An immunomicrofluidic chip that can broaden the range of β-hCG quantitative analysis and its application

Abstract

This invention proposes an immunomicrofluidic chip and its application that can broaden the quantitative analysis range of β-hCG. It includes a substrate and a cover plate pressed onto the substrate, with the substrate and cover plate forming a microchannel. The microchannel is provided with a labeling region and a detection region. The labeling region includes a first labeling region on the lower surface of the cover plate and a second labeling region on the upper surface of the substrate. The detection region includes a low-concentration β-hCG detection region on the lower surface of the cover plate and a high-concentration β-hCG detection region on the upper surface of the substrate. The first labeling region is coated with a labeled anti-human β-hCG antibody, and the second labeling region is coated with a labeled anti-human β-hCG antibody. The low-concentration β-hCG detection region is coated with a high-affinity anti-human β-hCG antibody, and the high-concentration β-hCG detection region is coated with a low-affinity anti-human β-hCG antibody. This enables accurate quantification of β-hCG with a wide concentration range.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostics and immunoassay technology, and specifically relates to an immunomicrofluidic chip that can broaden the quantitative analysis range of β-hCG. Background Technology

[0002] Human chorionic gonadotropin (hCG) is a glycoprotein hormone secreted by placental trophoblast cells. It consists of an α-subunit and a β-subunit, with the β-subunit conferring specific biological activity and an immune recognition site for hCG. In early pregnancy, syncytiotrophoblast cells in the placenta secrete large amounts of hCG, which helps maintain corpus luteum function during pregnancy, prevents endometrial shedding, and thus maintains early pregnancy. Furthermore, almost all trophoblastic tumors and most germ cell tumors of the gonads can also produce hCG. Therefore, dynamic monitoring of β-hCG is of significant value for early pregnancy confirmation and embryonic development monitoring.

[0003] In clinical practice, the analytical performance requirements for β-hCG detection are reflected in two aspects: high sensitivity and a wide detection range. Serum β-hCG levels are low in early pregnancy (often <5 IU / L), therefore, confirming early pregnancy requires extremely high detection sensitivity. High sensitivity is also crucial for predicting adverse outcomes in early pregnancy; a β-hCG level below 5 IU / L can essentially rule out pregnancy, while a lower-than-expected increase in β-hCG levels usually indicates an abnormal pregnancy, such as ectopic pregnancy, while a decrease in levels usually indicates pregnancy failure, such as miscarriage. Furthermore, in anti-doping surveillance, trace amounts of exogenous hCG and its metabolites are present, also requiring ultra-high sensitivity detection to identify illicit drug abuse.

[0004] On the other hand, during pregnancy, β-hCG levels increase exponentially, peaking between 8 and 10 weeks, then declining to moderate levels in the fourth month and remaining at that level until late pregnancy. Since serum β-hCG levels vary across different stages of pregnancy, dynamic monitoring of β-hCG helps to understand changes in fetal health in a timely manner. If the β-hCG concentration exceeds the measurement range of the detection system, a hook effect may occur, leading to false negative results or results lower than the actual concentration, which will seriously affect the clinician's judgment. Therefore, an ideal β-hCG detection method needs to have both high sensitivity (covering the needs of early pregnancy and doping monitoring) and a wide detection range (avoiding the hook effect at high concentrations).

[0005] β-hCG is a large-molecule protein hormone, and its immunoassay employs a double-antibody sandwich method: a capture antibody on a solid-phase support and a detection antibody labeled with a tracer specifically bind to different antigenic epitopes of β-hCG, forming a capture antibody-β-hCG-detection antibody complex. The affinity between the two paired antibodies in the double-antibody sandwich complex is a key factor determining the analytical sensitivity and detection range. Differences in the affinity between the detection antibody and the capture antibody result in different start and end points of the dose curve. Using a high-affinity capture antibody can improve detection sensitivity but is prone to a hook effect; using a low-affinity capture antibody results in lower sensitivity but a higher upper limit of detection.

[0006] Currently, chemiluminescence immunoassay has become a commonly used method for serum β-hCG detection in clinical laboratories, with a wide variety of detection products emerging. Most of these products can meet the analytical sensitivity requirements for detecting low levels of β-hCG. However, when detecting high-level β-hCG samples, it is usually necessary to dilute the sample and retest to achieve accurate quantification. This not only introduces operational errors but also increases the cost and time of single-sample testing. In contrast, urine β-hCG test strips based on colloidal gold immunochromatography, while portable and easy to read, have low sensitivity, only enabling qualitative / semi-quantitative detection, and cannot meet the clinical needs for precise quantification in pregnancy monitoring and disease management. Clearly, existing detection systems, limited by single-affinity antibody design, fail to achieve a balance between high sensitivity and a wide detection range. Therefore, there is an urgent need to develop a detection platform with synergistic effects of high and low affinity antibodies to overcome the two major technical bottlenecks of trace β-hCG detection and the anti-hook effect of ultra-high concentration β-hCG.

[0007] When implementing the co-detection of β-hCG with high- and low-affinity antibodies in conventional lateral flow microfluidic chips, two pairs of specific antibody combinations need to be screened: a high-affinity capture antibody and a low-affinity capture antibody, each coated onto its corresponding detection site (T1: high-affinity region; T2: low-affinity region). The design of the detection antibody faces two choices: a matching scheme (i.e., the high-affinity detection antibody corresponds to T1, and the low-affinity detection antibody corresponds to T2), and a universal scheme (a single detection antibody simultaneously recognizes both detection regions). In this case, the calibration curve will also incorporate two dose-response curves based on the specific combination. However, in conventional lateral flow microchips, all biomolecules are pre-immobilized in the biochip, and the reactions occur at the same layer of the chip. Therefore, regardless of whether a matching or universal scheme is chosen, the detection antibody can, to some extent, bind to the capture antibody-β-hCG complex in both detection regions (T1 high-affinity region / T2 low-affinity region), leading to deviations in the quantitative results. Summary of the Invention

[0008] This invention aims to provide an immunomicrofluidic chip and its application that can broaden the quantitative analysis range of β-hCG. By optimizing the chip design and detection method, it solves the problems existing in the prior art and proposes a layered microfluidic chip. By integrating the low-concentration detection area (coated with high-affinity capture antibody) and the high-concentration detection area (coated with low-affinity capture antibody) into independent fluid layers of the chip, sensitivity is ensured while the detection range is broadened, enabling accurate quantification of β-hCG with a wide concentration range.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows: an immunomicrofluidic chip that can broaden the quantitative analysis range of β-hCG 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. A flow control valve is provided at the right end of the microchannel, which 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 labeling area and a detection area. The labeling area includes a first labeling area on the lower surface of the cover plate and a second labeling area on the upper surface of the substrate. The detection area includes a low-concentration β-hCG detection area on the lower surface of the cover plate and a high-concentration β-hCG detection area on the upper surface of the substrate. The high-concentration β-hCG detection area and the low-concentration β-hCG detection area are arranged along the... The microchannels are arranged from left to right. 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 means of a magnet. The first labeling area is coated with a labeled anti-human β-hCG antibody one, the second labeling area is coated with a labeled anti-human β-hCG antibody two, the low-concentration β-hCG detection area is coated with a high-affinity anti-human β-hCG antibody, and the high-concentration β-hCG detection area is coated with a low-affinity anti-human β-hCG antibody.

[0010] In one embodiment of the present invention, the labeled anti-human β-hCG antibody one is matched with the high-affinity anti-human β-hCG antibody, and the labeled anti-human β-hCG antibody two is matched with the low-affinity anti-human β-hCG antibody.

[0011] In one embodiment of the present invention, the labeled anti-human β-hCG antibody one and the labeled anti-human β-hCG antibody two are homoantibodies.

[0012] In one embodiment of the present invention, the horizontal distance between the high-concentration β-hCG detection area and the buffer injection well is 30-35 mm, and the horizontal distance between the low-concentration β-hCG detection area and the buffer injection well is 40-45 mm.

[0013] 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.

[0014] In one embodiment of the present invention, the labeled anti-human β-hCG antibody one and labeled anti-human β-hCG antibody two are fluorescent microsphere labeled.

[0015] 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.

[0016] 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.

[0017] On the other hand, the present invention also provides an application of the microfluidic chip in any of the above technical solutions for β-hCG detection, comprising at least the following steps:

[0018] 1) Establishment of dual-zone calibration curves: The concentration gradient of β-hCG calibrator was detected using a microfluidic chip. Based on the signal response values ​​of the top low-concentration β-hCG detection zone and the bottom high-concentration β-hCG detection zone, calibration curves were established respectively. The calibration curve of the low-concentration β-hCG detection zone was denoted as CC1, and the calibration curve of the high-concentration β-hCG detection zone was denoted as CC2.

[0019] 2) Add the sample to be tested into the sample injection hole. At this time, turn the liquid flow control valve to the rightmost position so that it is not connected to the microchannel. The sample flows to the left and right. The liquid flowing to the right stays at the end of the microchannel under the action of surface tension. The liquid flowing to the left first flows through the low concentration β-hCG detection area and then flows through the high concentration β-hCG detection area. The liquid continues to flow and is blocked when it reaches the magnetic interception valve.

[0020] 3) After all the liquid flow in the microchannel has been collected into the magnetically controlled intercept valve, remove the magnetically controlled intercept valve. The microchannel will then be unobstructed. Move the liquid flow control valve to the far left so that it is embedded in the microchannel. Add buffer solution to the buffer injection well. The buffer solution will enter the microchannel under the action of capillary driving force and flow forward in a laminar flow state. The upper liquid flow will contact the lower surface of the cover plate, dissolve the labeled antibody in the first labeling area, and continue to flow to the low concentration β-hCG detection area to bind with the captured target analyte. The lower liquid flow will contact the upper surface of the substrate, dissolve the labeled antibody in the second labeling area, and continue to flow to the high concentration β-hCG detection area to bind with the captured target analyte. The remaining liquid flow will continuously wash the microchannel and be collected into the liquid flow control valve. After the reaction is complete, read the signal values ​​of the low concentration β-hCG detection area and the high concentration β-hCG detection area, respectively.

[0021] 4) Determine the concentration of the target analyte by comparing the dual-zone calibration curves: When a signal value is detected in the low-concentration β-hCG detection zone, while no signal value or only a weak signal value is detected in the high-concentration β-hCG detection zone, the concentration of the target analyte is determined based on the calibration curve CC1 and the signal value of the low-concentration β-hCG detection zone. When the signal value of the low-concentration β-hCG detection zone reaches the upper limit or the signal value of the high-concentration β-hCG detection zone is higher than the signal value of the low-concentration β-hCG detection zone, the concentration of the target analyte is determined based on the calibration curve CC2 and the signal value of the high-concentration β-hCG detection zone.

[0022] The immunomicrofluidic chip obtained through the above technical solution, which can broaden the quantitative analysis range of β-hCG, and its application, have the following beneficial effects:

[0023] 1. Balancing sensitivity and detection range: By placing high-affinity antibodies and low-affinity antibodies on different fluid layers of the same chip, different affinities of antibodies can play different roles. At the same time, combined with a hyperbolic quantification strategy, the detection range is broadened while ensuring sensitivity, enabling accurate quantification of β-hCG with a wide concentration range and reducing unnecessary dilution.

[0024] 2. Dual-layer design ensures quantitative accuracy: High-affinity antibodies and low-affinity antibodies are placed on different fluid layers of the same chip. The high / low concentration detection areas eliminate signal crosstalk through spatial stratification, and the reactions in the upper and lower layers proceed independently, enabling precise quantification. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the structure of the immunomicrofluidic chip (transparent) that can broaden the quantitative analysis range of β-hCG according to the present invention;

[0026] Figure 2 This is a schematic diagram of the cover plate (transparent) of the present invention;

[0027] Figure 3 This is a schematic diagram of the structure of the substrate described in this invention;

[0028] Figure 4 This is a cross-sectional view of the immunomicrofluidic chip that can broaden the quantitative analysis range of β-hCG according to the present invention;

[0029] 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.

[0030] Figure 6 This is a schematic diagram of the principle of adding buffer solution to the layered microfluidic chip described in this invention;

[0031] Figure 7 This is a biomolecular distribution map of the immunomicrofluidic chip described in this invention, which can broaden the quantitative analysis range of β-hCG.

[0032] Figure 8 This invention is in Figure 7 A schematic diagram illustrating the principle of adding the sample to be tested.

[0033] Figure 9 This invention is in Figure 8 A schematic diagram of the reaction stage of the sample under test on the chip;

[0034] 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.

[0035] Figure 11 This invention is in Figure 10 The schematic diagram of adding buffer solution onto the chip.

[0036] 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; L is the labeling area; L1 is the first labeling area; L2 is the second labeling area; T is the detection area; T1 is the low-concentration β-hCG detection area; T2 is the high-concentration β-hCG detection area. Detailed Implementation

[0037] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0038] 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.

[0039] This invention relates to the field of immunoassay technology, specifically to an immunomicrofluidic chip that broadens the quantitative analysis range of β-hCG and its applications. It integrates a low-concentration detection region (coated with a high-affinity capture antibody) and a high-concentration detection region (coated with a low-affinity capture antibody) into separate fluid layers on the chip, precisely controlling the microchannel height (20-50 μm) to maintain a stable laminar flow. The high-affinity antibody preferentially captures β-hCG in low-concentration samples, ensuring high sensitivity; the low-affinity antibody is less prone to saturation in high-concentration samples, effectively capturing β-hCG and reducing the hook effect, thus extending the detection upper limit. The synergistic effect of both achieves a wide dynamic range. Furthermore, the spatial stratification of the high / low concentration detection regions eliminates signal crosstalk, facilitating accurate quantification.

[0040] 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.

[0041] like Figure 1-4 As shown, this invention proposes an immunomicrofluidic chip that can broaden the quantitative analysis range of β-hCG, including a substrate 1 and a cover plate 2 pressed onto the substrate. The substrate 1 and the cover plate 2 enclose a microchannel 3, the height of which is 20-50 μm. The left end of the microchannel 3 is connected to a buffer injection hole 4 on the cover plate 2, and a flow control valve 5 is provided at the right end of the microchannel 3. 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 2 is provided with a labeling area L and a detection area T from left to right. The labeling area L includes a first labeling area L1 located on the lower surface of the cover plate 2 (top layer of the chip) and a second labeling area L2 located on the upper surface of the substrate 1 (bottom layer of the chip). The detection area T includes a low-concentration β-hCG detection area T1 located on the lower surface of the cover plate 2 (top layer of the chip) and a high-concentration β-hCG detection area T2 located on the upper surface of the substrate 1 (bottom layer of the chip). The low-concentration β-hCG detection zone T1 is arranged from left to right along the microchannel. The cover plate 2 is also provided with a sample injection hole 6, which is located between the detection zone T and the flow control valve 5. A magnetically controlled intercept valve 7 is provided between the labeling zone L and the detection zone T 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 zone L and the detection zone T in the microchannel. The first labeling zone L1 is coated with a labeled anti-human β-hCG antibody one, and the second labeling zone L2 is coated with a labeled anti-human β-hCG antibody two. The low-concentration β-hCG detection zone T1 is coated with a high-affinity anti-human β-hCG antibody, and the high-concentration β-hCG detection zone T2 is coated with a low-affinity anti-human β-hCG antibody.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] The workflow of the layered microfluidic chip is as follows:

[0046] 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. The liquid continues to flow and is intercepted at the magnetically controlled intercept valve. 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 intercept valve is removed, and the microchannel is restored to its original state, ensuring that subsequent reagent flow and reactions are not disturbed.

[0047] 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.

[0048] The labeled anti-human β-hCG antibody one is matched with the high-affinity anti-human β-hCG antibody, and the labeled anti-human β-hCG antibody two is matched with the low-affinity anti-human β-hCG antibody.

[0049] The labeled anti-human β-hCG antibody one and labeled anti-human β-hCG antibody two are the same antibody.

[0050] Since the microfluidic chip of this application is based on laminar flow, high-affinity antibodies and low-affinity antibodies are set on different fluid layers of the same chip. Therefore, whether different labeled antibodies are set according to the corresponding detection area or a universal labeled antibody is set, they will not affect each other.

[0051] The horizontal distance between the high-concentration β-hCG detection area T2 and the buffer injection hole 4 is 30-35 mm, and the horizontal distance between the low-concentration β-hCG detection area T1 and the buffer injection hole 4 is 40-45 mm.

[0052] The cover plate 2 has a groove on its lower surface (top layer of the chip) along its length. The cover plate 2 and the upper surface of the substrate 1 (bottom layer of the chip) are enclosed by the groove to form a microchannel 3. The width of the microchannel 3 is 2-3mm.

[0053] The labeled anti-human β-hCG antibody one and labeled anti-human β-hCG antibody two are labeled with fluorescent microspheres.

[0054] 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.

[0055] The flow control valve 7 is made of a movable absorbent material, which moves to contact or move away from the microchannel.

[0056] like Figure 7 As shown, the biomolecule distribution of the immunomicrofluidic chip, which can broaden the range of β-hCG quantitative analysis, is as follows:

[0057] L1: Fluorescent microsphere-labeled anti-human β-hCG antibody 1

[0058] L2: Fluorescent microsphere-labeled anti-human β-hCG antibody 2

[0059] T1: High-affinity anti-human β-hCG antibody

[0060] T2: Low-affinity anti-human β-hCG antibody

[0061] The detection principle is as follows:

[0062] 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 sample first encounters a high-affinity anti-human β-hCG antibody in the low-concentration β-hCG detection region, which is beneficial for identifying low-concentration β-hCG. If the β-hCG content is too high, the sample continues to flow to the left to the high-concentration β-hCG detection region, where it encounters a low-affinity anti-human β-hCG antibody. This pairing plateau occurs later, which is beneficial for identifying high-concentration β-hCG. 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.

[0063] Marking the binding stage of substances: such as Figure 11 As 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 state. The upper layer of the flow contacts the lower surface of the cover (top layer of the chip). Upon reaching the first labeling area, the fluorescent microspheres labeled with anti-human β-hCG antibody I dissolve. This labeled anti-human β-hCG antibody I then travels with the flow to the low-concentration β-hCG detection area, where it binds to the captured β-hCG, forming a double-antibody sandwich complex. The lower layer of the flow contacts the upper surface of the substrate (bottom layer of the chip). Upon reaching the second labeling area, the labeled anti-human β-hCG antibody II dissolves and subsequently binds to the captured β-hCG in the high-concentration β-hCG detection area. At this point, the upper and lower layer reactions do not interfere with each other, ensuring accurate quantification. The remaining flow continuously washes the microchannel and is collected in the flow control valve.

[0064] The β-hCG quantification method is as follows:

[0065] (1) Establishment of dual-zone calibration curves: The concentration gradient of β-hCG calibrator was detected using a layered microfluidic chip. Based on the signal response values ​​of the top low-concentration β-hCG detection zone (pre-coated with high-affinity anti-human β-hCG antibody) and the bottom high-concentration detection zone (pre-coated with low-affinity anti-human β-hCG antibody), calibration curves were established respectively. The calibration curve of T1 zone was denoted as CC1, and the calibration curve of T2 zone was denoted as CC2.

[0066] (2) β-hCG quantification strategy:

[0067] Low-concentration sample detection procedure: When the sample flows through zone T1, β-hCG in the supernatant is captured by high-affinity antibodies. Upon reaching zone T2, the signal is weak or absent due to the weak binding affinity of low-affinity antibodies to trace antigens. Next, the labeled antibodies in each layer bind to the complex in their respective layers, ultimately resulting in a significantly higher signal value in zone T1 compared to zone T2. At this point, a CC1 curve is used for quantification, and the concentration of β-hCG in the sample is determined based on the signal value in zone T1.

[0068] High-concentration sample detection procedure: When the sample flows through the T1 region, high-affinity antibodies rapidly saturate, causing the signal to plateau. Upon reaching the T2 region, low-affinity antibodies continuously capture β-hCG, are less prone to saturation, and can broaden the detection upper limit. Next, the labeled antibodies in each layer bind to the complex in their respective layers. Ultimately, it can be observed that the signal value in the T1 region reaches its upper limit, while the signal value in the T2 region does not reach its upper limit, or the signal value in the T2 region is higher than that in the T1 region. At this point, the CC2 curve is selected for quantification, and the concentration of β-hCG in the sample is determined based on the signal value in the T2 region.

[0069] It is understood that this invention does not limit the placement of the high-concentration β-hCG detection area on the upper surface of the substrate (bottom layer of the chip) and the low-concentration β-hCG detection area on the lower surface of the cover plate (top layer of the chip). If the labeled anti-human β-hCG antibody one coated at the position corresponding to the first labeled region on the lower surface of the cover plate (top layer of the chip) matches a low-affinity anti-human β-hCG antibody, and the labeled anti-human β-hCG antibody two coated at the position corresponding to the second labeled region on the upper surface of the substrate (bottom layer of the chip) matches a high-affinity anti-human β-hCG antibody, or if the labeled anti-human β-hCG antibody one and the labeled anti-human β-hCG antibody two are universal homologous antibodies, then the high-concentration β-hCG detection area is placed on the lower surface of the cover plate (top layer of the chip), and the low-concentration β-hCG detection area is placed on the upper surface of the substrate (bottom layer of the chip), as long as the low-concentration β-hCG detection area is closer to the sample injection well.

[0070] Example

[0071] 1. Chip fabrication

[0072] 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.

[0073] 2. Biomolecular coating program on the top layer of the chip

[0074] First, 1.5 μL of streptavidin was spotted at the T1 region on the top layer of the chip, incubated for 1 h, washed, and dried. Then, high-affinity biotinylated anti-human β-hCG antibody was spotted at the T1 region, and incubated for another 1 h. After washing again, fluorescent microspheres conjugated with anti-human β-hCG antibody 1 were spotted at the first labeled region and dried at 37°C.

[0075] 3. Biomolecular coating program at the chip's underlying layer

[0076] First, 1.5 μL of streptavidin was spotted at the T2 region on the bottom layer of the chip, incubated for 1 h, washed, and dried. Then, low-affinity biotinylated anti-human β-hCG antibody was spotted at the T2 region, and incubated for another 1 h. After washing again, fluorescent microspheres conjugated with anti-human β-hCG antibody were spotted at the second labeled region and dried at 37°C.

[0077] 4. Microfluidic chip assembly procedure

[0078] First, the magnetically controlled interceptor 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.

[0079] 5. Testing Procedure

[0080] Two samples, S1 and S2, with different concentrations, were selected for testing. First, the flow control valve was switched to the far right, disconnecting it from the microchannel. 10 μL of the sample was added to the sample injection port. The sample flowed to the left and right. Because the microchannel was not connected to the flow control valve, the liquid flowing to the right remained at the end of the microchannel due to surface tension and did not flow out spontaneously, while the liquid flowing to the left continued to flow. First, the sample flowed through T1, encountering a high-affinity anti-human β-hCG antibody in the low-concentration β-hCG detection zone, which is beneficial for identifying low-concentration β-hCG. If the β-hCG content was too high, the sample continued to flow to the left until it encountered a low-affinity anti-human β-hCG antibody in the high-concentration β-hCG detection zone. This pairing plateau occurred later, which is beneficial for identifying high-concentration β-hCG. The liquid flow continued to flow until it reached the magnetically controlled intercept valve, where it was intercepted. Because the suction force provided by the magnetically controlled intercept valve was greater than the capillary force within the microchannel, the liquid flow was immediately collected into the intercept valve and did not continue to flow to the left.

[0081] Secondly, after all the liquid flow in the microchannel has been collected into the magnetically controlled intercept valve, the intercept valve is removed, and the microchannel is restored to unobstructed flow, ensuring that subsequent reagent flow and reaction are not disturbed.

[0082] Next, the flow control valve is moved to the far left, embedding it within the microchannel. Buffer is added through the buffer injection well, entering the microchannel under capillary force and flowing forward in a laminar flow state. The upper flow contacts the inner surface of the chip's top layer. Upon reaching the first labeling area, the fluorescent microspheres labeled with anti-human β-hCG antibody 1 dissolve. When labeled antibody 1 reaches the low-concentration β-hCG detection area, it binds to the captured β-hCG, forming a double-antibody sandwich complex. The lower flow contacts the inner surface of the chip's bottom layer. Upon reaching the second labeling area, the labeled anti-human β-hCG antibody 2 dissolves and subsequently binds to the captured β-hCG in the high-concentration β-hCG detection area. The remaining flow continuously washes the microchannel and is collected into the flow control valve.

[0083] 6. Experimental Results

[0084] Table 1 CC1 calibration curve

[0085]

[0086] Table 2 CC2 Calibration Curve

[0087]

[0088] Table 3. β-hCG concentration in the test samples

[0089]

[0090] Sample S1 showed only a trace signal value in region T2, and the signal value in region T1 was much higher than that in region T2. ​​Therefore, it was a low-concentration sample, and calibration curve CC1 was selected to determine the concentration value of the sample. Sample S2 showed a higher signal value in region T2 than that in region T1. Therefore, it was a high-concentration sample, and calibration curve CC2 was selected to determine the concentration value of the sample.

[0091] 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.

[0092] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0093] 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.

[0094] 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 immunomicrofluidic chip capable of widening the quantitative analysis range of β-hCG, comprising a substrate and a cover plate pressed on the substrate, the substrate and the cover plate enclosing a microchannel, the height of the microchannel being 20-50 μm, the left end of the microchannel being in communication with a buffer injection hole opened on the cover plate, the right end of the microchannel being provided with a liquid flow control valve, the liquid flow control valve controlling the opening and closing of the flow path of the liquid in the microchannel to the liquid flow control valve, the microchannel being sequentially provided with a labeling zone and a detection zone from left to right, characterized in that, The marking area includes a first marking area arranged on the lower surface of the cover sheet and a second marking area arranged on the upper surface of the base sheet, the detection area includes a low-concentration β-hCG detection area arranged on the lower surface of the cover sheet and a high-concentration β-hCG detection area arranged on the upper surface of the base sheet, the high-concentration β-hCG detection area and the low-concentration β-hCG detection area are arranged along the microchannel from left to right, a sample injection hole is further arranged on the cover sheet, the sample injection hole is arranged between the detection area and the liquid flow control valve, a magnetic control intercept valve is arranged between the marking area and the detection area of the microchannel, the magnetic control intercept valve controls the opening and closing of the flow path between the marking area and the detection area in the microchannel through magnet adsorption, the first marking area is coated with a labeled anti-human β-hCG antibody one, the second marking area is coated with a labeled anti-human β-hCG antibody two, the low-concentration β-hCG detection area is coated with a high-affinity anti-human β-hCG antibody, and the high-concentration β-hCG detection area is coated with a low-affinity anti-human β-hCG antibody.

2. The immuno-microfluidic chip capable of broadening the quantitative analysis range of β-hCG according to claim 1, wherein, The labeled anti-human β-hCG antibody one and the high-affinity anti-human β-hCG antibody are matched, and the labeled anti-human β-hCG antibody two and the low-affinity anti-human β-hCG antibody are matched.

3. The immuno-microfluidic chip capable of broadening the quantitative analysis range of β-hCG according to claim 1, wherein, The labeled anti-human β-hCG antibody one and the labeled anti-human β-hCG antibody two are the same kind of antibodies.

4. The immuno-microfluidic chip capable of broadening the quantitative analysis range of β-hCG according to claim 1, wherein, The horizontal distance between the high-concentration β-hCG detection area and the buffer injection hole is 30-35 mm, and the horizontal distance between the low-concentration β-hCG detection area and the buffer injection hole is 40-45 mm.

5. The immuno-microfluidic chip capable of broadening the quantitative analysis range of β-hCG according to claim 1, wherein, A groove is arranged on the lower surface of the cover sheet along the length direction, the cover sheet and the upper surface of the base sheet form a microchannel through the groove, and the width of the microchannel is 2-3 mm.

6. The immuno-microfluidic chip capable of broadening the quantitative analysis range of β-hCG according to claim 1, wherein, The labeled anti-human β-hCG antibody one and the labeled anti-human β-hCG antibody two are fluorescent microsphere labels.

7. The immuno-microfluidic chip capable of broadening the quantitative analysis range of β-hCG according to claim 1, wherein, A flow guide hole is arranged on the cover sheet, the bottom of the magnetic control intercept valve passes through the flow guide hole and contacts the upper surface of the base sheet, the top of the magnetic control intercept valve is made of ferrous material that can be adsorbed by a magnet, the magnetic control intercept valve is adsorbed by the magnet and then the bottom is separated from the flow guide hole, and the bottom of the magnetic control intercept valve is made of water-absorbing material.

8. The immuno-microfluidic chip capable of broadening the quantitative analysis range of β-hCG according to claim 1, wherein, The liquid flow control valve is movable water-absorbing material, and the water-absorbing material moves to contact or move away from the microchannel.

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