A dry-type three-electrode electrochemiluminescence lateral flow immunosensor and detection application thereof

Abstract

This invention discloses a dry three-electrode electrochemiluminescence lateral flow immunosensor and its detection application, belonging to the field of microfluidic chip technology. The sensor includes a detection chip, which comprises a sample pad, a conjugate pad, at least one detection pad, a quality control pad, an absorbent pad, an electrode pad, and a base plate. The electrode pad has a common working electrode, a counter electrode, and a reference electrode. The counter electrode has a detection area counter electrode and a quality control area counter electrode. The reference electrode has a detection area reference electrode and a quality control area reference electrode. The sample pad, conjugate pad, detection pad, quality control pad, and absorbent pad all have hydrophilic regions. The sample pad, conjugate pad, each detection pad, quality control pad, and absorbent pad are arranged sequentially on top of the electrode pad. The hydrophilic regions are sequentially connected to form a single flow path. The hydrophilic regions of the detection pads are located directly above the corresponding detection area counter electrode and detection area reference electrode, respectively. The hydrophilic regions of the quality control pads are located directly above the quality control area counter electrode and quality control area reference electrode.

Description

Technical Field

[0001] This invention relates to the field of microfluidic chip technology, and more specifically, to a dry three-electrode electrochemiluminescence lateral flow immunosensor and its detection applications. Background Technology

[0002] With the development of ECL detection technology, the electrodes used for excitation reactions have expanded from the initial two-electrode system to a three-electrode system. Compared with the traditional two-electrode system, the three-electrode system, due to its lower required driving voltage and stable electrode potential during the reaction, has been successfully applied in the fields of electrochemistry and ECL, and has become a research hotspot.

[0003] A traditional three-electrode system includes a working electrode, a reference electrode, and a counter electrode. When a driving voltage is applied to the working electrode, current flows through both the working electrode and the counter electrode. The working electrode and the reference electrode form a feedback loop, with no current flowing between them, but a potential difference exists. This potential difference is constant to ensure that the potential on the working electrode remains constant during the reaction, avoiding voltage drops caused by the reaction and making the current in the loop formed by the working electrode and the counter electrode more stable.

[0004] ECL, as an emerging detection method, has gradually become a preferred method for detecting various biomarkers due to its advantages such as simple operation, low background signal, no need for isotopes, and high-throughput detection. Microfluidic three-electrode ECL biosensors, which combine a three-electrode system with ECL, exhibit good analytical performance and have been successfully applied to the detection of biomarkers such as genes, metal ions, proteases, and cells. However, some challenges remain. For example, the electrode materials used in the sensor's internal chip are often expensive materials such as silver / silver chloride, gold, platinum, and platinum-carbon; chip fabrication equipment is expensive; chip modification processes are complex; and traditional three-electrode ECLs contain only one reaction cell, making it difficult to achieve the lateral flow required for immunoassay. These factors significantly limit the application of three-electrode systems in the field of lateral flow immunoassay.

[0005] Dry chemistry analysis is an analytical technique based on dry chemical analysis methods. Compared to wet chemistry, dry chemistry pre-dries the required reactants within the reaction zone, simplifying the detection process. Currently, immunochromatographic reagent dry slides, developed based on lateral flow and antigen-antibody specific binding immunochromatography, utilize fluorescent labeling, gold nanoparticle labeling, etc., to achieve the detection of various biomarkers. These immunochromatographic reagent dry slides have advantages such as low cost, ease of use, rapid detection, or no need for expensive instruments, overcoming some of the shortcomings of traditional wet ECL microfluidic biosensors. However, they are either difficult to use for quantitative detection, difficult for high-throughput detection, or require relatively expensive detectors. Therefore, they are difficult to meet the demand for immunoassays that integrate quantitative, inexpensive, and high-throughput detection.

[0006] In summary, microfluidic biosensors that are quantitative, inexpensive, rapid, and simple, suitable for real-time detection in multiple scenarios, and capable of overcoming the difficulty of achieving dry immunoassay using traditional three-electrode systems, have become a pressing technical problem for engineers in this field. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to address the above-mentioned shortcomings of the prior art. The objective of the present invention is to provide a dry three-electrode electrochemiluminescence lateral flow immunosensor.

[0008] The second objective of this invention is to provide a detection application for a dry three-electrode electrochemiluminescence lateral flow immunosensor.

[0009] To achieve the above objective, this invention provides a dry three-electrode electrochemiluminescence lateral flow immunosensor, comprising a detection chip. The detection chip includes a sample application pad, a binding pad, at least one detection pad, a quality control pad, an absorption pad, electrode pads, and a base plate. A common working electrode is provided on the top of each electrode pad. Counter electrodes and reference electrodes are respectively provided on the electrode pads on both sides of the common working electrode. One end of each of the common working electrode, counter electrode, and reference electrode in the same direction serves as an electrical contact area. The other end of the counter electrode is sequentially provided with a detection area counter electrode and a quality control area counter electrode, each corresponding to the number of detection pads. Correspondingly, the other end of the reference electrode is sequentially provided with a detection area reference electrode and a quality control area reference electrode, each corresponding to the number of detection pads. The counter electrode, the quality control counter electrode, the detection reference electrode, and the quality control reference electrode all face the common working electrode. The sample pad has a hydrophilic area, the binding pad has a hydrophilic area, the detection pad has a hydrophilic area, the quality control pad has a hydrophilic area, and the absorption pad has a hydrophilic area. The sample pad, binding pad, each detection pad, quality control pad, and absorption pad are arranged sequentially on top of the electrode pad. The hydrophilic areas of the sample pad are sequentially connected to the hydrophilic areas of the binding pad, each detection pad, the quality control pad, and the absorption pad to form a single flow path. The hydrophilic areas of each detection pad are located directly above the corresponding detection counter electrode and detection reference electrode, and the hydrophilic area of ​​the quality control pad is located directly above the quality control counter electrode and quality control reference electrode.

[0010] As a further improvement, the electrode pad is made of hydrophobic fiber material as a base and is manufactured by screen printing with conductive material. The sample pad, bonding pad, detection pad, quality control pad, and absorption pad are all made of hydrophilic fiber material as a base and hydrophobic and hydrophilic areas are formed on the base by screen printing hydrophobic ink.

[0011] Furthermore, the electrode pad is bonded to the top of the base plate, and the middle parts of the sample pad, conjugate pad, detection pad, quality control pad, and absorption pad are all in close contact with the electrode pad, while both ends of the sample pad, conjugate pad, detection pad, quality control pad, and absorption pad are bonded to the base plate.

[0012] Furthermore, overlapping areas are provided between the sample application pad and the conjugate pad, between the conjugate pad and the detection pad, between the detection pad and the quality control pad, and between the quality control pad and the absorption pad.

[0013] Furthermore, it also includes an upper cover and a lower cover. The lower cover has a mounting slot for installing the detection chip. The upper cover covers the detection chip. The upper cover is provided with a sample application hole aligned with the hydrophilic area of ​​the sample application pad, a detection area observation window aligned with the hydrophilic area of ​​each of the detection pads, a quality control area observation window aligned with the hydrophilic area of ​​the quality control pad, and an opening slot aligned with the electrical contact area.

[0014] Furthermore, the hydrophilic areas of the sample application pad and the absorbent pad are both treated with Tween; the hydrophilic area of ​​the binding pad is treated with Tween and then dried and labeled with antibodies.

[0015] Furthermore, the hydrophilic region of the detection pad is immobilized with modified biomarkers coated with antibodies.

[0016] Furthermore, the hydrophilic region of the quality control pad is fixed with a quality control coated antibody.

[0017] Furthermore, the hydrophilic regions of the test pad and the quality control pad are fixed and modified using chitosan and glutaraldehyde.

[0018] To achieve the second objective mentioned above, this invention provides a detection application of a dry three-electrode electrochemiluminescence lateral flow immunosensor, comprising the following steps:

[0019] S1. A test sample solution containing a biomarker is dropped into the hydrophilic area of ​​the sample pad of the sensor. The test sample solution flows from the hydrophilic area of ​​the sample pad to the hydrophilic area of ​​the conjugate pad. The biomarker specifically binds to the labeled antibody dried in the hydrophilic area of ​​the conjugate pad to form a "labeled antibody-biomarker" complex. This complex further flows through the hydrophilic areas of each test pad and binds to the biomarker-coated antibody immobilized in the hydrophilic area of ​​the test pad to form an "labeled antibody-biomarker-coated antibody" immune sandwich complex.

[0020] S2. After the biomarker immune reaction is complete, add buffer solution to the hydrophilic area of ​​the sample pad to remove unbound labeled antibodies from the hydrophilic area of ​​the test pad.

[0021] S3. Start the ECL analyzer, place the sensor on the ECL analyzer's input / output module, the input / output module will transport the sensor to the underside of the imaging detection module, and connect the electrical contact area of ​​the electrode pad to the constant potential module of the ECL analyzer.

[0022] S4. Activate the detection button on the ECL analyzer. The constant potential module of the ECL analyzer provides the driving voltage to trigger the intramolecular reaction of the labeled antibody, generating an ECL signal. The ECL analyzer automatically collects and analyzes the ECL signal, and then performs quantitative detection of the biomarker based on the intensity of the ECL signal.

[0023] Beneficial effects

[0024] Compared with the prior art, the advantages of this invention are as follows:

[0025] 1. This invention ingeniously uses hydrophilic nonwoven fabric and hydrophobic electrode pads. Through the layered structure, each pad can be modified separately, avoiding the problems of direct modification of electrodes by traditional dry sheets and cross-contamination between different areas. Combined with lateral flow, it creatively realizes three-electrode ECL immunoassay.

[0026] 2. Based on the design of the hydrophilic regions of the sample pad, binding pad, detection pad, quality control pad and absorption pad, the present invention ensures that the electrodes will only form two parallel current loops when the chip layers are stacked and the sample solution to be tested fills the flow path, thereby improving the controllability of the chip triggering reaction.

[0027] 3. After the working electrode of the present invention is connected to the constant potential module of the analyzer, the detection circuit and the quality control circuit have different potentials and feedback signals under the premise of using the same working electrode, which avoids mutual interference between the detection area and the quality control area, and allows the detection area and the quality control area to perform ECL reaction independently.

[0028] 4. The dry three-electrode ECL lateral flow immunosensor proposed in this invention uses a series flow path, which enables the sensor to perform immunoassay with only one sample addition and rinsing, overcoming the problem that traditional three-electrode sensors require multiple sample additions and rinsings when performing wet immunoassay.

[0029] 5. This invention is the first to apply dry three-electrode ECL lateral flow technology to immunoassay. This technology has good sensitivity and dynamic range, meeting the requirements of ultrasensitive detection. Compared with conventional wet immunoassay, the detection technology of this invention has low cost, convenient operation and short detection time.

[0030] 6. This invention uses a ruthenium complex to combine polylysine with ruthenium, making it an intramolecular co-reactant. Compared with traditional ruthenium probes, no additional co-reactant is required, and the ECL signal intensity is greatly improved, thereby increasing the detection limit of biomarkers and expanding the dynamic detection range.

[0031] 7. The biomarkers detected by this invention can be replaced by changing the labeled antibodies as needed, realizing the ultrasensitive and quantitative detection of multiple biomarkers, which greatly increases the applicability of the sensor.

[0032] 8. The detection area and quality control area of ​​the sensor of the present invention share the same working electrode. By simply extending the working electrode, the counter electrode and the reference electrode, without increasing the total number of electrodes, multivariate immunoassay detection and quality control in a three-electrode system can be achieved.

[0033] 9. The non-woven fabric used in the sensor of the present invention is made of natural wood pulp and artificial fibers, and has the advantages of high toughness, good fluidity, high light transmittance, and easy ECL signal to pass through the detection pad and quality control pad from the electrode.

[0034] 10. This invention uses non-woven fabric, which greatly reduces the manufacturing cost of the sensor and simplifies the sensor processing. This perfectly meets the needs of chip manufacturing, reagent drying, solution flow, and signal release.

[0035] 11. The sensor of the present invention is easy to use and does not require professional personnel to operate, which is conducive to its use in primary medical units or homes and has the value of multi-scenario application. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the structure of the present invention;

[0037] Figure 2 This is a schematic diagram of the stacked structure of the detection chip in this invention;

[0038] Figure 3 This is a schematic diagram of the electrode pad structure with only one detection area for the electrode in this invention;

[0039] Figure 4 This is a schematic diagram of the structure of the electrode pad with two detection areas in this invention.

[0040] Figure 5 This is a schematic diagram of the detection chip in this invention, which has only one detection area electrode.

[0041] Figure 6 This is a schematic diagram of the detection chip in this invention, which has two detection areas and electrodes.

[0042] Figure 7 This is a graph showing the effect of exposure time on the ECL intensity value of the dry three-electrode circuit in this invention.

[0043] Figure 8 This is a diagram showing the effect of driving voltage on the ECL intensity value of the dry three-electrode circuit in this invention.

[0044] Figure 9This is a graph showing the effect of the amount of labeled antibody used on the ECL intensity value of the dry three-electrode in this invention;

[0045] Figure 10 This is a graph showing the effect of glutaraldehyde dosage on the ECL intensity value of the dry three-electrode circuit in this invention.

[0046] Figure 11 This is a graph showing the effect of cTnI-coated antibody concentration on the ECL intensity value of the dry three-electrode circuit in this invention.

[0047] Figure 12 This is a graph showing the effect of incubation time on the ECL intensity value of the dry three-electrode circuit in this invention.

[0048] Figure 13 This is a graph showing the relationship between the ECL intensity value and cTnI concentration of the dry three-electrode system in this invention.

[0049] The components are: 1-Detection chip, 2-Sample pad, 3-Binding pad, 4-Detection pad, 5-Quality control pad, 6-Absorbent pad, 7-Electrode pad, 8-Common working electrode, 9-Counter electrode, 10-Reference electrode, 11-Electrical contact area, 12-Detection area counter electrode, 13-Quality control area counter electrode, 14-Detection area reference electrode, 15-Quality control area reference electrode, 16-Sample pad hydrophilic area, 17-Binding pad hydrophilic area, 18-Detection pad hydrophilic area, 19-Quality control pad hydrophilic area, 20-Absorbent pad hydrophilic area, 21-Base plate, 22-Top cover, 23-Lower cover, 24-Sample hole, 25-Detection area observation window, 26-Quality control area observation window, 27-Open slot, 28-Detection area reaction cell, 29-Quality control area reaction cell. Detailed Implementation

[0050] The present invention will be further described below with reference to specific embodiments shown in the accompanying drawings.

[0051] See Figures 1 to 13 A dry three-electrode electrochemiluminescence lateral flow immunosensor includes a detection chip 1, which includes a sample application pad 2, a conjugation pad 3, at least one detection pad 4, a quality control pad 5, an absorption pad 6, and an electrode pad 7.

[0052] The electrode pad is used to drive the ECL reaction. The top of the electrode pad 7 is provided with a common working electrode 8, which is an I-shaped electrode. The electrode pads 7 on both sides of the common working electrode 8 are respectively provided with a counter electrode 9 and a reference electrode 10.

[0053] The common working electrode 8, counter electrode 9, and reference electrode 10 all have one end facing the same direction, which serves as the electrical contact area 11. The other end of the counter electrode 9 is sequentially provided with a detection area counter electrode 12, which corresponds to the number of detection pads 4 and is one-to-one with it, and a quality control area counter electrode 13. Correspondingly, the other end of the reference electrode 10 is sequentially provided with a detection area reference electrode 14, which corresponds to the number of detection pads 4 and is one-to-one with it, and a quality control area reference electrode 15. The detection area counter electrode 12, quality control area counter electrode 13, detection area reference electrode 14, and quality control area reference electrode 15 all face the common working electrode 8.

[0054] Preferably, the overall structure of the counter electrode 9 and the reference electrode 10 is symmetrical about the common working electrode 8.

[0055] Sample pad 2 has a hydrophilic region 16, binding pad 3 has a hydrophilic region 17, detection pad 4 has a hydrophilic region 18, quality control pad 5 has a hydrophilic region 19, and absorption pad 6 has a hydrophilic region 20. Sample pad 2, binding pad 3, each detection pad 4, quality control pad 5, and absorption pad 6 are arranged sequentially on top of electrode pad 7. The hydrophilic region 16 of the sample pad is sequentially connected to the hydrophilic regions 17, 18, 19, and 20 of the binding pad, forming a single flow path.

[0056] Preferably, there are overlapping areas between the sample pad 2 and the conjugate pad 3, between the conjugate pad 3 and the test pad 4, between the test pad 4 and the quality control pad 5, and between the quality control pad 5 and the absorbent pad 6. The width of the overlapping area is 1 to 3 mm. Preferably, the width of the overlapping area is 2 mm, which can facilitate communication between the hydrophilic area 16 of the sample pad, the hydrophilic area 17 of the conjugate pad, the hydrophilic areas 18 of each test pad, the hydrophilic area 19 of the quality control pad, and the hydrophilic area 20 of the absorbent pad.

[0057] Specifically, such as Figure 2 As shown, the hydrophilic area 20 of the absorbent pad has an L-shaped structure. Its thinner hydrophilic area is used to connect to the hydrophilic area 19 of the quality control pad, while its wider hydrophilic area is used to absorb excess solution.

[0058] The hydrophilic region 19 of the quality control pad has a Z-shaped structure. The hydrophilic region in the middle of the "Z" shape is used to form the ECL region, namely the quality control zone reaction cell 29 (the quality control zone reaction cell 29 is formed between the quality control zone counter electrode 13 and the common working electrode 8, and between the quality control zone reference electrode 15 and the common working electrode 8). It forms a flow path together with the hydrophilic regions at both ends of the "Z" shape, connecting the hydrophilic region 18 of the detection pad and the hydrophilic region 20 of the absorption pad.

[0059] The hydrophilic region 18 of the test pad has a Z-shaped structure. The hydrophilic region in the middle of the Z-shape is used to form the ECL region, namely the test zone reaction cell 28 (the test zone reaction cell 28 is formed between the test zone counter electrode 12 and the common working electrode 8, and between the test zone reference electrode 14 and the common working electrode 8). It forms a flow path together with the hydrophilic regions at both ends of the Z-shape, connecting the hydrophilic region 17 of the binding pad and the hydrophilic region 19 of the quality control pad.

[0060] Each test pad hydrophilic region 18 is located directly above the corresponding test area counter electrode 12 and test area reference electrode 14, while the quality control pad hydrophilic region 19 is located directly above the quality control area counter electrode 13 and quality control area reference electrode 15.

[0061] like Figure 3 , Figure 5 As shown, there is one detection pad 4, one corresponding reference electrode 14, and one hydrophilic region 18 of the detection pad. Figure 4 , Figure 6 As shown, there are two detection pads 4, corresponding to two detection area reference electrodes 14, which means there are two hydrophilic regions 18 of the detection pads. If more hydrophilic regions 18 of the detection pads are needed, simply increase the number of detection pads 4 and the number of detection area counter electrodes 12 accordingly.

[0062] The binding pad hydrophilic region 17 has an I-shaped structure, which is used to dry the labeled antibody and form a flow path. It connects the sample application pad hydrophilic region 16 and the test pad hydrophilic region 18. When the number of test pad hydrophilic regions 18 is greater than 1, the binding pad hydrophilic region 17 connects the sample application pad hydrophilic region 16 to the first test pad hydrophilic region 18. Each test pad hydrophilic region 18 is connected in sequence, and the last test pad hydrophilic region 18 is connected to the quality control pad hydrophilic region 19.

[0063] The hydrophilic area 16 of the sample loading pad has an L-shaped structure. Its thinner hydrophilic area is used to connect to the hydrophilic area 17 of the binding pad, and its wider hydrophilic area is used to add the sample solution to be tested.

[0064] The ECL region of the common working electrode 8 is located in the part where the hydrophilic region in the middle of the “Z” shape of the hydrophilic region 18 of the test pad and the hydrophilic region 19 of the quality control pad overlaps with the common working electrode 8.

[0065] The sample loading pad (hydrophilic area 16), the binding pad (hydrophilic area 17), each test pad (hydrophilic area 18), the quality control pad (hydrophilic area 19), and the absorbent pad (hydrophilic area 20) are connected in series to form a single flow path.

[0066] A quality control loop is formed between the shared working electrode 8, the hydrophilic area 19 of the quality control pad, and the counter electrode 13 of the quality control area. A detection loop is formed between the shared working electrode 8, the hydrophilic area 18 of the detection pad, and the counter electrode 12 of the detection area. The quality control loop and the detection loop are two parallel loops.

[0067] Electrode pad 7 is made of hydrophobic fiber material as base and is made by screen printing of conductive material, such as conductive carbon paste. Sample pad 2, bonding pad 3, detection pad 4, quality control pad 5, and absorption pad 6 are all made of hydrophilic fiber material as base and form hydrophobic and hydrophilic areas on the base by screen printing hydrophobic ink. Hydrophilic fiber material is such as polyester fiber nonwoven fabric, glass fiber, nitrocellulose membrane. Hydrophobic ink is made by mixing PP ink and diluent.

[0068] The detection chip 1 also includes a base plate 21, which is a rigid sheet with adhesive backing, such as any one of polyethylene terephthalate or polyvinyl chloride. The electrode pad 7 is bonded to the top of the base plate 21. The middle of the sample pad 2, the binding pad 3, the detection pad 4, the quality control pad 5, and the absorption pad 6 are all in close contact with the electrode pad 7. The two ends of the sample pad 2, the binding pad 3, the detection pad 4, the quality control pad 5, and the absorption pad 6 are bonded to the base plate 21. That is, the electrode pad 7, the absorption pad 6, the quality control pad 5, the detection pad 4, the binding pad 3, and the sample pad 2 are sequentially stacked and bonded to the base plate 21.

[0069] The sensor also includes an upper cover 22 and a lower cover 23. The lower cover 23 has a mounting slot for installing the detection chip 1. The upper cover 22 covers the detection chip 1. The upper cover 22 has a sample application hole 24 aligned with the hydrophilic area 16 of the sample application pad, a detection area observation window 25 aligned with the hydrophilic areas 18 of each detection pad, a quality control area observation window 26 aligned with the hydrophilic area 19 of the quality control pad, and an opening slot 27 aligned with the electrical contact area 11. The opening slot 27 is used to connect the electrical contact area 11 to electricity.

[0070] The hydrophilic region 16 of the sample loading pad is treated with Tween. Following Tween treatment, the hydrophilic region 17 of the binding pad is dried and labeled with antibody at a volume of 2–7 μL, preferably 5 μL. The hydrophilic region 18 of the detection pad is used to fix and modify the biomarker-coated antibody at a concentration of 40–140 μg / mL. -1 100 μg mL is preferred -1 The hydrophilic region 19 of the quality control pad is fixed with a quality control coated antibody, such as goat anti-chicken IgY. This coated antibody is a secondary antibody unrelated to the biomarker and serves a quality control function. The hydrophilic regions 18 of the detection pad and 19 of the quality control pad are fixed with chitosan and glutaraldehyde, with glutaraldehyde used in an amount of 3–6 μL, preferably 4.5 μL. The hydrophilic region 20 of the absorbent pad is treated with Tween. The labeled antibody is a complex of ruthenium tripyridine-intramolecular co-reactant-detection antibody (Ru(II)-PLL-Ab).

[0071] The specific process of chip modification is as follows: First, 0.2% Tween solution is added dropwise to the hydrophilic area 16 of the sample pad, the hydrophilic area 17 of the composite pad, and the hydrophilic area 20 of the absorbent pad. The dropwise amounts of the hydrophilic areas 16, 17, and 20 of the absorbent pad are 20, 5, and 13 μL, respectively. Then, the chips are placed in a 34°C oven to dry for 30 minutes. Subsequently, 7 μL of 2.5 mg / mL chitosan and 4.5 μL of 2.5% glutaraldehyde were sequentially added to the hydrophilic region 18 of the test pad and the hydrophilic region 19 of the control pad. The mixture was then incubated in a constant temperature and humidity chamber at 24°C and 50% for 30 minutes. Next, biomarker-coated antibody was added to the hydrophilic region 18 of the test pad, and quality control-coated antibody (IgY) was added to the hydrophilic region 19 of the control pad. Finally, the mixture was incubated in a constant temperature and humidity chamber at 24°C and 50% for 30 minutes to ensure that the biomarker-coated antibody and IgY were immobilized in the hydrophilic regions 18 and 19 of the control pad, respectively. Finally, 5 μL of labeled antibody was added to the hydrophilic region 17 of the binding pad, and the mixture was dried in a 34°C oven for 30 minutes to complete the modification of the internal chip.

[0072] A detection application of a dry three-electrode electrochemiluminescence lateral flow immunosensor includes the following steps:

[0073] S1. A test sample solution containing a biomarker is dropped into the hydrophilic area 16 of the sample pad of the sensor through the sample well 24. The test sample solution flows from the hydrophilic area 16 of the sample pad through the hydrophilic area 17 of the binding pad, where the biomarker specifically binds to the labeled antibody dried in the hydrophilic area 17 of the binding pad to form a "labeled antibody-biomarker" complex. This complex further flows through the hydrophilic areas 18 of each detection pad and binds to the biomarker-coated antibody immobilized in the hydrophilic area 18 of the detection pad to form an "labeled antibody-biomarker-coated antibody" immune sandwich complex.

[0074] S2. After the biomarker immune reaction is complete, add buffer solution to the hydrophilic area 16 of the sample application pad to remove the unbound labeled antibody from the hydrophilic area 18 of the test pad.

[0075] S3. Start the ECL analyzer, place the sensor on the ECL analyzer's input / output module, the input / output module will transport the sensor to the underside of the imaging detection module, and connect the electrical contact area 11 of the electrode pad 7 to the constant potential module of the ECL analyzer.

[0076] S4. Activate the detection button on the ECL analyzer. The constant potential module of the ECL analyzer provides the driving voltage to trigger the intramolecular reaction of the labeled antibody and generate an ECL signal. The ECL analyzer automatically collects and analyzes the ECL signal from the observation window 25 in the detection area and the observation window 26 in the quality control area, and then performs quantitative detection of biomarkers based on the intensity of the ECL signal.

[0077] The exposure time of the CMOS camera in the ECL analyzer is 100–600 ms, preferably 400 ms. The three-electrode ECL driving voltage is 2–4 V, preferably 3.5 V. The incubation time for the immune reaction is 2–8 min, preferably 5 min. The biomarker is cardiac troponin I (cTnI), and the intramolecular co-reactant is any one of polylysine, lysine, and cysteine, preferably polylysine.

[0078] Detection Application Example 1

[0079] The testing process is as follows:

[0080] 1. The sample solution containing cTnI is dropped into the hydrophilic area 16 of the sample pad of the detection chip 1 through the sample hole 24 of the top cover 22. The solution flows quickly through the hydrophilic area 17 of the binding pad and binds to the labeling antibody with specific affinity on it to form a "labeling antibody-cTnI" complex.

[0081] 2. The “labeled antibody-cTnI” complex further flows through the detection zone reaction cell 28 of the hydrophilic region 18 of the detection pad and binds to the cTnI-coated antibody with specific affinity thereon to form an “labeled antibody-cTnI-cTnI-coated antibody” immune sandwich complex.

[0082] 3. Wait several minutes until the immune reaction is complete, then add buffer solution to sample well 24 to remove unbound labeled antibodies from reaction chamber 28 in the detection area;

[0083] 4. The emission is captured by an ECL analyzer, and the light intensity is analyzed to achieve quantitative detection.

[0084] Detection Application Example 2

[0085] Several important factors affecting the ECL intensity value in Example 1 of the detection application were optimized: exposure time, driving voltage, amount of labeled probe, amount of glutaraldehyde, concentration of coating antibody, and incubation time (i.e., immune reaction time).

[0086] a) Optimal exposure time

[0087] 1. The concentration of cTnI to be tested is 0.01 ng / mL. -1 Exposure time to be determined, driving voltage 3V, labeled antibody volume 5μL, glutaraldehyde volume 5μL, cTnI coated antibody concentration 100μg / mL -1 The incubation time is 5 minutes.

[0088] 2. Set up several experimental groups: the exposure time is set to the following different values: 100ms, 200ms, 400ms, 500ms, and 600ms.

[0089] 3. The detection process is the same as in Example 1 of the detection application, and the experimental results are as follows: Figure 7 As shown.

[0090] from Figure 7 As can be seen, when the exposure time increases from 100ms to 600ms, the ECL intensity value continuously increases, while the signal-to-noise ratio gradually decreases. This phenomenon occurs because, with increasing exposure time, the integral of the ECL intensity value generated by the reaction tends to stabilize, while the integral of the ECL intensity value generated by the background continuously increases. Therefore, the total ECL intensity value continuously increases, and the signal-to-noise ratio continuously decreases. To obtain both a high ECL intensity value and a high signal-to-noise ratio simultaneously, an exposure time of 400ms is preferred.

[0091] b) Preferred driving voltage

[0092] 1. The concentration of cTnI to be tested is 0.01 ng / mL. -1 The exposure time was 400 ms, the driving voltage was to be determined, the amount of labeled antibody was 5 μL, the amount of glutaraldehyde was 5 μL, and the concentration of cTnI-coated antibody was 100 μg / mL. -1 The incubation time is 5 minutes.

[0093] 2. Set up several experimental groups: set the driving voltage to the following different values: 2V, 2.5V, 3V, 3.5V, 4V.

[0094] 3. The detection process is the same as in Example 1 of the detection application, and the experimental results are as follows: Figure 8 As shown.

[0095] from Figure 8 As can be seen, the ECL intensity value continuously increases as the driving voltage increases from 2V to 3.5V; however, after 3.5V, the ECL intensity value begins to decrease. This phenomenon may be due to background reactions (such as water oxidation) occurring at high driving voltages, which chemically and physically interfere with ECL emission. To obtain a higher ECL intensity value, 3.5V is preferred as the driving voltage.

[0096] c) Optimal dosage of labeled antibody

[0097] 1. The concentration of cTnI to be tested is 0.01 ng / mL. -1 The exposure time was 400 ms, the driving voltage was 3.5 V, the amount of labeled antibody was to be determined, the amount of glutaraldehyde was 5 μL, and the concentration of cTnI-coated antibody was 100 μg / mL. -1 The incubation time is 5 minutes.

[0098] 2. Set up several experimental groups: The amount of labeled antibody was set to the following different values: 2μL, 3μL, 4μL, 5μL, 6μL, and 7μL.

[0099] 3. The detection process is the same as in Example 1 of the detection application, and the experimental results are as follows: Figure 9 As shown.

[0100] from Figure 9 As can be seen, when the amount of labeled antibody increased from 2 μL to 5 μL, the ECL intensity value rose rapidly; after 5 μL, the ECL intensity value tended to remain constant. This phenomenon may be because, at a certain target concentration, the number of labeled antibody molecules gradually saturates. To obtain a higher ECL intensity value while controlling costs, 5 μL is the preferred amount of labeled antibody.

[0101] d) Optimize the dosage of glutaraldehyde

[0102] 1. The concentration of cTnI to be tested is 0.01 ng / mL. -1 The exposure time was 400 ms, the driving voltage was 3.5 V, the amount of labeled antibody was 5 μL, the amount of glutaraldehyde was to be determined, and the concentration of cTnI-coated antibody was 100 μg / mL. -1 The incubation time is 5 minutes.

[0103] 2. Set up several experimental groups: the amount of glutaraldehyde was set to the following different values: 3μL, 4μL, 4.5μL, 5μL, 5.5μL, and 6μL.

[0104] 3. The detection process is the same as in Example 1 of the detection application, and the experimental results are as follows: Figure 10 As shown.

[0105] from Figure 10 As can be seen, the ECL intensity value continuously increases when the glutaraldehyde dosage increases from 3 μL to 4.5 μL; however, the ECL intensity value begins to decrease after 4.5 μL. This phenomenon may be because excessive glutaraldehyde can affect the specific binding process between cTnI and the cTnI-coated antibody. To obtain a higher ECL intensity value, 4.5 μL is the preferred dosage of glutaraldehyde.

[0106] e) Preferred cTnI-coated antibody concentration

[0107] 1. The concentration of cTnI to be tested is 0.01 ng / mL. -1 The exposure time was 400ms, the driving voltage was 3.5V, the amount of labeled antibody was 5μL, the amount of glutaraldehyde was 4.5μL, the concentration of cTnI-coated antibody was to be determined, and the incubation time was 5min.

[0108] 2. Several experimental groups were set up: the concentration of the coated antibody was set to the following different values: 40 μg / mL -1 60μg mL -1 80μg mL -1 100μg mL -1 120μg mL-1 140μg mL -1 .

[0109] 3. The detection process is the same as in Example 1 of the detection application, and the experimental results are as follows: Figure 11 As shown.

[0110] from Figure 11 As can be seen from this, when the concentration of cTnI-coated antibody is increased from 40 μg / mL... -1 Increase to 100 μg / mL -1 As time progressed, the ECL intensity value continuously increased; 100 μg mL -1 Subsequently, the ECL intensity value began to decrease. This phenomenon may be due to excessive cTnI-coated antibody affecting the electron transfer process from the electrode to the labeled antibody. To obtain a higher ECL intensity value, 100 μg / mL is preferred. -1 The concentration of cTnI-coated antibody.

[0111] f) Preferred incubation time

[0112] 1. The concentration of cTnI to be tested is 0.01 ng / mL. -1 The exposure time was 400 ms, the driving voltage was 3.5 V, the amount of labeled antibody was 5 μL, the amount of glutaraldehyde was 4.5 μL, and the concentration of cTnI-coated antibody was 100 μg / mL. -1 The incubation period is yet to be determined.

[0113] 2. Set up several experimental groups: the incubation time is set to the following different values: 2 min, 3 min, 4 min, 5 min, 6 min, 8 min.

[0114] 3. The detection process is the same as in Example 1 of the detection application, and the experimental results are as follows: Figure 12 As shown.

[0115] from Figure 12 As can be seen, the ECL intensity value continuously increases when the incubation time increases from 2 min to 5 min; after 5 min, the ECL intensity value tends to stabilize. This phenomenon may be because the "labeled antibody-biomarker-coating antibody" immune sandwich complex is no longer generated after the immune reaction ends. To obtain higher ECL intensity values ​​while reducing the detection time, 5 min is the preferred incubation time.

[0116] Detection Application Example 3

[0117] The steps are as follows:

[0118] 1. According to the preferred parameters of the second example of the detection application: exposure time 400ms; driving voltage 3.5V; labeled probe volume 5μL; glutaraldehyde volume 4.5μL; cTnI coated antibody concentration 100μg / mL -1 The incubation time was 5 minutes. A dry three-electrode ECL lateral flow immunosensor was fabricated using a detection chip with one detection pad.

[0119] 2. Set up several experimental groups: set the cTnI concentration in the sample solution to be tested to several different values.

[0120] 3. The detection process is the same as in Example 1 of the detection application, and the experimental results are as follows: Figure 13 As shown.

[0121] As can be seen from the figure, the cTnI concentration ranges from 0.5 to 1000 pg / mL. -1 Within the specified range, the ECL intensity value increases with increasing cTnI concentration. A good linear relationship exists between the ECL intensity value (denoted by Y) and the logarithm of the cTnI concentration (denoted by X), expressed as Y = 5.998 + 1.801X. The estimated detection limit is 0.494 pg / mL. -1 The detection limit is calculated as follows: Y L =Y b +3S b , where Y b S represents the average ECL intensity value during the blank control period. b The standard deviation of the blank control (five replicates) was calculated using Y0. L The corresponding cTnI concentration is calculated and is the detection limit.

[0122] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the structure of the present invention, and these will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. A dry three-electrode electrochemiluminescence lateral flow immunosensor, characterized in that, The detection chip (1) includes a sample application pad (2), a binding pad (3), at least one detection pad (4), a quality control pad (5), an absorption pad (6), an electrode pad (7), and a base plate (21). A common working electrode (8) is provided on the top of the electrode pad (7). A counter electrode (9) and a reference electrode (10) are respectively provided on the electrode pads (7) on both sides of the common working electrode (8). The common working electrode (8), counter electrode (9), and reference electrode (10) are all positioned at one end in the same direction. For the electrical contact area (11), the other end of the counter electrode (9) is sequentially provided with a detection area counter electrode (12) that is equal in number to and corresponds one-to-one with the detection pad (4) and a quality control area counter electrode (13). Correspondingly, the other end of the reference electrode (10) is sequentially provided with a detection area reference electrode (14) that is equal in number to and corresponds one-to-one with the detection pad (4) and a quality control area reference electrode (15). The detection area counter electrode (12), the quality control area counter electrode (13), and the detection area reference electrode (14) are... The reference electrode (15) in the quality control area all faces the common working electrode (8); the sample pad (2) is provided with a sample pad hydrophilic area (16), the conjugate pad (3) is provided with a conjugate pad hydrophilic area (17), the detection pad (4) is provided with a detection pad hydrophilic area (18), the quality control pad (5) is provided with a quality control pad hydrophilic area (19), and the absorption pad (6) is provided with an absorption pad hydrophilic area (20). The sample pad (2), conjugate pad (3), each detection pad (4), quality control pad (5), and absorption pad (6) are arranged in sequence. Arranged on the top of the electrode pad (7), the hydrophilic area (16) of the sample pad is sequentially connected to the hydrophilic area (17) of the binding pad, the hydrophilic areas (18) of each detection pad, the hydrophilic area (19) of the quality control pad, and the hydrophilic area (20) of the absorption pad to form a single flow path. Each hydrophilic area (18) of the detection pad is located directly above the corresponding detection area counter electrode (12) and detection area reference electrode (14). The hydrophilic area (19) of the quality control pad is located directly above the quality control area counter electrode (13) and quality control area reference electrode (15). The overall structure of the counter electrode (9) and the reference electrode (10) is symmetrical about the common working electrode (8); Overlapping areas are provided between the sample application pad (2) and the conjugate pad (3), between the conjugate pad (3) and the detection pad (4), between the detection pad (4) and the quality control pad (5), and between the quality control pad (5) and the absorption pad (6); The hydrophilic area (20) of the absorbent pad has an L-shaped structure. Its thinner hydrophilic area is used to connect to the hydrophilic area (19) of the quality control pad, and its wider hydrophilic area is used to absorb excess solution. The hydrophilic area (19) of the quality control pad has a Z-shaped structure. The hydrophilic area in the middle of the "Z" shape is used to form the ECL area, namely the quality control area reaction cell (29). The quality control area reaction cell (29) is formed between the quality control area counter electrode (13) and the common working electrode (8), and between the quality control area reference electrode (15) and the common working electrode (8). Together with the hydrophilic areas at both ends of the "Z" shape, it forms a flow path, connecting the hydrophilic area (18) of the detection pad and the hydrophilic area (20) of the absorption pad. The hydrophilic area (18) of the detection pad has a Z-shaped structure. The hydrophilic area in the middle of the "Z" shape is used to form the ECL area, namely the detection zone reaction pool (28). The detection zone reaction pool (28) is formed between the detection zone counter electrode (12) and the common working electrode (8), and between the detection zone reference electrode (14) and the common working electrode (8). Together with the hydrophilic areas at both ends of the "Z" shape, it forms a flow path, connecting the hydrophilic area (17) of the binding pad and the hydrophilic area (19) of the quality control pad.

2. The dry three-electrode electrochemiluminescence lateral flow immunosensor according to claim 1, characterized in that, The electrode pad (7) is made of hydrophobic fiber material as a base and is made by screen printing of conductive material. The sample pad (2), binding pad (3), detection pad (4), quality control pad (5), and absorption pad (6) are all made of hydrophilic fiber material as a base and form hydrophobic and hydrophilic areas on the base by screen printing hydrophobic ink.

3. The dry three-electrode electrochemiluminescence lateral flow immunosensor according to claim 1, characterized in that, The electrode pad (7) is bonded to the top of the base plate (21). The middle parts of the sample pad (2), the binding pad (3), the detection pad (4), the quality control pad (5), and the absorption pad (6) are all in close contact with the electrode pad (7). The two ends of the sample pad (2), the binding pad (3), the detection pad (4), the quality control pad (5), and the absorption pad (6) are all bonded to the base plate (21).

4. The dry three-electrode electrochemiluminescence lateral flow immunosensor according to claim 1, characterized in that, It also includes an upper cover (22) and a lower cover (23). The lower cover (23) has an installation groove for installing the detection chip (1). The upper cover (22) covers the detection chip (1). The upper cover (22) is provided with a sample application hole (24) aligned with the hydrophilic area (16) of the sample application pad, a detection area observation window (25) aligned with each of the hydrophilic areas (18) of the detection pad, a quality control area observation window (26) aligned with the hydrophilic area (19) of the quality control pad, and an opening groove (27) aligned with the electrical contact area (11).

5. A dry three-electrode electrochemiluminescence lateral flow immunosensor according to claim 1, characterized in that, The hydrophilic area (16) of the sample loading pad and the hydrophilic area (20) of the absorption pad are both treated with Tween; the hydrophilic area (17) of the binding pad is treated with Tween and then dried and labeled with antibodies.

6. The dry three-electrode electrochemiluminescence lateral flow immunosensor according to claim 1, characterized in that, The hydrophilic region (18) of the test pad is fixed with modified biomarkers coated with antibodies.

7. A dry three-electrode electrochemiluminescence lateral flow immunosensor according to claim 1, characterized in that, The hydrophilic region (19) of the quality control pad is fixed with modified quality control coated antibodies.

8. A dry three-electrode electrochemiluminescence lateral flow immunosensor according to claim 1, characterized in that, The hydrophilic region (18) of the test pad and the hydrophilic region (19) of the quality control pad are fixed and modified with chitosan and glutaraldehyde.

9. A detection application of the dry three-electrode electrochemiluminescence lateral flow immunosensor according to any one of claims 1-8, characterized in that, Includes the following steps: S1. A test sample solution containing a biomarker is dropped into the hydrophilic area (16) of the sample pad of the sensor. The test sample solution flows from the hydrophilic area (16) of the sample pad through the hydrophilic area (17) of the conjugate pad. The biomarker specifically binds to the labeled antibody dried in the hydrophilic area (17) of the conjugate pad to form a "labeled antibody-biomarker" complex. The complex further flows through the hydrophilic areas (18) of each test pad and binds to the biomarker-coated antibody fixed in the hydrophilic area (18) of the test pad to form an "labeled antibody-biomarker-coated antibody" immune sandwich complex. S2. After the biomarker immune reaction is completed, add buffer solution to the hydrophilic area (16) of the sample application pad to remove the unbound labeled antibody from the hydrophilic area (18) of the test pad. S3. Start the ECL analyzer, place the sensor on the ECL analyzer's inlet / outlet module, the inlet / outlet module will transport the sensor to the underside of the imaging detection module, and connect the electrical contact area (11) of the electrode pad (7) to the constant potential module of the ECL analyzer. S4. Activate the detection button on the ECL analyzer. The constant potential module of the ECL analyzer provides the driving voltage to trigger the intramolecular reaction of the labeled antibody, generating an ECL signal. The ECL analyzer automatically collects and analyzes the ECL signal, and then performs quantitative detection of the biomarker based on the intensity of the ECL signal.

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