Application of a three-dimensional closed-loop bipolar electrochemiluminescence lateral flow chip in immunoassay

Abstract

This application discloses the application of a three-dimensional closed-loop bipolar electrochemiluminescence lateral flow chip in immunoassay. The chip includes an electrode sheet, a test strip, and a connecting pad. The electrode sheet includes two sets of electrodes: one set includes a positive driving electrode I, a bipolar electrode, and a negative driving electrode; the other set includes a positive driving electrode II. The two sets of electrodes are distributed on the front and back surfaces of a hydrophobic substrate, respectively. The positive driving electrode I and the positive driving electrode II are connected by a conductive material. This invention is the first to construct CBPE on the front and back surfaces of a hydrophobic substrate, forming a three-dimensional CBPE-ECL electrode sheet, reducing the errors introduced by manual assembly of multilayer electrodes and significantly improving the consistency and reliability of the electrode sheet.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical sensors, specifically relating to the application of a three-dimensional closed-loop bipolar electrochemiluminescence lateral flow chip in immunoassay. Background Technology

[0002] Closed-cell bipolar electrochemiluminescence (CBPE-ECL) electrode sheets utilize insulating materials to physically isolate the CBPE cathode and anode, offering advantages such as high reaction independence, low background interference, and strong controllability of the reaction process. However, in these electrode sheets, the CBPE cathode and anode are on the same plane. When the sample volume is too large or external interference occurs, the sample solution is prone to cross-regional conduction. This phenomenon can cause a short circuit in the driving current, leading to significant attenuation or even complete disappearance of the ECL signal. Furthermore, existing CBPE-ECL detection devices lack integrated signal acquisition and analysis functions, making operation cumbersome and limiting their application in home self-testing.

[0003] Dry chemistry analysis (also known as dry analysis) involves drying and immobilizing test reagents on a solid support, followed by chromatographic reaction to generate a signal. Dry lateral flow immunoassay strips offer advantages such as low cost, ease of use, and fast detection speed. However, existing lateral flow immunoassay strips often suffer from low detection sensitivity and difficulty in quantification, hindering their application in point-of-care testing for home use.

[0004] CBPE-ECL lateral flow immunoassay strips combine two technologies and, while successfully applied to the detection of various biomarkers, still have some limitations. First, existing technologies often utilize intramolecularly enhanced ECL signal antibodies. Although these have high electron transfer efficiency, their synthesis involves multiple steps, and interfering substances are difficult to separate, leading to false positives and low sensitivity. Second, the two-dimensional CBPE-ECL electrode pads require isolation between the CBPE cathode and anode, resulting in a much larger area than lateral flow immunoassay strips. Furthermore, manual assembly is necessary, leading to significant errors and low accuracy. Third, there is no positioning device between the chip and the cartridge; each time, the chip's luminescent area must be manually aligned with the cartridge's observation window, making the operation complex and requiring a high level of operator skill. These limitations significantly restrict the widespread application of this type of test strip. Summary of the Invention

[0005] The purpose of this invention is to provide an application of a three-dimensional closed-loop bipolar electrochemiluminescence lateral flow chip in immunoassay.

[0006] The objective of this invention is achieved through the following technical solution: Application of a three-dimensional closed-loop bipolar electrochemiluminescence lateral flow chip in immunoassay, wherein the three-dimensional closed-loop bipolar electrochemiluminescence lateral flow chip includes an electrode sheet, a test strip, and a connecting pad; The electrode sheet includes two sets of electrodes: one set includes a positive driving electrode, a bipolar electrode, and a negative driving electrode; the other set includes a positive driving electrode. The two sets of electrodes are distributed on the front and back surfaces of the hydrophobic substrate, respectively. One end of the positive driving electrode 1 and one end of the positive driving electrode 2 are connected by a conductive material; specifically, one end of the positive driving electrode 2 is connected to the positive driving electrode 1 by a conductive tape located on the substrate end side; one end of the positive driving electrode 2 and the positive driving electrode 1 can also be connected by drilling or injecting conductive material; the other end of the positive driving electrode 2 has a blank area of ​​several millimeters without an electrode, so that the driving electrode contact points are spatially staggered to prevent short circuit. The bipolar electrode includes a control anode, at least one detection anode, and a shared cathode, wherein one end of the control anode is close to the negative driving electrode; the detection anode and the control anode are connected in series and are on the same straight line as the shared cathode; the control anode and the negative driving electrode, as well as the shared cathode and the positive driving electrode, are naturally separated by a hydrophobic substrate. The test strip includes a sample pad, a conjugate pad, a detection pad, and an absorbent pad; the test strip and the connecting pad are arranged in a straight line; the widths of the test strip, electrode sheet, and connecting pad are consistent, which facilitates the large-scale production of the sensor; the length ratio of the absorbent pad to the connecting pad is preferably 10:11. The aforementioned binding pad is dried to fix high-purity ECL signaling antibodies; The detection pad is a nitrocellulose membrane (NC membrane) with a backing, and has T lines and C lines; the T lines are coated with T line capture antibodies, and the C lines are coated with C line capture antibodies; the backing side of the detection pad faces upward and the front side faces downward in contact with the electrode sheet, wherein the T lines on the detection pad are in contact with the detection anode, and the C lines are in contact with the quality control anode; The binding pad is located between the detection pad and the sample pad, and the sample pad is located between the shared cathode and the detection anode; the absorbent pad is close to the quality control anode and is in contact with a portion of the negative drive electrode; During preparation, the detection pad, conjugate pad, sample pad, and absorbent pad are stacked and fixed on a transparent adhesive plate in sequence, and then inverted on the front of the electrode sheet to form the above-mentioned correspondence; the transparent adhesive plate has a hole near the sample pad end to facilitate the addition of liquid; The connecting pad is located between the shared cathode and the first positive driving electrode, and maintains partial contact with both the first positive driving electrode and the shared cathode; the connecting pad is spatially separated from the test strip to prevent cross-contamination of the reaction solutions and ensure that the electrochemical reaction takes place in the CBPE system; During preparation, the test strips, connecting pads and electrode sheets can all be prepared in large form, and after being bonded together, they can be cut into individual three-dimensional closed bipolar electrochemiluminescence lateral flow chips. Hydrophobic double-sided tape is adhered between the shared cathode and the detection anode of the electrode sheet to position and fix the sample pad, conjugate pad, and part of the detection pad, while ensuring that the electrode area between the shared cathode and the detection anode and the lateral flow immunoassay strip remain isolated; hydrophobic double-sided tape is adhered between the quality control anode and the negative driving electrode to fix part of the absorption pad and part of the detection pad; hydrophobic double-sided tape is adhered between the shared cathode and the first positive driving electrode to fix the connecting pad; The hydrophobic substrate material is preferably a PET plastic sheet with a thickness of 0.8 mm; the transparent adhesive plate is preferably a PET plastic sheet with adhesive backing and a thickness of 0.1 mm; the sample pad and bonding pad are preferably made of glass fiber; the absorbent pad and connecting pad are preferably made of absorbent paper; the electrode sheet is constructed on the hydrophobic substrate by screen printing with conductive paste, and the conductive paste is preferably conductive carbon paste; the casing is prepared by a 3D printer. The sample pad preparation process is as follows: the glass fibers cut by the laser cutting machine are first treated with the sample pad treatment solution, and then baked at 37 ℃ for 1 h; then treated with the intermolecular co-reactant BIS-TRIS solution, and then dried in an oven at 37 ℃ for 1 h to obtain the sample pad. The conjugate pad preparation process is as follows: the glass fibers cut by the laser cutting machine are first treated with the conjugate pad treatment solution, and then dried in an oven at 37 ℃ for 1 h; then, the ECL signal antibody mixed solution is uniformly sprayed onto the surface of the dried glass fibers using a gold spraying film scribing instrument, and then dried in an oven at 37 ℃ for 1 h to obtain the conjugate pad. The detection pad preparation process is as follows: T-line capture antibody solution and C-line capture antibody solution are sprayed onto the corresponding positions of the same NC membrane using a gold spray coating instrument to form T-lines and C-lines; then the NC membrane is placed in a 37 ℃ oven to dry for 2 h to obtain the detection pad; Both the absorbent pad and the connecting pad are made by cutting absorbent paper with a laser cutting machine; The transparent adhesive board is prepared by cutting a 0.1 mm thick PET plastic sheet with adhesive backing using a laser cutting machine; The sample pad treatment solution and conjugate pad treatment solution can adopt existing technologies, such as paragraph 20 of CN118688451 A specification. The intermolecular co-reactant BIS-TRIS solution is PBS (0.1 M, pH 7.5) containing 10.5 mg / mL BIS-TRIS. The T-line capture antibody solution is PBS (0.1 M, pH 7.2-7.4) comprising 10 mg / mL sucrose, 0.05% S9 and 0.11-0.18 mg / mL T-line capture antibody, preferably with a T-line capture antibody concentration of 0.16 mg / mL; The C-line capture antibody solution is PBS (0.1 M, pH 7.2-7.4) comprising 10 mg / mL sucrose, 5 mg / mL trehalose, 0.05% S9 and 0.11-0.18 mg / mL T-line capture antibody, preferably with a C-line capture antibody concentration of 0.16 mg / mL; The ECL signaling antibody mixture is composed of a 4.9-8.9 ng / mL ECL signaling antibody solution conjugated with a C-line labeled antibody (i.e., C-line ECL signaling antibody), a 4.9-8.9 ng / mL ECL signaling antibody solution conjugated with a T-line labeled antibody (i.e., T-line ECL signaling antibody), and a signaling antibody treatment solution. The signaling antibody treatment solution comprises 50 mg / mL sucrose, 5 mg / mL PVP, 2.5 mg / mL casein, 0.25% S9, 50 mg / mL trehalose, 3 mg / mL sorbitol, and 1 mg / mL PEG-20000 in PBS (0.1 M, pH 7.2-7.4). Preferably, the concentrations of the C-line ECL signaling antibody and the T-line ECL signaling antibody are 7.4 ng / mL. The C-line ECL signal antibody (or T-line ECL signal antibody) is prepared by direct coupling of a terpyridine ruthenium derivative with a C-line labeled antibody (or a T-line labeled antibody) followed by purification using a dextran gel chromatography column; the dextran gel chromatography column packing material is preferably dextran gel. ® G-50.

[0007] The aforementioned three-dimensional closed-loop bipolar electrochemiluminescence lateral flow chip is installed in a housing to form a sensor; The casing is elongated and includes a top cover and a bottom plate; the top cover is provided with an observation window, a sample application port, and a buffer solution addition port, all three of which are located on the same straight line; The base plate is provided with an electrode contact area, three sets of longitudinal protrusions and one transverse protrusion; The electrode contact area corresponds to the first end of the negative driving electrode and the second positive driving electrode; The longitudinal protrusion is used to restrict the path and direction of chip insertion, and the transverse protrusion is used for precise positioning of the sensor with the observation window, sample loading port, and buffer addition port of the top cover. The observation window corresponds to the T-line and C-line; the sample application well corresponds to the sample pad; the buffer addition well corresponds to the connection pad. The sensor can be inserted into a fully automatic ECL real-time detector for automatic acquisition and analysis of ECL signals; The fully automated ECL instantaneous detection instrument includes a voltage excitation module, an ECL signal acquisition module, an ECL signal processing module, and a dark reaction chamber. During detection, the user only needs to click the detection button on the instrument screen to automatically complete the entire process of driving voltage excitation, ECL signal acquisition and signal processing, and directly provide feedback on the biomarker concentration to the user. The electrode contact points of the voltage excitation module are located in the dark reaction chamber and can contact the electrode contact area of ​​the casing base plate. The camera of the ECL signal acquisition module is installed in the dark reaction chamber and faces the observation window on the top cover of the casing. It can automatically record ECL video on the T-line and C-line. The ECL signal processing module is used to analyze and process the recorded video: extract the corresponding frames where the luminescence intensity on the T-line and C-line reaches the maximum value in the video; accurately extract the corresponding luminescent area; calculate the ECL signal values ​​of the T-line and C-line, and calculate their ratio (T / C value); substitute the T / C value into the standard curve for biomarker concentration detection, thereby automatically obtaining the concentration value of the biomarker being tested.

[0008] The aforementioned immune detection is used to detect multiple biomarkers; Depending on the antibody, the biomarker may be one of luteinizing hormone (LH), cardiac troponin I, C-reactive protein, peptidin, serum amyloid, or Alzheimer's disease-associated neuronal linein.

[0009] The immune detection includes the following steps: First, 25-45 μL of sample solution containing the biomarker to be tested is added to the sample pad, and the reaction is allowed to proceed for 2.5-4.5 minutes. During this time, the solution flows through the binding pad via capillary action and specifically recognizes the T-line ECL signal antibody, forming an immune complex of the T-line ECL signal antibody and the biomarker (i.e., the T-line ECL signal antibody-biomarker complex). The solution then flows to the detection pad, where the T-line ECL signal antibody-biomarker complex is captured by the T-line capture antibody, further forming a sandwich immune complex (i.e., the T-line ECL signal antibody-biomarker-T-line capture antibody). The preferred sample solution containing the biomarker to be tested is added in an amount of 35 μL, and the immune reaction time is 4 min. Next, 30-50 μL of PBS was added to the sample pad to further promote the immune response and wash away any unreacted substances into the absorbent pad. This process was maintained for 3 minutes. Subsequently, 40 μL of PBS was added to the connection pad. Finally, the three-dimensional closed-loop bipolar electrochemiluminescence lateral flow chip is placed into an automated ECL instantaneous detection instrument for detection. The ECL reaction is carried out under a certain driving voltage (12-18 V) to achieve quantitative detection of the biomarker to be tested; the preferred driving voltage is 14 V.

[0010] The present invention has the following advantages and effects compared with the prior art: 1. This invention is the first to construct CBPE on the front and back sides of a hydrophobic substrate to form a three-dimensional CBPE-ECL electrode sheet, which reduces the error introduced by manual assembly of multilayer electrodes and significantly improves the consistency and reliability of the electrode sheet.

[0011] 2. In this invention, the front and back sides of the three-dimensional CBPE-ECL electrode sheet can be conveniently connected by a conductive tape located on the substrate end side, or by drilling holes or injecting conductive material.

[0012] 3. In this invention, the quality control anode and the detection anode are distributed in series and are located on the same straight line as the shared cathode and the driving electrode; the connecting pad and the lateral flow immunoassay strip are also distributed along the same straight line. This layout allows the connecting pad and the lateral flow immunoassay strip to be effectively separated in space, preventing the reaction solutions from flowing together and ensuring that the electrochemical reaction takes place in the CBPE system.

[0013] 4. This invention normalizes the width of the lateral flow immunoassay strip, the connecting pad, and the three-dimensional CBPE-ECL electrode sheet and manufactures them into a large-format sheet, which can be efficiently cut using a high-speed chopping machine, thereby enabling large-scale, standardized production of the three-dimensional CBPE-ECL lateral flow immunoassay chip.

[0014] 5. The base plate of the card case of the present invention is provided with a protrusion, which can realize the rapid and accurate assembly of the chip and ensure the precise positioning between the three-dimensional CBPE-ECL lateral flow immunoassay chip and the observation window, sample application port and buffer addition port on the top cover.

[0015] 6. This invention uses high-purity ECL signal antibody purified by dextran gel chromatography column, which significantly improves detection sensitivity and effectively reduces background signal and non-specific adsorption.

[0016] 7. This invention uses BIS-TRIS as an intermolecular co-reactant. Its electrochemical performance is similar to that of tripropylamine, and it has the advantages of low toxicity, low cost, low volatility, and high stability, making it more suitable for drying and storing on lateral flow immunoassay strips.

[0017] 8. This invention dries the intermolecular co-reactant on the sample pad, avoiding microenvironmental fluctuations caused by the addition of co-reactant during the reaction process, making ECL detection more stable and further simplifying the operation steps.

[0018] 9. This invention uses a self-developed fully automatic ECL instantaneous detection instrument, which integrates functions such as voltage excitation, ECL signal acquisition and ECL signal processing. It can directly obtain the concentration of the biomarker to be tested based on the T / C value, solving the problems of single function, scattered and complex operation of existing equipment, and realizing "one-click" rapid automatic detection.

[0019] 10. The entire process from sample addition to detection completion takes only about 7 minutes, meeting the application requirements for rapid on-site detection.

[0020] 11. The three-dimensional CBPE-ECL lateral flow immunosensor of the present invention has high detection sensitivity, wide dynamic linear range and good storage stability, which is beneficial for applications such as home self-testing of biomarkers and on-site instant detection.

[0021] 12. The sensor of the present invention is easy to operate and has wide applicability. Users only need to add sample solution and buffer solution, put the sensor into the detector, and the instrument will automatically complete the analysis after one-button operation, without the need for professional training.

[0022] 13. The chip material of this invention has low cost and simple structure, conforms to the concept of green environmental protection, and has good market promotion potential. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the overall three-dimensional closed bipolar electrochemiluminescence lateral flow chip of the present invention; wherein, 1-1: electrode sheet, 1-2: test strip, 1-3: connecting pad.

[0024] Figure 2 This is a schematic diagram of the electrode sheet composition; where 1-1-1: Positive driving electrode one, 1-1-2: Bipolar electrode, 1-1-2-1: Quality control anode, 1-1-2-2: Detection anode, 1-1-2-3: Shared cathode, 1-1-3: Negative driving electrode, 1-1-4: Positive driving electrode two, 1-1-5: Hydrophobic substrate, 1-1-6: Conductive tape, 1-1-7: Perforation, 1-1-8: Blank area, 1-1-9: Hydrophobic double-sided adhesive.

[0025] Figure 3 This is a schematic diagram of the test strip composition; where 1-2-1: sample pad, 1-2-2: conjugate pad, 1-2-3: detection pad, 1-2-3-1: T line, 1-2-3-2: C line, 1-2-4: absorption pad, 1-2-5: transparent adhesive plate, 1-2-5-1: well.

[0026] Figure 4 This is an exploded schematic diagram of the three-dimensional closed-loop bipolar electrochemiluminescence lateral flow chip of the present invention.

[0027] Figure 5This is a schematic diagram of the components of the casing; where 2-1: top cover, 2-1-1: observation window, 2-1-2: sample application hole, 2-1-3: buffer solution application hole, 2-2: base plate, 2-2-1: electrode contact area, 2-2-2: longitudinal protrusion, 2-2-3: transverse protrusion.

[0028] Figure 6 This is a schematic diagram of the sensor of the present invention.

[0029] Figure 7 For T / C value and L c Relationship diagram.

[0030] Figure 8 For T / C value and L a The relationship diagram.

[0031] Figure 9 For T / C value and E tot The relationship diagram.

[0032] Figure 10 This is a graph showing the relationship between the T / C value and [CAb].

[0033] Figure 11 This is a graph showing the relationship between T / C values ​​and [SAb].

[0034] Figure 12 For T / C value and V s The relationship diagram.

[0035] Figure 13 For T / C value and t i Relationship diagram.

[0036] Figure 14 For T / C value and V w Relationship diagram.

[0037] Figure 15 The graph shows the analytical curves for detecting different concentrations of LH (the inset is a double logarithmic fitting curve of the data).

[0038] Figure 16 This is a graph for evaluating real sample detection.

[0039] Figure 17 This is a graph used to evaluate the storage stability of LH.

[0040] Figure 18 This is a schematic diagram of the large-format chip.

[0041] Figure 19 This is a schematic diagram of the front of the large electrode sheet.

[0042] Figure 20 This is a schematic diagram of the back of the large electrode sheet.

[0043] Figure 21 This is a schematic diagram of the large-format test strip.

[0044] Figure 22 This is a schematic diagram of the large-format connecting pad.

[0045] Figure 23 This is an exploded view of the large-format test strip structure; where A: large-format transparent adhesive plate with backing, B: large-format sample pad, C: large-format binding pad, D: large-format detection pad, and E: large-format absorbent pad. Detailed Implementation

[0046] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0047] Example 1 Application of a three-dimensional closed-loop bipolar electrochemiluminescence lateral flow chip in immunoassay, the composition and structure of the chip are as follows: Figures 1-4 As shown, it includes electrode sheet 1-1, test strip 1-2, and connecting pad 1-3; The electrode sheet 1-1 includes two sets of electrodes. One set includes a positive driving electrode 1-1-1, a bipolar electrode 1-1-2, and a negative driving electrode 1-1-3. The other set includes a positive driving electrode 1-1-4. The two sets of electrodes are distributed on the front and back surfaces of the hydrophobic substrate 1-1-5. The positive driving electrode 1-1-1 and the positive driving electrode 1-1-4 are connected by a conductive material; specifically, one end of the positive driving electrode 1-1-4 is connected to the positive driving electrode 1-1-1 through a conductive tape 1-1-6 located on the substrate end side; or, one end of the positive driving electrode 1-1-4 and the positive driving electrode 1-1-1 can also be connected by drilling a hole 1-1-7 and injecting conductive material. The other end of the positive driving electrode 1-1-4 is provided with a blank area 1-1-8 (several millimeters) where no electrode is constructed, so that the contact points of the driving electrodes are spatially staggered to prevent short circuits. The bipolar electrode 1-1-2 includes a control anode 1-1-2-1, at least one detection anode 1-1-2-2, and a shared cathode 1-1-2-3, wherein the control anode 1-1-2-1 is close to the negative driving electrode 1-1-3; the detection anode 1-1-2-2 and the control anode 1-1-2-1 are connected in series and are on the same straight line as the shared cathode 1-1-2-3; the control anode 1-1-2-1 and the negative driving electrode 1-1-3, as well as the shared cathode 1-1-2-3 and the positive driving electrode 1-1-1, are naturally separated by a hydrophobic substrate; The test strip 1-2 includes a sample pad 1-2-1, a conjugate pad 1-2-2, a detection pad 1-2-3, and an absorption pad 1-2-4; the test strip 1-2 and the connecting pad 1-3 are distributed in a straight line; the widths of the test strip 1-2, the electrode sheet 1-1, and the connecting pad 1-3 are consistent, which facilitates the large-scale production of the sensor. The aforementioned binding pad 1-2-2 is dried and immobilized with high-purity ECL signaling antibody; The detection pad 1-2-3 is a nitrocellulose membrane (NC membrane) with a backing, and has a T line 1-2-3-1 and a C line 1-2-3-2. The T line 1-2-3-1 is coated with T line capture antibody, and the C line 1-2-3-2 is coated with C line capture antibody. The backing side of the detection pad 1-2-3 faces upward and the front side faces downward, and it contacts the electrode sheet 1-1. The T line 1-2-3-1 on the detection pad 1-2-3 is in contact with the detection anode 1-1-2-2, and the C line 1-2-3-2 is in contact with the quality control anode 1-1-2-1. The binding pad 1-2-2 is located between the detection pad 1-2-3 and the sample pad 1-2-1, and the sample pad 1-2-1 is located between the shared cathode 1-1-2-3 and the detection anode 1-1-2-2; the absorption pad 1-2-4 is close to the quality control anode 1-1-2-1 and is in contact with part of the negative driving electrode 1-1-3. During preparation, the detection pad 1-2-3, the binding pad 1-2-2, the sample pad 1-2-1, and the absorption pad 1-2-4 are stacked sequentially and fixed on the transparent adhesive plate 1-2-5, and then inverted on the front of the electrode sheet 1-1 to form the above-mentioned correspondence; the transparent adhesive plate 1-2-5 has a hole 1-2-5-1 at one end near the sample pad 1-2-1 to facilitate the addition of liquid; The connecting pad 1-3 is located between the shared cathode 1-1-2-3 and the positive driving electrode 1-1-1, and maintains partial contact with the positive driving electrode 1-1-1 and the shared cathode 1-1-2-3; the connecting pad 1-3 is spatially separated from the test strip 1-2 to prevent the reaction solution from flowing together and to ensure that the electrochemical reaction is carried out in the CBPE system. During preparation, the test strips 1-2, connecting pads 1-3 and electrode sheets 1-1 can all be prepared in large form, and after being bonded together, they can be cut into individual three-dimensional closed bipolar electrochemiluminescence lateral flow chips. Hydrophobic double-sided tape 1-1-9 is adhered between the shared cathode 1-1-2-3 and the detection anode 1-1-2-2 of electrode sheet 1-1 for positioning and fixing sample pad 1-2-1, conjugate pad 1-2-2, and part of detection pad 1-2-3, while ensuring that the electrode area between the shared cathode 1-1-2-3 and the detection anode 1-1-2-2 remains isolated from the test strip 1-2; hydrophobic double-sided tape 1-1-9 is adhered between the quality control anode 1-1-2-1 and the negative driving electrode 1-1-3 for fixing part of the absorption pad 1-2-4 and part of the detection pad 1-2-3; hydrophobic double-sided tape 1-1-9 is adhered between the shared cathode 1-1-2-3 and the positive driving electrode 1-1-1 for fixing the connecting pad 1-3; The hydrophobic substrate 1-1-5 is preferably made of PET plastic sheet with a thickness of 0.8 mm; the transparent adhesive plate 1-2-5 is preferably made of PET plastic sheet with adhesive backing with a thickness of 0.1 mm; the sample pad 1-2-1 and bonding pad 1-2-2 are preferably made of glass fiber; the absorbent pad 1-2-4 and connecting pad 1-3 are preferably made of absorbent paper; the electrode sheet 1-1 is constructed on the hydrophobic substrate 1-1-5 by screen printing of conductive paste, preferably conductive carbon paste; the casing is prepared by 3D printer. The absorbent pads 1-2-4 and connecting pads 1-3 are both made by cutting absorbent paper with a laser cutting machine; The transparent adhesive board 1-2-5 is prepared by cutting a 0.1 mm thick PET plastic board with adhesive backing using a laser cutting machine.

[0048] A sensor comprising the aforementioned three-dimensional closed-loop bipolar electrochemiluminescence lateral flow chip ( Figure 6 ), also includes jamming ( Figure 5 ); The card is elongated and includes an upper cover 2-1 and a bottom plate 2-2; the upper cover 2-1 is provided with an observation window 2-1-1, a sample application hole 2-1-2, and a buffer solution addition hole 2-1-3, all of which are located on the same straight line; The base plate 2-2 is provided with an electrode contact area 2-2-1, three sets of longitudinal protrusions 2-2-2 and one transverse protrusion 2-2-3; The electrode contact area 2-2-1 corresponds to the first end of the negative driving electrode 1-1-3 and the positive driving electrode 1-1-4; The longitudinal protrusion 2-2-2 is used to restrict the path and direction of chip insertion, and the transverse protrusion 2-2-3 is used for precise positioning of the chip between the observation window 2-1-1 on the top cover, the sample application hole 2-1-2, and the buffer addition hole 2-1-3. The observation window 2-1-1 corresponds to the T line 1-2-3-1 and the C line 1-2-3-2; the sample application well 2-1-2 corresponds to the sample pad 1-2-1; and the buffer solution addition well 2-1-3 corresponds to the connection pad 1-3.

[0049] Example 2 The LH detection process of the three-dimensional closed-loop bipolar electrochemiluminescence lateral flow chip of the present invention is as follows: First, 25-45 μL of sample solution containing LH is added to the sample pad, and the reaction is allowed to proceed for 2.5-4.5 minutes. During this time, the solution flows through the binding pad via capillary action and specifically recognizes the T-line signal antibody, forming an immune complex of T-line ECL signal antibody and LH (i.e., T-line ECL signal antibody-LH). The solution then flows to the detection pad, where the T-line ECL signal antibody-LH is captured by the T-line capture antibody, further forming a sandwich immune complex (i.e., T-line ECL signal antibody-LH-T-line capture antibody). Next, 30-50 μL of PBS was added to the sample pad to further promote the immune response and wash away any unreacted substances into the absorbent pad. This process was maintained for 3 minutes. Subsequently, 40 μL of PBS was added to the connection pad. Finally, the chip is placed in a fully automatic ECL instant detection instrument for detection. The ECL reaction is carried out under a certain driving voltage (12-18V). During the detection process, the instrument automatically excites and collects the ECL signals on the C line and T line. The LH is quantitatively detected based on the ratio of the collected signals (i.e., the T / C value). The length of the connection pad (L) is tested using a sample solution containing 1 mIU / mL LH and the three-dimensional CBPE-ECL lateral flow immunoassay chip of this invention as an example. c The influence of the sensor's emission signal value.

[0050] 1. The length of the connecting pad is to be determined, the length of the absorbent pad is 7 mm, the driving voltage is 18 V, the concentration of the capture antibody is 0.16 mg / mL, the concentration of the signal antibody is 7.4 ng / mL, the sample loading volume is 40 μL, the immune reaction time is 3.5 min, and the washing volume is 40 μL.

[0051] 2. Set up experimental groups: Select five L... c The optimal values ​​(7, 9, 11, 13, 15 mm) were selected through an optimization experiment.

[0052] Experimental results are as follows Figure 7 As shown, when L c As the T / C value increases from 7 mm to 15 mm, it first increases and then decreases; when L... cAs the T / C value increased from 11 mm to 15 mm, it maintained a stable trend, and the relative standard deviation (RSD) of the T / C value gradually decreased. Therefore, 11 mm was chosen as the optimal L. c value.

[0053] Example 3 Other important factors affecting the T / C value in Example 2 (absorbent pad length L) a Drive voltage E tot Capture antibody concentration [CAb], signal antibody concentration [SAb], and sample solution loading volume V. s Immune response time t i and flushing volume V w ) to select the best option.

[0054] (1) Preferred absorbent pad length L a 1. The LH concentration to be tested is 1 mIU / mL, the length of the connecting pad is 11 mm, the length of the absorbent pad is to be determined, the driving voltage is 18V, the T-line capture antibody concentration is 0.16 mg / mL, the signal antibody concentration is 7.4 ng / mL, the sample volume is 40 μL, the immune reaction time is 3.5 min, and the washing volume is 40 μL.

[0055] 2. Set up experimental groups: Select five L... a The optimal values ​​(7, 8, 9, 10, 11 mm) were selected through an optimization experiment.

[0056] 3. The three-dimensional CBPE-ECL lateral flow immunoassay chip detection process is as described in Example 2, and the experimental results are as follows: Figure 8 As shown.

[0057] The experimental results show that when L a When the diameter was increased from 7 mm to 8 mm, the ECL signal values ​​of both the T-line and C-line decreased. This indicates that increasing the diameter by L... a This increases the absorbency of the absorbent pad, allowing ECL signal antibodies that have not undergone specific recognition to be fully absorbed by the absorbent pad as they flow laterally, significantly reducing non-specific residues on the test pad.

[0058] With L a Increasing the diameter further to 10 mm resulted in improved ECL signal values ​​for both the T and C lines. This is because, with the increase in L... a This increases the contact between the absorber pad and the negative drive electrode, resulting in a more complete contact and a decrease in the total circuit resistance. Further, L... a Increasing the diameter from 10 mm to 11 mm did not significantly improve the T / C value. Therefore, 10 mm is selected as the optimal L. a value.

[0059] (2) Preferred driving voltage E tot 1. The LH concentration to be tested is 1 mIU / mL, the length of the cathode connection pad is 11 mm, the length of the absorption pad is 10 mm, the driving voltage is to be determined, the concentration of the capture antibody is 0.16 mg / mL, the concentration of the signal antibody is 7.4 ng / mL, the sample volume is 40 μL, the immune reaction time is 3.5 min, and the washing volume is 40 μL.

[0060] 2. Set up experimental groups: Select five E tot Optimization experiments were conducted using numerical values ​​(12, 13, 14, 15, 16, 18 V).

[0061] 3. The three-dimensional CBPE-ECL lateral flow immunoassay chip detection process is as described in Example 2, and the experimental results are as follows: Figure 9 As shown.

[0062] The experimental results show that when E tot As the voltage is gradually increased from 12 V to 18 V, the ECL signal values ​​of both the T and C lines increase accordingly. Further analysis of the T / C value variation reveals that when E... tot When the voltage is increased from 12 V to 14 V, the T / C value gradually increases, reaching its maximum at 14 V; further increasing the voltage above 14 V does not significantly change the T / C value. Therefore, 14 V is chosen as the optimal value for E. tot value.

[0063] (3) Optimal capture antibody concentration [CAb] 1. The LH concentration to be tested is 1 mIU / mL, the length of the cathode connection pad is 11 mm, the length of the absorption pad is 10 mm, the driving voltage is 14 V, the concentration of the capture antibody is to be determined, the concentration of the signal antibody is 7.4 ng / mL, the sample volume is 40 μL, the immune reaction time is 3.5 min, and the washing volume is 40 μL.

[0064] 2. Set up experimental groups: Select five [CAb] values ​​(0.11, 0.12, 0.14, 0.16, 0.18 mg / mL) for optimization experiments.

[0065] 3. The three-dimensional CBPE-ECL lateral flow immunoassay chip detection process is as described in Example 2, and the experimental results are as follows: Figure 10 As shown.

[0066] The experimental results show that within the range of 0.11-0.18 mg / mL, the ECL signal values ​​of both the T and C lines decrease as the [CAb] concentration decreases. When the [CAb] concentration is greater than 0.16 mg / mL, the T / C ratio remains relatively constant. Therefore, 0.16 mg / mL is the optimal [CAb] concentration.

[0067] (4) Optimal signal antibody concentration [SAb] 1. The LH concentration to be tested is 1 mIU / mL, the length of the cathode connection pad is 11 mm, the length of the absorption pad is 10 mm, the driving voltage is 14 V, the concentration of the capture antibody is 0.16 mg / mL, the concentration of the signal antibody is to be determined, the sample volume is 40 μL, the immune reaction time is 3.5 min, and the washing volume is 40 μL.

[0068] 2. Set up experimental groups: Select five [SAb] values ​​(8.9, 7.4, 6.3, 5.6, 4.9 ng / mL) for optimization experiments.

[0069] 3. The three-dimensional CBPE-ECL lateral flow immunoassay chip detection process is as described in Example 2, and the experimental results are as follows: Figure 11 As shown.

[0070] The experimental results show that as [SAb] increases from 4.9 ng / mL to 8.9 ng / mL, the ECL signal values ​​of both the T and C lines increase, and the T / C value also increases accordingly. When [SAb] is between 7.4 ng / mL and 8.9 ng / mL, the T / C value remains at a relatively high level and is relatively stable. Therefore, the optimal [SAb] is selected as 7.4 ng / mL.

[0071] (5) Optimal sample loading volume V s 1. The LH concentration to be tested is 1 mIU / mL, the length of the cathode connection pad is 11 mm, the length of the absorption pad is 10 mm, the driving voltage is 14 V, the concentration of the capture antibody is 0.16 mg / mL, the concentration of the signal antibody is 7.4 ng / mL, the sample volume is to be determined, the immune reaction time is 3.5 min, and the washing volume is 40 μL.

[0072] 2. Set up experimental groups: Select five V groups. s The optimal values ​​(25, 30, 35, 40, 45 μL) were used in the optimization experiment.

[0073] 3. The three-dimensional CBPE-ECL lateral flow immunoassay chip detection process is as described in Example 2, and the experimental results are as follows: Figure 12 As shown.

[0074] The experimental results show that when V s Increasing V from 25 μL to 35 μL resulted in a significant increase in the ECL signal value of the T line; further increasing V... s At 45 μL, the ECL signal value of the T line did not show a significant increase. Combined with the relatively stable ECL signal value of the C line, when V... s The T / C value increases with increasing V from 25 μL to 35 μL; further increasing V s The T / C value remained stable. Therefore, 35 μL was chosen as the optimal volume. s .

[0075] (6) Optimal immune response time t i 1. The LH concentration to be tested is 1 mIU / mL, the length of the cathode connection pad is 11 mm, the length of the absorption pad is 10 mm, the driving voltage is 14 V, the concentration of the capture antibody is 0.16 mg / mL, the concentration of the signal antibody is 7.4 ng / mL, the sample volume is 35 μL, the immune reaction time is to be determined, and the washing volume is 40 μL.

[0076] 2. Set up experimental groups: Select five t groups. i The optimal values ​​(2.5, 3.0, 3.5, 4.0, 4.5 min) were selected through an optimization experiment.

[0077] 3. The three-dimensional CBPE-ECL lateral flow immunoassay chip detection process is as described in Example 2, and the experimental results are as follows: Figure 13 As shown.

[0078] The experimental results show that as t i Extending the time from 2.5 min to 4 min, the ECL signal values ​​of both the T and C lines gradually increased; when t i After 4 minutes, the ECL signal values ​​of both the T and C lines plateaued, and the T / C ratio also remained stable. Therefore, 4 minutes was chosen as the optimal t. i .

[0079] (7) Preferred flushing volume V w 1. The LH concentration to be tested is 1 mIU / mL, the length of the cathode connection pad is 11 mm, the length of the absorption pad is 10 mm, the driving voltage is 14 V, the concentration of the capture antibody is 0.16 mg / mL, the concentration of the signal antibody is 7.4 ng / mL, the sample volume is 35 μL, the immune reaction time is 4 min, and the washing volume is to be determined.

[0080] 2. Set up experimental groups: Select five V groups. w The optimal values ​​(30, 35, 40, 45, 50 μL) were used in the optimization experiment.

[0081] 3. The three-dimensional CBPE-ECL lateral flow immunoassay chip detection process is as described in Example 2, and the experimental results are as follows: Figure 14 As shown.

[0082] The experimental results show that as V w As the volume was gradually increased from 30 μL to 50 μL, the ECL signal values ​​of both the T-line and C-line showed a decreasing trend. When V w Within the range of 30 to 40 μL, the T / C value remains relatively stable; however, as the volume increases further to above 40 μL, the T / C value gradually decreases. Therefore, 40 μL is chosen as the optimal volume. w .

[0083] Example 4 LH detection was performed using the optimized conditions explored in Example 3 and the three-dimensional CBPE-ECL lateral flow immunosensor from Example 1.

[0084] 1. The cathode connection pad is 11 mm long, the absorption pad is 10 mm long, the driving voltage is 14 V, the capture antibody concentration is 0.16 mg / mL, the signal antibody concentration is 7.4 ng / mL, the sample volume is 35 μL, the immune reaction time is 4 min, and the rinsing volume is 40 μL.

[0085] 2. Set up experimental groups: Set up several different [LH] values ​​(0.01, 0.1, 0.5, 1, 5, 10, 15 and 24 mIU / mL), and use 0 mIU / mL LH solution as a blank control.

[0086] 3. The three-dimensional CBPE-ECL lateral flow immunoassay chip detection process is as described in Example 2, and the experimental results are as follows: Figure 15 As shown.

[0087] The experimental results show that when [LH] is in the range of 0.01–24 mIU / mL, the T / C value increases with increasing [LH]. There is a certain linear relationship between the logarithm of the T / C value (denoted by Y) and the logarithm of [LH] (denoted by X), with the linear equation being Y = 0.4755X - 0.3404 (R²). 2 = 0.9964, n = 5), the limit of detection (LOD) is estimated to be 0.011 mIU / mL.

[0088] The method for calculating the detection limit is: Y L = Y b + 3S b , where Y b S represents the average T / C value in the blank control group. b The standard deviation is expressed as a blank control, using the obtained Y... LThe corresponding [LH] value is calculated as the detection limit.

[0089] Example 5 Using the optimized conditions explored in Example 3, a real sample detection experiment was conducted using the three-dimensional CBPE-ECL lateral flow immunosensor from Example 1.

[0090] 1. The cathode connection pad is 11 mm long, the absorption pad is 10 mm long, the driving voltage is 14 V, the capture antibody concentration is 0.16 mg / mL, the signal antibody concentration is 7.4 ng / mL, the sample volume is 35 μL, the immune reaction time is 4 min, and the rinsing volume is 40 μL.

[0091] 2. Set up the experimental group: The five urine samples collected were diluted 10 times, and the diluted sample solutions were tested.

[0092] 3. The three-dimensional CBPE-ECL lateral flow immunoassay chip detection process is as described in Example 2, and the experimental results are as follows: Figure 16 As shown.

[0093] The [LH] values ​​of the five diluted sample solutions obtained using the conventional ELISA method were 9.18, 0.78, 3.48, 7.66, and 1.80 mIU / mL, respectively. Plotting the ELISA results on the x-axis and the concentration values ​​measured by the sensor of this invention on the y-axis, a linear fitting analysis was performed, yielding the linear equation Y = 0.9709X – 0.3581 (R² + π / 2)². 2 = 0.9961, n = 5). The results show that the concentration values ​​obtained by this invention have a good linear correlation and consistency with the ELISA measurements, and the chip of this invention has the potential to become a reliable method for detecting LH in complex biological samples.

[0094] Example 6 Storage stability experiments were conducted using the optimized conditions explored in Example 3 and the three-dimensional CBPE-ECL lateral flow immunochip from Example 1.

[0095] 1. The cathode connection pad is 11 mm long, the absorption pad is 10 mm long, the driving voltage is 14 V, the capture antibody concentration is 0.16 mg / mL, the signal antibody concentration is 7.4 ng / mL, the sample volume is 35 μL, the immune reaction time is 4 min, and the rinsing volume is 40 μL.

[0096] 2. Set up experimental groups: The prepared three-dimensional CBPE-ECL lateral flow immunoassay chip was sealed and stored in a 45 ℃ oven and tested every 3 days.

[0097] 3. The three-dimensional CBPE-ECL lateral flow immunoassay chip detection process is as described in Example 2, and the experimental results are as follows: Figure 17 As shown.

[0098] The experimental results show that as the accelerated aging time increases, the T / C value gradually decreases after initially remaining stable. This can usually be determined using the Arrhenius equation (K=A×e). -Ea / RT The stability equivalence relationship at different temperatures was calculated. Based on the experimental results, it was estimated that the sensor accelerated at 45 °C for 9 days is equivalent to being stored for approximately 3 years under refrigeration conditions at 4 °C. Therefore, the three-dimensional CBPE-ECL lateral flow chip of this invention has good storage stability.

[0099] Example 7 A three-dimensional CBPE-ECL lateral flow immunosensor, the composition and structure of which are as follows: Figure 6 As shown, it includes a three-dimensional CBPE-ECL lateral flow immunochip and a casing; The independent, individual three-dimensional CBPE-ECL lateral flow immunoassay chip consists of a large-format three-dimensional CBPE-ECL lateral flow immunoassay chip ( Figure 18 It is prepared by cutting using an induction strip cutter (model HGS220S, Hangzhou Fenghang Technology Co., Ltd.); The large-format three-dimensional CBPE-ECL lateral flow immunoassay chip includes a large-format three-dimensional CBPE-ECL electrode sheet ( Figure 19 , Figure 20 Large-format lateral flow immunoassay strips ( Figure 21 ) and large connecting pad ( Figure 22 ); The large-format three-dimensional CBPE-ECL electrode sheet includes the front side of the large-format electrode sheet constructed on a large-format hydrophobic substrate. Figure 19 ) and the reverse side of the large electrode sheet constructed on the large hydrophobic substrate ( Figure 20 The large electrode sheet includes 20 horizontally arranged three-dimensional CBPE-ECL electrode sheets, with a 2 mm interval between them; The large-format lateral flow immunoassay strip consists of a large-format transparent adhesive pad A, a large-format sample pad B, a large-format conjugation pad C, a large-format detection pad D, and a large-format absorbent pad E. Figure 23The chip is constructed from 20 horizontally arranged independent lateral flow immunoassay strips. A large-format three-dimensional CBPE-ECL electrode sheet is bonded to the large-format lateral flow immunoassay strips via double-sided adhesive on the front side of the large-format hydrophobic substrate. The large-format connecting pad is adhered to the shared cathode and positive driving electrode on the front side of the hydrophobic substrate via double-sided adhesive, and is arranged parallel to the large-format lateral flow immunoassay strips. Through the above process, the required large-format three-dimensional CBPE-ECL lateral flow immunoassay chip is obtained for cutting with an inductive strip cutter, thereby producing independent, individual three-dimensional CBPE-ECL lateral flow immunoassay chips.

[0100] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of a three-dimensional closed-loop bipolar electrochemiluminescence lateral flow chip in the preparation of an immunoassay device, characterized in that: The three-dimensional closed-loop bipolar electrochemiluminescence lateral flow chip includes an electrode sheet, a test strip, and a connecting pad; The electrode sheet includes two sets of electrodes. One set includes a positive driving electrode, a bipolar electrode, and a negative driving electrode, which are arranged in a straight line. The other set is a positive driving electrode. The two sets of electrodes are distributed on the front and back surfaces of the hydrophobic substrate, respectively. One end of the positive driving electrode and the positive driving electrode are connected by a conductive material. The bipolar electrode includes a control anode, at least one detection anode, and a shared cathode, wherein one end of the control anode is close to the negative drive electrode. The test strip includes a sample pad, a conjugate pad, a detection pad, and an absorbent pad; the detection pad has a T line and a C line; the T line is in contact with the detection anode, and the C line is in contact with the quality control anode. The binding pad is located between the detection pad and the sample pad, and the sample pad is located between the shared cathode and the detection anode; the absorbent pad is close to the quality control anode and is in contact with a portion of the negative drive electrode; The detection pad, conjugate pad, sample pad, and absorbent pad are stacked and fixed on a transparent adhesive plate in sequence, and then inverted onto the front of the electrode sheet; The connecting pad is located between the shared cathode and the first positive driving electrode, and maintains partial contact with both the first positive driving electrode and the shared cathode.

2. The application according to claim 1, characterized in that: One end of the second positive driving electrode is connected to the first positive driving electrode via a conductive tape located on the substrate end side.

3. The application according to claim 1, characterized in that: One end of the second positive driving electrode is connected to the first positive driving electrode by drilling a hole and injecting conductive material.

4. The application according to claim 1, characterized in that: The aforementioned binding pad is used to dry and fix ECL signaling antibodies.

5. The application according to claim 1, characterized in that: The T-line package is captured by T-line antibodies.

6. The application according to claim 1, characterized in that: The C-line package is captured by the C-line antibody.

7. The application according to claim 1, characterized in that: The chip is installed in a housing to form a sensor.

8. The application according to claim 1, characterized in that: The aforementioned immune detection is used to detect multiple biomarkers.

9. The application according to claim 8, characterized in that: The biomarker is one of luteinizing hormone, cardiac troponin I, C-reactive protein, peptidin, serum amyloid protein, or Alzheimer's disease-associated neuronal line protein.

10. The application according to claim 8, characterized in that: The immune detection includes the following steps: First, the sample solution containing the biomarker to be tested is dropped onto the sample pad to initiate an immune reaction; Next, PBS was added to the sample pad, followed by PBS to the connection pad; Finally, the three-dimensional closed-loop bipolar electrochemiluminescence lateral flow sensor was placed into the detector for detection.

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