and FT4 as described in this invention. Figure 5 This is a schematic diagram illustrating the principle of adding a sample to be tested using the layered microfluidic chip described in this invention. Figure 6 This is a schematic diagram of the principle of adding buffer solution to the layered microfluidic chip described in this invention; Figure 7 This is a biomolecular distribution map of the microfluidic chip for combined quantitative analysis of TSH, FT3, and FT4 as described in this invention; Figure 8 This invention is in Figure 7 A schematic diagram illustrating the principle of adding the sample to be tested. Figure 9 This invention is in Figure 8 A schematic diagram of the reaction stage of the sample under test on the chip; Figure 10 This invention is in Figure 9 A schematic diagram showing how a sample to be tested on a chip adsorbs waste liquid through a magnetically controlled interception valve. Figure 11 This invention is in Figure 10 The schematic diagram of adding buffer solution onto the chip.

[0025] In the figure, 1 is the substrate; 2 is the cover plate; 3 is the microchannel; 4 is the buffer injection port; 5 is the flow control valve; 6 is the sample injection port; 7 is the magnetically controlled intercept valve; 31 is the labeling area; 31a is the first labeling area; 31b is the second labeling area; 32 is the detection area; 32a is the FT4 detection area; 32b is the FT3 detection area; and 32c is the TSH detection area. Detailed Implementation

[0026] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0027] 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.

[0028] This invention relates to the field of immunoassay technology, specifically to a microfluidic chip for combined quantitative analysis of TSH, FT3, and FT4, and its application. The chip employs a vertically layered design, integrating FT4 competitive detection on the top layer and FT3 competitive and TSH sandwich detection on the bottom layer. It precisely controls the microchannel height (20-50 μm) to maintain a stable laminar flow, enabling simultaneous and non-interfering detection of FT3, FT4, and TSH after a single sample injection. This addresses the problems of existing detection methods, such as the need for multiple sample additions, high workload, complex and cumbersome detection processes, waste of reagents and raw materials, and high detection costs.

[0029] 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.

[0030] like Figures 1-4As shown, this invention proposes a microfluidic chip for combined quantitative analysis of TSH, FT3, and FT4, comprising a substrate 1 and a cover plate 2 pressed onto the substrate 1. The substrate 1 and the cover plate 2 enclose a microchannel 3, the height of which is 20-50 mm. μm, the left end of the microchannel 3 is connected to the buffer injection hole 4 on the cover plate 2, and the right end of the microchannel 3 is provided with a flow control valve 5. The flow control valve 5 controls the opening and closing of the flow path of liquid in the microchannel 3 to the flow control valve 5. The microchannel 3 is provided with a marking area 31 and a detection area 32 from left to right. The marking area 31 includes a first marking area 31a on the lower surface of the cover plate 2 (chip top layer) and a second marking area 31b on the upper surface of the substrate 1 (chip bottom layer). The detection area 32 includes an FT4 detection area 32a on the lower surface of the cover plate 2 (chip top layer), an FT3 detection area 32b and a TSH detection area 32c on the upper surface of the substrate 1 (chip bottom layer). 2a, FT3 detection area 32b, and TSH detection area 32c do not overlap on the vertical plane. The cover plate 2 is also provided with a sample injection hole 6, which is located between the detection area 32 and the liquid flow control valve 5. A magnetically controlled intercept valve 7 is provided between the labeling area 31 and the detection area 32 of the microchannel 3. The magnetically controlled intercept valve 7 uses a magnet to attract and control the opening and closing of the flow path between the labeling area 31 and the detection area 32 in the microchannel 3. The first labeling area 31a is coated with T3, the second labeling area 31b is coated with T2 and TSH antibody, the FT4 detection area 32a is coated with anti-FT4 antibody, the FT3 detection area 32b is coated with anti-FT3 antibody, and the TSH detection area 32c is coated with anti-TSH antibody.

[0031] In fluid mechanics, the Reynolds number (Re) is a key parameter for determining the flow state of a liquid. When Re is below a critical value, the flow is laminar. In microfluidic channels, Re < 100 can be considered laminar flow.

[0032] According to the formula for calculating the Reynolds number: Re = (ρ is the fluid density, v is the average flow velocity, Dh is the hydraulic diameter, and μ is the fluid dynamic viscosity). For a rectangular microchannel, Dh = (w is the channel width, h is the channel height). Since the width of the microchannel is much greater than its height, Dh can be simplified to Dh = 2h. Therefore, the height of the microchannel is the key parameter that determines the liquid flow state in the microchannel.

[0033] For common fluids (such as water and buffer solutions), at typical flow rates (0.1-10 mm / s), the channel height h is usually set to 10-100 μm to ensure Re < 100. However, for other liquids with faster flow rates, if the microchannel height is not designed properly, the internal flow will exhibit an irregular turbulent state, causing reactions at different levels to interfere with each other. Therefore, limiting the microchannel height to 20-50 μm ensures that any liquid flow exhibits a stable laminar flow state within the microchannel.

[0034] The workflow diagram of the layered microfluidic chip is as follows: A. Detection molecule capture stage: such as Figure 5 As shown, the flow control valve is switched to the far right, disconnecting it from the microchannel. After adding the sample to be tested into the sample injection port, the sample flows to the left and right. Because the microchannel is not connected to the flow control valve, the liquid flowing to the right remains at the end of the microchannel due to the surface tension at the tip and does not flow out spontaneously, while the liquid flowing to the left continues to flow. When it reaches the detection zone, the analyte binds to the capture antibody in the detection zone. When the liquid continues to flow and reaches the magnetically controlled intercept valve, it is intercepted. Because the suction force provided by the magnetically controlled intercept valve is greater than the capillary force in the microchannel, the liquid is immediately collected into the intercept valve and will not continue to flow to the left. After all the liquid in the microchannel has been collected into the magnetically controlled intercept valve, the magnetically controlled intercept valve is removed, and the microchannel is restored to its unobstructed state, ensuring that subsequent reagent flow and reactions are not disturbed.

[0035] B. The stage of marker substance binding: such as Figure 6 As shown, move the flow control valve to the far left so that it is embedded in the microchannel. Add buffer solution to the buffer injection well. Under the action of capillary driving force, the buffer solution enters the microchannel and dissolves the labeled antibody in the labeled area as it flows through it. After the labeled antibody reaches the detection area with the flow, it is captured by the corresponding substance in the detection area. The remaining flow is collected in the flow control valve.

[0036] The FT3 detection area 32b, TSH detection area 32c, and FT4 detection area 32a are arranged sequentially from left to right along the microchannel 3.

[0037] The cover plate 2 has a groove along its length on its lower surface. The cover plate 2 and the upper surface of the substrate 1 are enclosed by the groove to form a microchannel 3. The width of the microchannel 3 is 2-3 mm.

[0038] The labeled T3, labeled T2, and labeled TSH antibodies are fluorescent microsphere labeled.

[0039] The cover plate 2 is provided with a flow guide hole. The bottom of the magnetically controlled intercept valve 7 passes through the flow guide hole and contacts the upper surface of the substrate 1. The top of the magnetically controlled intercept valve 7 is provided with an iron material that can be attracted by a magnet. After the magnetically controlled intercept valve 7 is attracted by magnetic force, the bottom of the magnetically controlled intercept valve 7 is separated from the flow guide hole. The bottom of the magnetically controlled intercept valve 7 is provided with a water-absorbing material.

[0040] The flow control valve 5 is made of a movable absorbent material, which moves to contact or move away from the microchannel 3.

[0041] like Figure 7 As shown, the biomolecule distribution of the microfluidic chip used for combined quantitative analysis of TSH, FT3, and FT4 is as follows: Labeled region 1: fluorescent microspheres labeled T3 Labeling region 2: Fluorescent microspheres labeled with T2, fluorescent microspheres labeled with anti-TSH antibody Detection area 1: Anti-FT4 antibody Detection area 2: Anti-FT3 antibody Detection area 3: Anti-TSH antibody The detection principle is as follows: Detection molecule capture stage: such as Figure 8 As shown, the flow control valve is switched to the far right, disconnecting it from the microchannel. After adding the sample to be tested through the sample injection port, the sample flows to the left and right. Since the microchannel is not connected to the flow control valve at this time, under the action of surface tension at the end, the liquid flowing to the right remains at the end of the microchannel and will not flow out spontaneously, while the liquid flowing to the left can continue to flow. Figure 9 As shown, the upper liquid stream contacts the lower surface of the cover plate (top layer of the chip). When it reaches the FT4 detection area, the FT4 in the sample is captured by the anti-human FT4 antibody solidified there, and the remaining substances are collected into the magnetically controlled intercept valve. The lower liquid stream contacts the upper surface of the substrate (bottom layer of the chip). The TSH in the sample binds to the anti-TSH antibody solidified in the TSH detection area, while the FT3 in the sample is captured by the anti-FT3 antibody solidified in the FT3 detection area. Figure 10 As shown, after all the liquid flow in the channel has been collected into the magnetically controlled intercept valve, the intercept valve is removed, and the microchannel is restored to its unobstructed state.

[0042] Marking the stage of substance binding: such as Figure 11As shown, the flow control valve is switched to the far left, embedding it within the microchannel. Buffer is added to the buffer injection well, entering the microchannel under capillary force and flowing forward in a laminar flow. The upper flow contacts the top layer of the chip, dissolving the T3 label in the labeled area. When the T3 label reaches the FT4 detection area, it binds to the remaining antibody sites, while excess labeled antigen is collected into the control valve. The lower flow contacts the bottom layer of the chip, dissolving the T2 label and anti-TSH antibody in the labeled area. When the T2 label reaches the FT3 detection area, it binds to the remaining antibody sites; when the anti-TSH antibody reaches the TSH detection area, it binds to the captured TSH. The remaining flow continuously washes the microchannel and is collected into the flow control valve.

[0043] It is understood that this invention does not limit the placement of the FT3 detection region and the TSH detection region on the upper surface of the substrate (bottom layer of the chip) and the placement of the FT4 detection region on the lower surface of the cover plate (top layer of the chip). It only limits the placement of the FT3 detection region and the FT4 detection region on different horizontal levels. That is, if the FT4 detection region is placed on the upper surface of the substrate (bottom layer of the chip), then the FT3 detection region must be placed on the lower surface of the cover plate (top layer of the chip). At the same time, the second marking region corresponding to the upper surface of the substrate must be coated with T3, and the first marking region corresponding to the lower surface of the cover plate must be coated with T2. The location of the TSH detection region on the upper surface of the substrate (bottom layer of the chip) or the lower surface of the cover plate (top layer of the chip) is not limited. It is only required that the corresponding marking region on the upper surface of the substrate (bottom layer of the chip) or the lower surface of the cover plate (top layer of the chip) is coated with TSH antibody.

[0044] Example

[0045] 1. Chip fabrication Both the substrate and the coverslip were made of PMMA material. The microchannel structure, sample injection port, buffer injection port, drainage port, and other structures of the coverslip were designed using CAD software, and then the PMMA surface was processed using a CO2 laser etching machine.

[0046] 2. Biomolecular coating program on the top layer of the chip First, 1.5 μL of streptavidin was spotted on the top layer of the chip corresponding to the FT4 detection area, incubated for 1 h, washed, and dried. Then, 1.6 μL of anti-FT4 antibody was spotted on the same detection area, and incubation continued for 1 h. After another wash, fluorescent microspheres conjugated with T3 competitive antigen were spotted on the labeled area and dried at 37°C.

[0047] 3. Biomolecular coating program at the chip's underlying layer First, 1.5 μL of streptavidin was spotted at the locations corresponding to the FT3 and TSH detection areas on the bottom layer of the chip. After incubation for 1 h and washing, the sample was dried and then 1.6 μL of anti-FT3 antibody and anti-TSH antibody were spotted at the aforementioned detection areas. Incubation was continued for another 1 h. After washing again, fluorescent microspheres conjugated with anti-T2 competitive antigen and anti-TSH antibody were spotted at the labeled areas and dried at 37°C.

[0048] 4. Microfluidic chip assembly procedure First, the liquid flow interception valve is embedded in the liquid guide hole of the top layer of the chip; second, the liquid flow control valve is placed in the waste liquid chamber of the top layer of the chip; finally, the top layer and the bottom layer of the chip are tightly bonded together.

[0049] 5. Testing Procedure Three different types of samples were selected for testing: First, move the flow control valve to the far right, disconnecting it from the microchannel. Add 10 μL of the sample to be tested into the sample injection port. The sample flows to the left and right. Because the microchannel is not connected to the flow control valve, the liquid flowing to the right remains at the end of the microchannel due to surface tension and will not flow out spontaneously, while the liquid flowing to the left continues to flow. The upper liquid flow contacts the inner surface of the top layer of the chip. When it reaches the FT4 detection area, the FT4 in the sample is captured by the anti-human FT4 antibody solidified there, and the remaining substances are collected into the flow interception valve. The lower liquid flow contacts the inner surface of the bottom layer of the chip. The TSH in the sample binds to the anti-TSH antibody solidified in the TSH detection area, and the FT3 in the sample is captured by the anti-FT3 antibody solidified in the FT3 detection area. After all the liquid in the channel has been collected into the interception valve, remove the interception valve. At this point, the microchannel is reopened.

[0050] Next, move the flow control valve to the far left, embedding it within the microchannel. Add buffer solution to the buffer injection well; it enters the microchannel under capillary force and flows forward in a laminar flow. The upper layer of the flow contacts the inner surface of the top layer of the chip, dissolving the T3 label in the labeled area. When the T3 label reaches the FT4 detection area, it binds to the remaining antibody sites, while excess labeled antigen is collected into the control valve. The lower layer of the flow contacts the inner surface of the bottom layer of the chip, dissolving the T2 label and anti-TSH antibody in the labeled area. When the T2 label reaches the FT3 detection area, it binds to the remaining antibody sites; when the anti-TSH antibody reaches the TSH detection area, it binds to the captured TSH. The remaining flow continuously washes the microchannel and is collected into the flow control valve.

[0051] 6. Experimental Results

[0052] Using the chip in this invention to test different types of samples, the obtained CV value is less than 10%, which meets the precision requirements of the detection.

[0053] 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.

[0054] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0055] 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.

[0056] 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. A microfluidic chip for combined quantitative analysis of TSH, FT3, and FT4, comprising a substrate and a cover plate pressed onto the substrate, wherein the substrate and the cover plate enclose a microchannel with a height of 20-50 μm, the left end of the microchannel communicating with a buffer injection port on the cover plate, and a flow control valve disposed at the right end of the microchannel, the flow control valve controlling the opening and closing of the flow path of liquid in the microchannel to the flow control valve, wherein the microchannel is provided with a labeling area and a detection area from left to right, characterized in that... The labeling area includes a first labeling area disposed on the lower surface of the cover plate and a second labeling area disposed on the upper surface of the substrate. The detection area includes an FT4 detection area disposed on the lower surface of the cover plate, an FT3 detection area disposed on the upper surface of the substrate, and a TSH detection area disposed on the upper surface of the substrate. The FT4 detection area, FT3 detection area, and TSH detection area do not overlap in the vertical plane. The cover plate is also provided with a sample injection hole, which is disposed between the detection area and the flow control valve. A magnetically controlled intercepting valve is disposed between the labeling area and the detection area of ​​the microchannel. The magnetically controlled intercepting valve attracts and controls the opening and closing of the flow path between the labeling area and the detection area in the microchannel by means of a magnet. The first labeling area is coated with T3 label, the second labeling area is coated with T2 label and TSH antibody label, the FT4 detection area is coated with anti-FT4 antibody, the FT3 detection area is coated with anti-FT3 antibody, and the TSH detection area is coated with anti-TSH antibody.

2. The microfluidic chip for combined quantitative analysis of TSH, FT3, and FT4 according to claim 1, characterized in that, The FT3 detection area, TSH detection area, and FT4 detection area are arranged sequentially from left to right along the microchannel.

3. The microfluidic chip for combined quantitative analysis of TSH, FT3, and FT4 according to claim 1, characterized in that, The cover plate has a groove along its length on its lower surface. The cover plate forms a microchannel by enclosing the upper surface of the substrate through the groove. The width of the microchannel is 2-3 mm.

4. The microfluidic chip for combined quantitative analysis of TSH, FT3, and FT4 according to claim 1, characterized in that, The labeled T3, labeled T2, and labeled TSH antibodies are fluorescent microsphere labeled.

5. A microfluidic chip for combined quantitative analysis of TSH, FT3, and FT4 according to claim 1, characterized in that, The cover plate is provided with a flow guide hole. The bottom of the magnetically controlled intercept valve passes through the flow guide hole and contacts the upper surface of the substrate. The top of the magnetically controlled intercept valve is provided with an iron material that can be attracted by a magnet. After the magnetically controlled intercept valve is attracted by magnetic force, the bottom of the magnetically controlled intercept valve is separated from the flow guide hole. The bottom of the magnetically controlled intercept valve is provided with a water-absorbing material.

6. The microfluidic chip for combined quantitative analysis of TSH, FT3, and FT4 according to claim 1, characterized in that, The flow control valve is made of a movable absorbent material, which moves to contact or move away from the microchannel.

7. An application of a microfluidic chip for combined quantitative analysis of TSH, FT3, and FT4, characterized in that, The detection using the microfluidic chip according to any one of claims 1-6 includes at least the following steps: 1) Move the flow control valve to the far right so that it is not connected to the microchannel. After adding the sample to be tested into the sample injection hole, the sample flows to the left and right. The liquid flowing to the right stops at the end of the microchannel due to surface tension, while the liquid flowing to the left can continue to flow. When it reaches the detection area, the upper liquid flows into contact with the lower surface of the cover plate. When it reaches the FT4 detection area, the FT4 contained in the sample is captured by the anti-human FT4 antibody solidified here, and the remaining substances are collected into the magnetically controlled intercept valve. The lower liquid flows into contact with the upper surface of the substrate. The TSH contained in the sample binds to the anti-TSH antibody solidified in the TSH detection area, and the FT3 in the sample is captured by the anti-FT3 antibody solidified in the FT3 detection area. After all the liquid in the channel has been collected into the magnetically controlled intercept valve, remove the intercept valve. At this time, the microchannel is restored to an unobstructed state. 2) Move the flow control valve to the far left so that it is embedded in the microchannel. Add buffer solution to the buffer injection well. Under the action of capillary driving force, the buffer solution enters the microchannel and flows forward in a laminar flow state. The upper layer of liquid contactes the lower surface of the coverslip. When it reaches the first labeling area, it dissolves the T3 label. When the T3 label reaches the FT4 detection area, it binds to the remaining antibody sites. The excess liquid is collected into the control valve. The lower layer of liquid contacts the upper surface of the substrate. When it reaches the second labeling area, it dissolves the T2 label and the labeled anti-TSH antibody. When the T2 label reaches the FT3 detection area, it binds to the remaining antibody sites. When the labeled anti-TSH antibody reaches the TSH detection area, it binds to the TSH that has been captured there. The remaining liquid continuously washes the microchannel and is collected into the flow control valve. 3) Read the signal values ​​of the FT3 detection area, TSH detection area, and FT4 detection area using conventional microfluidics.

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