Microfluidic chip for combined detection of C-reactive protein and ferritin and its application

Abstract

The present invention proposes a microfluidic chip for the combined detection of C-reactive protein and ferritin and its application. The chip comprises a substrate and a cover sheet pressed onto the substrate. The substrate and cover sheet enclose a microchannel. The left end of the microchannel is connected to a buffer injection hole provided on the cover sheet, and the right end is connected to a waste liquid area. The waste liquid area is provided with a removable absorbent paper sheet. The microchannel is provided with a labeling area, a detection area, and a sample injection area from left to right. The labeling area is provided with a labeled anti-human CRP antibody, a labeled anti-human SF antibody, a labeled second antibody, and a labeled antigen. The first detection site is provided with an anti-human CRP antibody, the first reference point is provided with a first antibody that reacts with the labeled second antibody, the second detection site is provided with an anti-human SF antibody, and the second reference point is provided with an antibody that reacts with the labeled antigen. This can simplify the detection process of C-reactive protein and ferritin and shorten the detection time.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro diagnosis and immune detection, and particularly relates to a microfluidic chip for detecting C-reactive protein and ferritin and applications thereof. Background Art

[0002] C-reactive protein (CRP) is an acute phase protein primarily synthesized by the liver. It significantly increases in various pathological conditions, including inflammation, infection, and tissue damage, and is a commonly used inflammatory marker in clinical practice. Studies have found that CRP can not only reflect mild inflammatory responses but can also indicate the presence of severe infection. Furthermore, in certain conditions of chronic inflammation or recurrent infection, CRP levels may remain elevated or fluctuate repeatedly, impacting disease diagnosis and assessment of treatment efficacy.

[0003] Ferritin (FT) is an iron storage protein widely present in human tissues and body fluids. Changes in ferritin levels are closely associated with a variety of diseases, including inflammation, infection, anemia, and cancer, and have become a key biomarker for assessing a wide range of conditions. In certain circumstances, abnormally elevated ferritin levels may also indicate the presence of severe inflammation or chronic disease and may be associated with sequelae such as organ dysfunction (e.g., liver dysfunction and renal insufficiency).

[0004] CRP and ferritin are clinically valuable in the diagnosis and monitoring of various diseases. Combined detection of these two markers can more comprehensively reflect the body's inflammatory status and underlying pathology. However, in clinical testing, CRP and ferritin are typically measured using different assays, such as immunoturbidimetry and enzyme-linked immunosorbent assay (ELISA). These methods have drawbacks such as complex operation, lengthy testing times, and high costs. Furthermore, because the concentration of ferritin is much higher than that of C-reactive protein, rapid and accurate combined detection of CRP and ferritin is difficult. Using the immunoturbidimetric assay commonly used for C-reactive protein detection presents two challenges for ferritin detection: first, high ferritin concentrations can exceed the upper limit of detection, leading to false negatives (the hook effect); second, high-concentration samples require significant dilution, amplifying errors. Conversely, using the chemiluminescence assay used for ferritin to detect C-reactive protein may not be sensitive enough to accurately measure low C-reactive protein concentrations. Summary of the Invention

[0005] The present invention aims to provide a microfluidic chip for the combined detection of C-reactive protein and ferritin and its application. By optimizing the chip design and detection method, the problems existing in the prior art are solved, and rapid, accurate, and sensitive combined detection of CRP and ferritin is achieved, providing an efficient and reliable tool for clinical diagnosis and disease monitoring.

[0006] To achieve the above-mentioned object, the technical solution of the present invention is implemented as follows: a microfluidic chip for the combined detection of C-reactive protein and ferritin, comprising a substrate and a cover sheet pressed onto the substrate, the substrate and the cover sheet enclosing a microchannel, the left end of the microchannel being connected to a buffer injection hole provided on the cover sheet, and the right end being connected to a waste liquid area, the waste liquid area being provided with a movable absorbent paper sheet, the absorbent paper sheet contacting or moving away from the microchannel when moving, the microchannel being provided with a marking area, a detection area, and a sample injection area in sequence from left to right, the sample injection area comprising a sample injection hole provided on the cover sheet, the sample injection hole being provided in the detection area. The device is located above the microchannel between the labeling area and the waste liquid area and is connected to the microchannel. A buffer zone for slowing down the liquid flow rate is provided between the labeling area and the detection area. The detection area is provided with a first detection site, a first reference point, a second detection site, and a second reference point. The labeling area is provided with a labeled anti-human CRP antibody, a labeled anti-human SF antibody, a labeled second antibody, and a labeled antigen. The first detection site is provided with an anti-human CRP antibody, the first reference point is provided with a first antibody reacting with the labeled second antibody, the second detection site is provided with an anti-human SF antibody, and the second reference point is provided with an antibody reacting with the labeled antigen.

[0007] In one embodiment of the present invention, the lower surface of the cover sheet is provided with grooves along its length direction, and the cover sheet and the upper surface of the substrate are enclosed by the grooves to form microchannels.

[0008] In one embodiment of the present invention, the marking area, detection area, sample addition area, and waste liquid area are all arranged on the upper surface of the substrate, and the first detection site, first reference point, second detection site, and second reference point are distributed in the detection area from left to right.

[0009] In one embodiment of the present invention, the labeled anti-human CRP antibody, labeled anti-human SF antibody, labeled secondary antibody, and labeled antigen are labeled with fluorescent microspheres.

[0010] In one embodiment of the present invention, anticoagulant dry powder is provided on the upper surface of the substrate at a position corresponding to the sample addition area.

[0011] In one embodiment of the present invention, the labeled second antibody of the labeled region is labeled goat anti-chicken IgY, and the first antibody of the first reference point is chicken IgY.

[0012] In one embodiment of the present invention, the labeled antigen in the labeled region is labeled DNP-BSA, and the antibody of the second reference point is DNP-rabbit antibody.

[0013] In one embodiment of the present invention, the buffer zone includes a plurality of pillars disposed on the microchannel, and the pillars are fixed to the upper surface of the substrate.

[0014] On the other hand, the present invention also provides an application of the microfluidic chip in any of the above technical solutions for the combined detection of C-reactive protein and ferritin, comprising at least the following steps:

[0015] 1) The sample to be tested is added to the sample injection well. Driven by the capillary action of the liquid in the microchannel, the liquid moves from right to left. The target analyte in the sample preferentially binds to the anti-human CRP antibody and anti-human SF antibody in the detection area in a lateral flow manner.

[0016] 2) Buffer is added to the buffer injection well. At the same time, the absorbent paper in the waste liquid area is moved to the left to contact the liquid in the microchannel. The liquid moves from left to right, dissolving the labeled anti-human CRP antibody, labeled anti-human SF antibody, labeled second antibody, and labeled antigen in the labeled area. The antibody and antigen in the labeled area continue to move to the detection area and bind to the first detection site, the first reference site, the second detection site, and the second reference site. The excess antibody and antigen in the labeled area are collected in the waste liquid area along with the liquid flow.

[0017] 3) Read the signal value of the detection area according to conventional microfluidics.

[0018] The microfluidic chip for combined detection of C-reactive protein and ferritin obtained by the above technical solution and its application have the following beneficial effects:

[0019] It can simplify the detection process of C-reactive protein and ferritin and shorten the detection time. The tiny size and precision manufacturing of the microfluidic chip can achieve accurate fluid manipulation and reaction control, and can achieve micron-level accuracy, which is more accurate than traditional experimental methods and more suitable for POCT analysis mode.

[0020] This invention, based on the unique bidirectional reaction of dual-drive microfluidics, effectively overcomes the negative effect of high CRP concentrations in blood while retaining the sensitivity of low SF concentrations, enabling simultaneous joint detection of these two indicators without sample dilution. This significantly shortens experimental time, improves work efficiency, and provides portability for clinical diagnosis. Results are presented as signal values, unaffected by the subjective judgment of the operator. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the structure of the microfluidic chip of the present invention;

[0022] Figure 2 is a schematic structural diagram of the upper surface of the substrate of the present invention;

[0023] Figure 3 Schematic diagram of the structure of the lower surface of the cover sheet of the present invention;

[0024] Figure 4Schematic diagram of the absorbent paper sheet driving the liquid flow in the waste liquid area of ​​the present invention;

[0025] Figure 5 This is a schematic structural diagram of a microfluidic chip for combined detection of C-reactive protein and ferritin, one of the technical solutions of the present invention;

[0026] Figure 6 The present invention Figure 5 Schematic diagram after the sample to be tested is added to the chip;

[0027] Figure 7 The present invention Figure 5 Schematic diagram of the chip after adding buffer. DETAILED DESCRIPTION

[0028] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0029] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which this invention belongs. The experimental reagents used in the following examples, unless otherwise specified, are conventional biochemical reagents; the experimental methods described, unless otherwise specified, are conventional methods.

[0030] The present invention generally relates to the field of immunoassay technology, and particularly to a combined detection of C-reactive protein and ferritin. The core of this technical invention is based on microfluidic technology, and designs a "bidirectional + dual-drive" immune microfluidic chip that uses an indirect method to simultaneously detect C-reactive protein and ferritin. The chip integrates the complex processes of conventional C-reactive protein and ferritin detection, significantly reducing the time required for C-reactive protein and ferritin detection, making the detection work faster and more convenient.

[0031] The present invention is further explained below with reference to the embodiments and the accompanying drawings. It should be understood that the present invention is not limited to the specific embodiments described.

[0032] like Figure 1 、 Figure 3As shown, the present invention proposes a microfluidic chip for the combined detection of C-reactive protein and ferritin, comprising a substrate and a cover sheet pressed onto the substrate, the substrate and the cover sheet enclose a microchannel, the left end of the microchannel is connected to the buffer injection hole B provided on the cover sheet, and the right end is connected to the waste liquid area W, the waste liquid area W is provided with a movable absorbent paper, the absorbent paper contacts or moves away from the microchannel when moving, and is used to control the flow of liquid in the microchannel to the waste liquid area W, the microchannel is provided with a marking area L, a detection area, and a sample injection area S from left to right, the sample injection area S includes a sample injection hole provided on the cover sheet, the sample injection hole is provided above the microchannel between the detection area and the waste liquid area, and is connected to the microchannel, the marking area L is provided, and the detection area is provided, and the sample injection area S is provided. A buffer zone D is provided between the recording area L and the detection area to slow down the flow rate of the liquid. When there is a slight excess of sample, the buffer zone slows down the flow and blocks it from flowing to the pre-dried marking area. The detection area is provided with a first detection site T1, a first reference point C1, a second detection site T2, and a second reference point C2. The marking area is provided with a labeled anti-human CRP antibody, a labeled anti-human SF antibody (where SF is serum ferritin), a labeled second antibody, and a labeled antigen. The first detection site T1 is provided with an anti-human CRP antibody, the first reference point C1 is provided with a first antibody that reacts with the labeled second antibody, the second detection site T2 is provided with an anti-human SF antibody, and the second reference point C2 is provided with an antibody that reacts with the labeled antigen.

[0033] Among them, the CRP in the target analyte combines with the labeled anti-human CRP antibody set in the labeling area and the anti-human CRP antibody set at the first detection site to form a double antibody sandwich structure, and the ferritin in the target analyte combines with the labeled anti-human SF antibody set in the labeling area and the anti-human SF antibody set at the second detection site to form a double antibody sandwich structure.

[0034] Among them, the labeled second antibody set in the marking area is combined with the first antibody set at the first reference point to determine the effectiveness of the first detection site, and the labeled antigen set in the marking area is combined with the antibody set at the second reference point to determine the effectiveness of the second detection site.

[0035] For example, the labeled second antibody of the labeling area L is labeled goat anti-chicken IgY, and the first antibody of the first reference point C1 is chicken IgY; the labeled antigen of the labeling area L is labeled DNP-BSA, and the antibody of the second reference point C2 is DNP-rabbit anti-.

[0036] like Figure 4As shown, the absorbent paper sheet set in the waste liquid area can control the flow of liquid in the microchannel. When the absorbent paper sheet moves to the right and is not in contact with the microchannel, the liquid entering through the sample injection hole will flow from right to left under the capillary force of the microchannel. When the absorbent paper sheet moves to the left and contacts the microchannel, the liquid in the microchannel will flow from left to right due to the suction force of the absorbent paper sheet.

[0037] The sample to be tested is added to the microchannel through the sample injection hole. The sample to be tested first binds to the anti-human CRP antibody and anti-human SF antibody in the detection zone. Then the buffer dissolves the labeled antibody\antigen in the labeling zone. Under the combined action of capillary force and the suction of the absorbent paper, the buffer at the front end pushes the excess sample to the waste liquid zone first. Then the labeled antibody in the labeling zone binds to the target analyte in the detection zone to form a double antibody sandwich structure, avoiding the HOOK effect caused by high-concentration CRP. The low-concentration ferritin passes through the second detection zone twice (flowing from right to left under the action of capillary force and from left to right under the suction of the absorbent paper) and fully binds to the capture antibody, resulting in good sensitivity.

[0038] The lower surface of the cover sheet is provided with a groove along its length direction, and the cover sheet and the upper surface of the substrate are enclosed by the groove to form a microchannel.

[0039] like Figure 2 As shown, the marking area L, detection area, sample addition area S, and waste liquid area W are all arranged on the upper surface of the substrate, and the first detection site T1, the first reference point C1, the second detection site T2, and the second reference point C2 are distributed in the detection area from left to right.

[0040] The labeled anti-human CRP antibody, the labeled anti-human SF antibody, the labeled secondary antibody, and the labeled antigen are labeled with fluorescent microspheres.

[0041] Anticoagulant dry powder is provided at a position on the upper surface of the substrate corresponding to the sample addition area S.

[0042] The buffer zone includes multiple columns positioned on the microchannel and fixed to the upper surface of the substrate. When a slight excess of sample is present, the columns in the buffer zone slow the flow of liquid to the right. Due to the small amount of sample added, the slightly excessive sample is trapped in the buffer zone due to the slowed flow rate and prevented from flowing into the labeling zone. As buffer is added, the microchannel is flushed by the continuous addition of buffer, preventing antibodies / antigens in the labeling zone from being retained in the buffer zone.

[0043] Example

[0044] (1) Chip production

[0045] Polymethyl methacrylate (PMMA) material is used to make the dual-drive bidirectional immune microfluidic chip. The microchannel structure of the chip is designed using CAD software, and then the PMMA surface is processed using a CO2 laser etcher.

[0046] (2) Chip surface modification

[0047] Conventional treatment methods, such as plasma treatment, are used to modify the surface of the PMMA microfluidic chip, thereby modifying the originally hydrophobic surface into a more hydrophilic surface.

[0048] (3) Preparation of substrate

[0049] First, activated avidin was spotted on the T1, C1, T2, and C2 areas of the substrate, incubated, and the film was washed. After drying, biotinylated anti-human CRP antibody, biotinylated chicken IgY, biotinylated anti-human SF antibody, and biotinylated DNP-rabbit antibody were spotted on the T1, C1, T2, and C2 areas respectively. The incubation was continued, and after washing the substrate again, fluorescent microsphere-labeled anti-human CRP antibody, anti-human SF antibody, goat anti-chicken IgY, and DNP-BSA were spotted on the corresponding positions of the L area, and anticoagulant was spotted on the corresponding positions of the S well. The film was dried at 37°C. The biochip preparation was now complete.

[0050] (4) Assembly of dual-drive bidirectional immune microfluidic chip

[0051] First, the water-absorbing material is placed in the corresponding waste liquid area, and then the prepared biochip is bonded to the chip cover containing the microchannel structure, and the microfluidic chip is completed.

[0052] Distribution of biomolecules in microfluidic chips Figure 5 As shown,

[0053] Labeling area L: fluorescent microsphere-labeled anti-human CRP antibody, anti-human SF antibody, goat anti-chicken IgY, and DNP-BSA.

[0054] The first detection site T1: biotinylated anti-human CRP antibody.

[0055] First reference point C1: biotinylated chicken IgY.

[0056] Second detection site T2: biotinylated anti-human SF antibody.

[0057] Second reference point C2: biotinylated DNP-rabbit antibody.

[0058] Five test samples S1, S2, S3, S4, and S5 of different concentrations were respectively subjected to immunoturbidimetric analysis of C-reactive protein, chemiluminescence analysis of ferritin, and microfluidic chip detection of the present application. The immunoturbidimetric analysis of C-reactive protein and the chemiluminescence analysis of ferritin are both existing technologies, and the detection process is omitted here.

[0059] When using microfluidic chips for detection, Figure 4 、 Figure 6 As shown, the absorbent paper in the waste liquid area is first placed on the right side, not connected to the channel, and the whole blood sample to be tested is added to the sample addition hole S. Under the action of the microchannel driving force, it flows from right to left and combines with the detection area.

[0060] like Figure 4 、 Figure 7 As shown, the absorbent paper in the waste liquid area is pushed to the left and combined with the microfluidic channel. At this time, buffer solution is added to the buffer solution injection hole B. The liquid moves to the right under the action of the microchannel driving force, dissolves the marked area, and continues to flow to the right through the detection area and quality control area. The remaining liquid flows to the waste liquid area.

[0061] At this time, a detection instrument is used to detect the signal intensity of the detection area and output the detection result according to the calibration function (the detection instrument is conventional technology, such as a fluorescence detector, which is consistent with the signal carried by the labeled antibody).

[0062] The calibration functions are CRP concentration calibration function and ferritin concentration calibration function.

[0063] Taking the CRP concentration calibration function as an example, standard solutions of a series of CRP concentrations were selected for the above experiment. The signal values ​​T1 and R1 of the first detection site and the first reference point were read, and the inhibition rate T1 / R1 was calculated. A functional relationship was established with the inhibition rate as the vertical axis and the standard concentration value as the horizontal axis.

[0064] The experimental method for the calibration function of ferritin concentration is the same as that of the CRP concentration calibration function. The above experiment is performed on standard solutions of a series of ferritin concentrations. The signal values ​​T2 and R2 of the second detection site and the second reference point are read, and the inhibition ratio T2 / R2 is calculated. A functional relationship is established with the inhibition ratio as the vertical axis and the standard concentration value as the horizontal axis.

[0065] The test results are shown in Table 1 and Table 2.

[0066] Table 1 C-reactive protein test results

[0067]

[0068] Table 2 Ferritin test results

[0069]

[0070] Detection conclusion: The conventional method is used to detect C-reactive protein and ferritin respectively, and the detection process is cumbersome and the detection time is long. The present invention is based on dual-drive microfluidic technology, which simplifies the detection process, shortens the detection time, and compares the detection results with the conventional method, and the data is accurate.

[0071] The above technical solutions only reflect the preferred technical solutions of the technical solutions of the present invention. Any changes that may be made to certain parts thereof by those skilled in the art all reflect the principles of the present invention and fall within the scope of protection of the present invention.

[0072] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0073] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, features defined as "first", "second", etc. may explicitly or implicitly include one or more of the features. In the description of the present invention, unless otherwise specified, "multiple" means two or more.

[0074] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "installed," "connected," "connected," and "disposed" should be understood in a broad sense. For example, they may refer to fixed, detachable, or integral connections; mechanical or electrical connections; direct or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

Claims

1. A microfluidic chip for the combined detection of C-reactive protein and ferritin, comprising a substrate and a cover sheet pressed onto the substrate, wherein the substrate and the cover sheet enclose a microchannel, characterized in that: The left end of the microchannel is connected to the buffer injection hole provided on the cover plate, and the right end is connected to the waste liquid area. The waste liquid area is provided with a movable absorbent paper sheet, and the absorbent paper sheet contacts or moves away from the microchannel when moving. The microchannel is provided with a marking area, a detection area, and a sample injection area from left to right. The sample injection area includes a sample injection hole provided on the cover plate. The sample injection hole is provided above the microchannel between the detection area and the waste liquid area and is connected to the microchannel. A buffer zone for slowing down the flow rate of the liquid is provided between the marking area and the detection area. The detection area is provided with a first detection site, a first reference point, a second detection site, and a second reference point. The marking area is provided with a labeled anti-human CRP antibody, a labeled anti-human SF antibody, a labeled second antibody, and a labeled antigen. The first detection site is provided with an anti-human CRP antibody, the first reference point is provided with a first antibody that reacts with the labeled second antibody, the second detection site is provided with an anti-human SF antibody, and the second reference point is provided with an antibody that reacts with the labeled antigen. The buffer zone includes a plurality of columns provided on the microchannel, and the columns are fixed on the upper surface of the substrate.

2. The microfluidic chip for combined detection of C-reactive protein and ferritin according to claim 1, characterized in that: The lower surface of the cover sheet is provided with a groove along its length direction, and the cover sheet and the upper surface of the substrate are enclosed by the groove to form a microchannel.

3. The microfluidic chip for combined detection of C-reactive protein and ferritin according to claim 1, characterized in that: The marking area, detection area, sample addition area, and waste liquid area are all arranged on the upper surface of the substrate, and the first detection site, first reference point, second detection site, and second reference point are distributed in the detection area from left to right.

4. The microfluidic chip for combined detection of C-reactive protein and ferritin according to claim 1, characterized in that: The labeled anti-human CRP antibody, the labeled anti-human SF antibody, the labeled secondary antibody, and the labeled antigen are labeled with fluorescent microspheres.

5. The microfluidic chip for combined detection of C-reactive protein and ferritin according to claim 1, characterized in that: Anticoagulant dry powder is provided at a position on the upper surface of the substrate corresponding to the sample adding area.

6. The microfluidic chip for combined detection of C-reactive protein and ferritin according to claim 1, characterized in that: The labeled second antibody in the labeled area is labeled goat anti-chicken IgY, and the first antibody in the first reference point is chicken IgY.

7. The microfluidic chip for combined detection of C-reactive protein and ferritin according to claim 1, characterized in that: The labeled antigen in the labeled region is labeled DNP-BSA, and the antibody at the second reference point is DNP-rabbit antibody.

8. An application of a microfluidic chip for the combined detection of C-reactive protein and ferritin for non-diagnostic and non-therapeutic purposes, characterized in that: The detection using the microfluidic chip according to any one of claims 1 to 7 comprises at least the following steps: 1) The sample to be tested is added to the sample injection well. Driven by the capillary action of the liquid in the microchannel, the liquid moves from right to left. The target analyte in the sample preferentially binds to the anti-human CRP antibody and anti-human SF antibody in the detection area in a lateral flow manner. 2) Buffer is added to the buffer injection well. At the same time, the absorbent paper in the waste liquid area is moved to the left to contact the liquid in the microchannel. The liquid moves from left to right, dissolving the labeled anti-human CRP antibody, labeled anti-human SF antibody, labeled second antibody, and labeled antigen in the labeled area. The antibody and antigen in the labeled area continue to move to the detection area and bind to the first detection site, the first reference site, the second detection site, and the second reference site. The excess antibody and antigen in the labeled area are collected in the waste liquid area along with the liquid flow. 3) Read the signal value of the detection area according to conventional microfluidics.

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