Dry-type electrochemical luminescence lateral flow immune test strip and kit

Abstract

The utility model discloses a dry-type electrochemical luminescence lateral flow immune test strip and a kit. The dry-type electrochemical luminescence lateral flow immune test strip comprises a lateral flow immune test strip, an electrode pad and a connecting pad, the lateral flow immune test strip is formed by laminating a sample pad, a combination pad, a detection pad with a backing NC membrane and an absorption pad on a transparent sticking plate, the electrode pad comprises an integrated closed bipolar electrode and a driving electrode; the integrated closed bipolar electrode comprises a closed bipolar electrode detection anode, a quality control anode and a shared cathode of the two anodes; the lateral flow immune test strip is inversely buckled on the electrode pad; and the T line and the C line on the detection pad are respectively contacted with the detection anode and the quality control anode on the electrode pad. According to the utility model, T / C is adopted to quantitatively detect biomarkers in the ECL lateral flow immunodetection for the first time, so that the problem that quality control signals are not fully utilized in the existing ECL lateral flow immunodetection is solved, and the detection stability and the storage stability are improved.

Description

Technical Field

[0001] The utility model relates to a dry type electrochemiluminescence lateral flow immunoassay strip and a kit. Background Art

[0002] The closed bipolar electrode electrochemiluminescence (ECL) cloth-based microfluidic chip is a combination of closed bipolar electrode ECL and cloth-based microfluidic chip. This kind of chip has gradually developed into a detection means for various biomarkers, but still faces some problems. For example, the chip material is generally cloth material, and the material and source are unstable; the electrodes are printed on rough cloth, with large and unstable resistance; it is necessary to construct a hydrophobic dam on the cloth material to isolate the reporting channel and the support channel, and it is necessary to press the chip with a transparent plastic sheet, which is easy to cause the reaction solution to collude, and the chip becomes an open bipolar electrode; the detection area and the quality control area are separately modified and then combined, and the function of the latter is not fully reflected. These have greatly limited the application scenarios of the closed bipolar electrode ECL cloth-based microfluidic chip.

[0003] The ECL lateral flow immunoassay strip is a combination of a lateral flow immunoassay strip and ECL. Its preparation materials are nitrocellulose membrane (NC membrane), glass fiber and absorbent paper, which combines the advantages of ECL and lateral flow immunoassay strip and has been successfully applied to various immunoassays, but still faces some problems. First, its detection pad is a backless NC membrane, which is relatively brittle and easy to break during use, which increases the processing and experimental operation difficulty and affects the accuracy and reliability of the detection results. Second, during assembly, it is necessary to first attach the electrode to the plastic bottom plate and then assemble it in turn, which increases the processing difficulty and is difficult to carry out large-scale production. Third, different detection methods are used for the test line (T line) and the quality control line (C line), and quantitative detection of T / C (the ratio of the signal value on the T line to the signal value on the C line) of biomarkers cannot be carried out. Finally, an unstable tripropylamine needs to be added as a co-reactant during detection, and the operation process is complex. These have greatly limited the application of this type of test strip.

[0004] Dry chemical analysis technology is an analysis technology based on wet chemical analysis methods. More and more detection technologies have been applied to the field of dry chemical analysis. For example, the lateral flow immunoassay strip is an analysis technology that combines certain labels and is based on the specific recognition of biomarkers and antibodies. At present, the lateral flow immunoassay strips on the market have the advantages of low price, convenient use, rapid detection, and no need for expensive instruments, and are a conventional in vitro diagnostic method. However, these lateral flow immunoassay strip technologies have the disadvantages of high background, low sensitivity, long detection time, and difficult to accurately quantitatively detect. In addition, the detection of some biomarkers (such as luteinizing hormone (LH), cardiac troponin, etc.) still cannot meet the needs of home self-testing.

[0005] In summary, a technology that is new, fast, simple, accurately detectable, and easy to mass-produce, applicable to hospital and home self-tests, and can effectively solve the respective drawbacks of existing closed bipolar electrode ECL cloth-based microfluidic chips, ECL lateral flow immunoassay strips, and lateral flow immunoassay strips has become a technical problem that technicians in this field urgently need to solve. Summary of the Invention

[0006] The purpose of the present utility model is to provide a dry-type electrochemiluminescence lateral flow immunoassay strip and a kit. The immunoassay strip of the present utility model integrates reaction reagent components on the strip, enabling dry quantitative detection of biomarkers. Its detection method is fast, convenient to operate, and highly sensitive.

[0007] The purpose of the present utility model is achieved through the following technical solutions:

[0008] A dry-type electrochemiluminescence lateral flow immunoassay strip, comprising a lateral flow immunoassay strip, an electrode pad, and a connection pad;

[0009] The lateral flow immunoassay strip is composed of a sample pad, a conjugate pad, a nitrocellulose (NC) membrane detection pad with a backing, and an absorbent pad laminated on a transparent adhesive plate; the detection pad is provided with a T line and a C line; the T line is coated with a T line capture antibody, and the C line is coated with a C line capture antibody; the conjugate pad is dried and fixed with an ECL signal antibody;

[0010] The electrode pad is formed by a conductive material on a hydrophobic bottom plate; the electrode pad includes an integrated closed bipolar electrode and a driving electrode; the integrated closed bipolar electrode includes a closed bipolar electrode detection anode and a quality control anode, as well as a shared cathode for the two anodes; the driving electrode includes a negative driving electrode corresponding to the anode and a positive driving electrode corresponding to the shared cathode; between the two anodes and the corresponding negative driving electrodes, and between the shared cathode and the corresponding positive driving electrodes, they are naturally separated by the hydrophobic bottom plate;

[0011] Double-sided tape is provided between the detection anode, the shared cathode, and the driving electrode of the electrode pad to fix part of the absorbent pad and part of the detection pad, and double-sided tape is provided on one side of the quality control anode and the shared cathode to fix part of the sample pad and part of the conjugate pad;

[0012] The lateral flow immunoassay strip is buckled on the electrode pad; the NC membrane of the detection pad faces down and the backing face faces up; the T line and the C line on the detection pad are respectively in contact with the detection anode and the quality control anode on the electrode pad; one end of the conjugate pad close to the quality control anode (far from the detection anode) is in contact with the non-electrode area of the electrode pad; the sample pad is close to the conjugate pad (far from the quality control anode) and is in contact with the non-electrode area of the electrode pad; one end of the absorbent pad close to the detection anode is in contact with the negative driving electrode corresponding to the detection anode;

[0013] The connection pad is located above the hydrophobic bottom plate between the shared cathode and between the shared cathode and the positive driving electrode, and is in partial contact with the positive driving electrode. This design can prevent the reaction solution from colluding to form an open bipolar electrode;

[0014] The sample pad and the conjugate pad are made of glass fiber, the absorbent pad and the connection pad are made of absorbent paper; the electrode pad is constructed on a hydrophobic PET plastic plate by screen printing with conductive carbon ink; the transparent adhesive plate is a transparent PET plastic plate;

[0015] The preparation process of the conjugate pad: The glass fiber is first treated with the conjugate pad treatment solution, then the ECL signal antibody mixed solution (6 μL) is evenly sprayed on it, and then baked in an oven at 37 °C for 0.5 - 2 h to obtain the conjugate pad;

[0016] The preparation process of the sample pad: The glass fiber is first treated with the sample pad treatment solution, and then baked in an oven at 37 °C for 1 - 2 h to obtain the sample pad;

[0017] The preparation process of the detection pad: The T-line capture antibody solution and the C-line capture antibody solution are used to modify and fix the capture antibody on the same NC membrane by a gold-spraying and membrane-drawing instrument, and then baked in an oven at 37 °C for 1 - 3 h to obtain the detection pad;

[0018] The absorbent pad and the connection pad are both prepared by cutting absorbent paper with a laser cutting machine;

[0019] The transparent adhesive plate is prepared by cutting a transparent PET plastic plate with a laser cutting machine;

[0020] The conjugate pad treatment solution and the sample pad treatment solution are both phosphate buffer solution (PBS) including 0.05% polyvinylpyrrolidone (PVP), 0.025% casein, 0.1% - 1% Tetronic1307 (abbreviation S9), preferably 0.25% S9;

[0021] The T-line capture antibody solution includes PBS (pH value is 6.0 - 10.0) of 15 mg / mL sucrose, 0.1% S9 and 0.1 - 1 mg / mL T-line capture antibody, preferably the pH value is 8.0 and the concentration of the T-line capture antibody is 0.5 mg / mL;

[0022] The C-line capture antibody solution includes PBS (pH value is 6.0 - 10.0) of 5 mg / mL sucrose, 5 mg / mL trehalose, 0.1% S9, 0.25 mg / mL C-line capture antibody (such as mouse IgG), preferably the pH value is 7.0.

[0023] The ECL signal antibody mixed solution is formed by mixing a solution of an intramolecular self-enhanced ECL signal antibody conjugated with a C-line labeled antibody (i.e., the C-line ECL signal antibody) and a solution of an intramolecular self-enhanced ECL signal antibody conjugated with a T-line labeled antibody (i.e., the T-line ECL signal antibody);

[0024] The preparation process of the T-line ECL signal antibody (or C-line ECL signal antibody) solution is as follows:

[0025] (1) The preparation process of the ruthenium terpyridine derivative (Ru(II)) is similar to the prior art (Patent Application No. 202310708705.8).

[0026] (2) The preparation process of the complex of Ru(II) and L-lysine (Ru(II)-L-Lys) is as follows:

[0027] Weigh 20 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and 0.5 mg of N-hydroxysuccinimide (NHS) into 500 μL of PBS; then weigh 3.5 mg of Ru(II) and add it to the above solution and mix well by shaking; then activate the carboxyl group of Ru(II) in a constant temperature shaker at 25 °C for 30 min to obtain an intermediate product (Ru(II)-NHS);

[0028] Weigh 1 mg of L-Lys and dissolve it in 500 μL of PBS; then add 500 μL of the Ru(II)-NHS synthesized in the above step to this solution and mix well by shaking; then place it in a constant temperature shaker at 25 °C for 8 h to complete the amide linkage; finally, use a Nanosep ultrafiltration centrifugal tube (1K MWCO, 8000 rpm, 5 min) to purify the solution after amide linkage to obtain a purified product (Ru(II)-L-Lys).

[0029] (3) The preparation process of the T-line ECL signal antibody (or C-line ECL signal antibody) solution

[0030] First, weigh 20 mg of EDC and 0.5 mg of NHS and add them to 500 μL of the Ru(II)-L-Lys solution, and activate the carboxyl group of Ru(II)-L-Lys in a constant temperature shaker at 25 °C for 30 min;

[0031] Next, add the T-line labeled antibody (or C-line labeled antibody) to the activated Ru(II)-L-Lys solution to make its concentration 0.2 mg / mL, and incubate it in a constant temperature shaker at 25 °C for 10 h to covalently conjugate Ru(II)-L-Lys with the T-line labeled antibody (or C-line labeled antibody);

[0032] Finally, use a Nanosep ultrafiltration centrifuge tube (3K MWCO, 8000 rpm, 5 min) to purify the solution after the above-mentioned covalent coupling reaction to obtain a solution of the T-line ECL signal antibody (or the C-line ECL signal antibody).

[0033] A dry-type electrochemiluminescence lateral flow immunoassay kit contains the above-mentioned dry-type electrochemiluminescence lateral flow immunoassay strip.

[0034] The application of the above-mentioned dry-type ECL lateral flow immunoassay strip in immunoassay includes the following steps:

[0035] First, drop 20 - 40 μL of a sample solution containing the biomarker to be detected onto the sample pad of the test strip, and wait for an immunoassay reaction time of 1.5 - 9.5 min. The solution flows through the conjugate pad and forms a complex with the T-line ECL signal antibody (T-line ECL signal antibody - biomarker). Subsequently, this immunocomplex continues to flow to the detection pad and binds to the T-line capture antibody on it, forming a "T-line ECL signal antibody - biomarker - T-line capture antibody" immunological sandwich complex. Preferably, use 35 μL of a sample solution containing the biomarker to be detected and an immunoassay reaction time of 3.5 min.

[0036] Next, drop 25 - 50 μL of PBS onto the sample pad to wash away the excess residues, and the washing time is 3 min. Subsequently, drop 15 - 40 μL of PBS onto the conjugate pad.

[0037] Finally, place the dry-type electrochemiluminescence lateral flow immunoassay strip into an ECL instant detector (such as that disclosed in application number: 2024210642259) for detection. Perform an ECL trigger reaction under a certain camera exposure time (50 - 450 ms), camera white balance (2800 - 3200 K), and driving voltage (9 - 13 V). During the reaction process, the ECL signals on the T-line and C-line are automatically collected by the camera in the detector, and the ratio T / C of the collected signal values is used to quantitatively detect the biomarker. Preferably, the camera exposure time is 166 ms, the camera white balance is 2900 K, and the driving voltage is 11 V.

[0038] The biomarker is one of LH, cardiac troponin I, or C-reactive protein.

[0039] The present utility model has the following advantages and effects compared with the prior art:

[0040] 1. This patent invented a dry-type electrochemiluminescence lateral flow immunoassay strip. By integrating the reaction reagent components on the test strip, it overcomes the disadvantages of the existing closed bipolar electrode ECL immunoassay chip and ECL lateral flow immunoassay strip, which are cumbersome to prepare and operate.

[0041] 2. The test strip of the present utility model uses NC membrane, glass fiber and absorbent paper as materials, which are materials with mature manufacturing processes in the market. The use of these materials further improves the practicability and universality of the test strip of the present utility model.

[0042] 3. The present utility model first uses a back-lined NC membrane to prepare an ECL lateral flow immunoassay test strip, solving a series of problems encountered in the use of non-back-lined NC membranes in existing ECL lateral flow immunoassay test strips (such as being fragile, difficult to process, difficult to perform experimental operations and mass production).

[0043] 4. The electrodes of the present utility model are printed on a hydrophobic PET plastic plate, which not only avoids the disadvantages of uneven paper-based electrodes and cloth-based electrodes, but also simplifies the assembly of the ECL lateral flow immunoassay test strip.

[0044] 5. The lateral flow immunoassay test strip of the present utility model is inverted on the electrode pad, eliminating the need for a bottom plate to assemble the test strip, simplifying the chelation of the test strip and the electrode pad, and solving the problems of poor bonding when using a PVC plastic plate to press-fit in a closed bipolar electrode ECL cloth-based microfluidic chip and easy leakage of the reaction solution during detection.

[0045] 6. The electrode pad of the present utility model ingeniously solves the problem of easy leakage of the reaction solution between the reporting channel and the supporting channel in the existing closed bipolar electrode ECL cloth-based microfluidic chip.

[0046] 7. The present utility model uses an intramolecular self-enhanced ECL signal antibody. Compared with existing ECL lateral flow immunoassay test strips, there is no need to add a co-reactant reagent again, avoiding the influence of the microenvironment, making the ECL detection more accurate and the operation simpler.

[0047] 8. The present utility model first applies the Ru(II)-L-Lys luminophore to the detection of dry-type electrochemiluminescence lateral flow immunoassay test strips.

[0048] 9. The present utility model first uses T / C to quantitatively detect biomarkers in ECL lateral flow immunoassay, solving the problem of insufficient utilization of quality control signals in existing ECL lateral flow immunoassay, and improving the detection stability and storage stability.

[0049] 10. It takes about 6.5 minutes from sample addition to the completion of detection in the present utility model, enabling rapid quantitative detection.

[0050] 11. The test strip of the present utility model is easy to operate. Only by dropping the sample solution to be tested and the buffer solution can it be put into the detector for analysis, without the need for professional operation, and is suitable for on-site point-of-care testing.

[0051] 12. The test strip of the present utility model is environmentally friendly, low in cost, and can be batch-prepared using traditional test strip production processes. Description of the Drawings

[0052] Figure 1 This is the overall structural schematic diagram of the dry electrochemical luminescence lateral flow immunoassay strip of the present utility model;

[0053] Among them, 1 - lateral flow immunoassay strip, 2 - electrode pad, 3 - connection pad.

[0054] Figure 2 This is the structural schematic diagram of the lateral flow immunoassay strip of the present utility model;

[0055] Among them, 1-1 - sample pad, 1-2 - conjugate pad, 1-3 - detection pad, 1-3-1 - front side of NC membrane, 1-3-2 - back side, 1-3-3 - T line, 1-3-4 - C line, 1-4 - absorbent pad, 1-5 - transparent adhesive plate.

[0056] Figure 3 This is the schematic diagram of the composition structure of the electrode pad of the present utility model;

[0057] Among them, 2-1 - hydrophobic bottom plate, 2-2 - integrated closed bipolar electrode, 2-2-1 - detection anode, 2-2-2 - quality control anode, 2-2-3 - shared cathode, 2-3 - driving electrode, 2-3-1 - negative driving electrode, 2-3-2 - positive driving electrode.

[0058] Figure 4 This is the exploded structural schematic diagram of the dry electrochemical luminescence lateral flow immunoassay strip of the present utility model;

[0059] Figure 5 This is the relationship diagram of T / C with different types of C-line antibodies.

[0060] Figure 6 This is the relationship diagram of T / C with C-line antibodies of the same type from different manufacturers.

[0061] Figure 7 This is the relationship diagram of T / C with different types of surfactants in the conjugate pad treatment solution.

[0062] Figure 8 This is the relationship diagram of T / C with different concentrations of surfactants in the conjugate pad treatment solution.

[0063] Figure 9 This is the relationship diagram of T / C with the buffer system of the sample pad treatment solution.

[0064] Figure 10 This is the relationship diagram of T / C with different types of surfactants in the sample pad treatment solution.

[0065] Figure 11 This is the relationship diagram of T / C with different concentrations of surfactants in the sample pad treatment solution.

[0066] Figure 12 It is a relationship diagram between T / C and the types of NC membranes.

[0067] Figure 13 It is a relationship diagram between T / C and the pH value of PBS of the T-line capture antibody.

[0068] Figure 14 It is a relationship diagram between T / C and the pH value of PBS of the C-line capture antibody.

[0069] Figure 15 It is a relationship diagram between T / C and the concentration of the T-line capture antibody.

[0070] Figure 16 It is a relationship diagram between T / C and the camera white balance.

[0071] Figure 17 It is a relationship diagram between T / C and the camera exposure time.

[0072] Figure 18 It is a relationship diagram between T / C and the driving voltage.

[0073] Figure 19 It is a relationship diagram between T / C and the immune reaction time.

[0074] Figure 20 It is a relationship diagram between T / C and the sample loading volume.

[0075] Figure 21 It is a relationship diagram between T / C and the sample dilution ratio.

[0076] Figure 22 It is an analysis curve graph for detecting LH at different concentrations (the inset is a data linear fitting curve graph).

[0077] Figure 23 It is an evaluation of the storage stability when detecting LH. Specific implementation mode

[0078] The present utility model will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the implementation modes of the present utility model are not limited thereto.

[0079] Embodiment 1

[0080] A dry-type electrochemiluminescence lateral flow immunoassay strip, whose composition and structure are as Figures 1 to 4 shown, including a lateral flow immunoassay strip 1, an electrode pad 2, and a connection pad 3;

[0081] The lateral flow immunoassay strip 1 is composed of a sample pad 1-1, a conjugate pad 1-2, a detection pad 1-3, and an absorbent pad 1-4 laminated on a transparent adhesive plate 1-5; the detection pad 1-3 is provided with a T line 1-3-3 and a C line 1-3-4; the T line 1-3-3 is coated with a T line capture antibody, and the C line 1-3-4 is coated with a C line capture antibody; the conjugate pad 1-2 is dried and fixed with an ECL signal antibody;

[0082] The electrode pad 2 is formed by a conductive material on a hydrophobic bottom plate 2-1; the electrode pad 2 includes an integrated closed bipolar electrode 2-2 and a driving electrode 2-3; the integrated closed bipolar electrode 2-2 includes a closed bipolar electrode detection anode 2-2-1 and a quality control anode 2-2-2 and a shared cathode 2-2-3 of the two anodes; the driving electrode 2-3 includes a negative driving electrode 2-3-1 corresponding to the anodes 2-2-1 and 2-2-2 and a positive driving electrode 2-3-2 corresponding to the shared cathode 2-2-3; between the two anodes 2-2-1 and 2-2-2 and the corresponding negative driving electrode 2-3-1 and between the shared cathode 2-2-3 and the corresponding positive driving electrode 2-3-2 are naturally separated by the hydrophobic bottom plate 2-1;

[0083] The electrode pad 2 is provided with double-sided tapes 2-4 between the detection anode 2-2-1, the shared cathode 2-2-3 and the driving electrode 2-3 for fixing a part of the absorbent pad 1-4 and a part of the detection pad 1-3, and double-sided tapes 2-5 are provided on one side of the quality control anode 2-2-2 and the shared cathode 2-2-3 for fixing a part of the sample pad 1-1 and a part of the conjugate pad 1-2;

[0084] The lateral flow immunoassay strip 1 is inverted on the electrode pad 2; the front side 1-3-1 of the NC membrane of the detection pad 1-3 faces downwards and the back side 1-3-2 faces upwards; the T line 1-3-3 and the C line 1-3-4 on the detection pad 1-3 are respectively in contact with the detection anode 2-2-1 and the quality control anode 2-2-2 on the electrode pad 2; the end of the conjugate pad 1-2 close to the quality control anode 2-2-2 (away from the detection anode 2-2-1) is in contact with the non-electrode area of the electrode pad 2; the sample pad 1-1 is close to the conjugate pad 1-2 (away from the quality control anode 2-2-2) and is in contact with the non-electrode area of the electrode pad 2; the end of the absorbent pad 1-4 close to the detection anode 2-2-1 is in contact with the negative driving electrode 2-3-1 corresponding to the detection anode 2-2-1;

[0085] The connection pad 3 is located on the hydrophobic bottom plate 2-1 between the shared cathode 2-2-3, the shared cathode 2-2-3 and the positive driving electrode 2-3-2, and is in partial contact with the positive driving electrode 2-3-2. Such a design can prevent the reaction solution from colluding to form an open bipolar electrode;

[0086] The sample pad 1-1 and the conjugate pad 1-2 are made of glass fiber, the absorbent pad 1-4 and the connection pad 3 are made of absorbent paper; the conductive material of the electrode pad 2 is conductive carbon ink, and the hydrophobic bottom plate 2-1 for constructing the electrode is a hydrophobic PET plastic plate; the transparent adhesive plate 1-5 is a transparent PET plastic plate.

[0087] Example 2

[0088] Application of the dry-type electrochemiluminescence lateral flow immunoassay strip of the present invention in detecting LH, the detection process is as follows:

[0089] First, 30 μL of a sample solution containing LH is dropped onto the sample pad 1-1 of the test strip, and after waiting for 3.5 min for the immunoreaction time, the solution flows through the conjugate pad 1-2 to form a complex with the T-line ECL signal antibody (T-line ECL signal antibody-LH). Subsequently, this immune complex continues to flow to the detection pad 1-3 and binds to the T-line capture antibody thereon to form a "T-line ECL signal antibody-LH-T-line capture antibody" immune sandwich complex;

[0090] Next, 30 μL of PBS is dropped onto the sample pad 1-1 to wash away the excess residues, and the washing time is 3 min; subsequently, 20 μL of PBS is dropped onto the connection pad 3;

[0091] Finally, the dry-type ECL lateral flow immunoassay strip is placed in a self-built ECL instant detector (application number: 2024210642259) for detection. An ECL trigger reaction is carried out under certain camera exposure time (166 ms), camera white balance (2800 K), and driving voltage (11 V). During the reaction process, the ECL signals on the T-line 1-3-3 and the C-line 1-3-4 are automatically collected by the camera in the detector, and the ratio T / C of the collected signal values is used to quantitatively detect LH;

[0092] Now, taking a test sample solution containing 1 mIU / mL LH and the test strip of the present invention as an example, the relationship between the types of C-line antibody pairs (i.e., the C-line capture antibody and the corresponding C-line labeled antibody form the C-line antibody pair) and T / C in the dry-type electrochemiluminescence lateral flow immunoassay strip is tested.

[0093] Set up experimental groups: Select five C-line antibody pairs (goat anti-chicken IgY-chicken IgY (GC), goat anti-mouse IgG-mouse IgG (GM), goat anti-rabbit IgG-rabbit IgG (GR), rabbit anti-mouse IgG-mouse IgG (RM), and rabbit anti-goat IgG-goat IgG (RG)) for optimization experiments.

[0094] The test results are as Figure 5As shown, it can be seen that the ECL signals and T / C stabilities obtained by GC, GM, and RG are not good, which may be due to the poor immunoreactivity of these C-line antibody pairs in the ECL detection system. The detection results of RM and GR are good, but RM is better in terms of stability, and its ECL signal and T / C on T line 1-3-3 are higher than those of GR. Therefore, RM is preferably selected in this study.

[0095] Example 3

[0096] Optimize the important factors affecting T / C in Example 2 (C-line antibody pairs of the same type from different manufacturers, different types and concentrations of surfactants in the binding pad 1-2 treatment solution, sample pad 1-1 treatment solution buffer system, different types and concentrations of surfactants in the sample pad 1-1 treatment solution, NC membrane type, pH value of PBS for T-line and C-line capture antibodies, T-line capture antibody concentration, camera white balance, camera exposure time, driving voltage, immunoreaction time, sample addition volume, sample dilution ratio):

[0097] (1) Optimize C-line antibody pairs of the same type from different manufacturers

[0098] 1. The concentration of LH to be measured is 1 mIU / mL, the camera white balance is 2800K, the camera exposure time is 166 ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6 μL, the concentrations of T-line and C-line capture antibodies are both 0.25 mg / mL, and the immunoreaction time is 3.5 min, and the sample addition volume is 30 μL.

[0099] 2. Set up experimental groups: Select C-line antibody pairs (RM) from five manufacturers (RM1 from Hangzhou Qitai Biotechnology Co., Ltd., RM2 from Beijing Solarbio Science & Technology Co., Ltd., RM3 from Luoyang Bai'ao Tong Experimental Materials Center, RM4 from Jiaozuo Yunzhiyu Biotechnology Co., Ltd., RM5 from Nanjing XinFan Biotechnology Co., Ltd.).

[0100] 3. The detection process of the dry-type electrochemiluminescence lateral flow immunoassay strip is the same as that in Example 2, and the test results are as Figure 6 shown.

[0101] It can be seen from the experimental results that the ECL signals and T / C stabilities of RM2, RM3, and RM5 are not good, which may be due to the poor compatibility of the antibody production process and storage conditions of these manufacturers with the ECL detection system. In addition, the ECL signals of the T-line and C-line of RM4 are relatively stable, but its T / C stability is poor. In contrast, the antibody pair RM1 has good stability in both ECL signal and T / C, and the T / C is relatively high. Therefore, the RM1 antibody pair is preferably selected.

[0102] (2) Optimize different types of surfactants in the binding pad 1-2 treatment solution

[0103] 1. The LH concentration to be measured is 1 mIU / mL and 5 mIU / mL, the camera white balance is 2800K, the camera exposure time is 166 ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6 μL, the capture antibody concentrations of both the T line and the C line are 0.25 mg / mL, the immunoreaction time is 3.5 min, and the sample loading volume is 30 μL.

[0104] 2. Set up the experimental group: Select five surfactants (S9, S14, S16, S19, and S21) for the optimization experiment.

[0105] 3. The detection process of the dry electrochemical luminescence lateral flow immunoassay strip is the same as that in Example 2, and the test results are as Figure 7 shown.

[0106] It can be seen from the experimental results that for the conjugate pads 1-2 treated with S9, S14, and S16 and the control group (Control), both the ECL signal and T / C show relatively large standard deviations, indicating poor stability. The possible reason for this phenomenon is that neither the conjugate pads 1-2 treated with these surfactants nor those untreated can well redissolve and release the ECL signal antibody, which affects the binding of the biomarker to the ECL signal antibody, and further affects the formation of "T line ECL signal antibody - LH - T line capture antibody" on the detection pad 1-3, resulting in unstable detection results. In contrast, the detection results shown by the conjugate pads 1-2 treated with S19 and S21 are more stable. Especially when the LH concentration is 1 mIU / mL, the T / C of S19 is higher than that of S21, while at 5 mIU / mL, the difference between the two is not significant. Therefore, S19 is preferred in this study.

[0107] (3) Optimize the surfactant concentration in the conjugate pad 1-2 treatment solution

[0108] 1. The LH concentration to be measured is 1 mIU / mL and 5 mIU / mL, the camera white balance is 2800K, the camera exposure time is 166 ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6 μL, the capture antibody concentrations of both the T line and the C line are 0.25 mg / mL, the immunoreaction time is 3.5 min, and the sample loading volume is 30 μL.

[0109] 2. Set up the experimental group: Set different concentrations of S19 (0, 0.1%, 0.25%, 0.5%, 0.75%, and 1%) for the optimization experiment.

[0110] 3. The detection process of the dry electrochemical luminescence lateral flow immunoassay strip is the same as that in Example 2, and the test results are as Figure 8 shown.

[0111] It can be seen from the experimental results that when the S19 concentration in the treatment solution of the conjugate pad 1-2 reaches 0.5%, 0.75% and 1%, and the LH concentration is 1 mIU / mL and 5 mIU / mL, in 7 repeated detections, there will be at least 1 time when the luminescence is almost not observed, and both the ECL signal and T / C are low, with extremely poor stability. The possible reason for this phenomenon is that the conjugate pad 1-2 is treated with S19 at too high a concentration, which will cause the hindrance of electric conduction and prevent the normal generation of the ECL signal. In contrast, when the S19 concentration is 0, 0.1% and 0.25%, the ECL signal can be generated normally, but when the S19 concentration is 0.25%, it shows more excellent stability. The possible reason for this phenomenon is that when the S19 concentration is 0 and 0.1%, the concentration is too low to effectively promote the re-dissolution and release of the ECL signal antibody and the binding of the biomarker to the ECL signal antibody, thus affecting the stability. Therefore, the preferred concentration of S19 in the treatment solution of the conjugate pad 1-2 is 0.25%.

[0112] (4) Optimize the buffer system of the sample pad 1-1 treatment solution

[0113] 1. The concentration of the LH to be measured is 1 mIU / mL and 5 mIU / mL, the white balance of the camera is 2800K, the exposure time of the camera is 166 ms, the driving voltage is 11V, the mixed solution of the ECL signal antibody is 6 μL, the capture antibody concentrations of the T line and C line are both 0.25 mg / mL, the immunoreaction time is 3.5 min, and the sample loading volume is 30 μL.

[0114] 2. Set up the experimental groups: Select five buffer systems (disodium hydrogen phosphate-citric acid buffer (CASDP), PBS, Tris-HCl, glycine-NaOH buffer (GSH), borax-NaOH (BSH)) for the optimization experiment.

[0115] 3. The detection process of the dry type electrochemiluminescence lateral flow immunoassay strip is the same as that in Example 2, and the test results are as Figure 9 shown.

[0116] It can be seen from the experimental results that when the LH concentration is 1 mIU / mL and 5 mIU / mL, the stabilities of the corresponding ECL signals and T / C obtained with the CASDP, Tris-HCl, GSH and BSH buffers are not good, probably because the ionic environments and pH values of these four buffers are not conducive to the immunoreaction of the biomarker and the antibody or the generation of the ECL signal. Relatively speaking, when the buffer system is PBS, the generated ECL signals and T / C both show excellent stability. Therefore, this study preferably selects PBS as the buffer system for the sample pad 1-1 treatment solution.

[0117] (5) Optimize the type of surfactant in the sample pad 1-1 treatment solution

[0118] 1. The LH concentration to be measured is 1 mIU / mL and 5 mIU / mL, the camera white balance is 2800K, the camera exposure time is 166 ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6 μL, the capture antibody concentrations of both the T line and the C line are 0.25 mg / mL, the immunoreaction time is 3.5 min, and the sample loading volume is 30 μL.

[0119] 2. Set up the experimental group: Select five surfactants (S9, S14, S16, S19, and S21) for the optimization experiment.

[0120] 3. The detection process of the dry-type electrochemiluminescence lateral flow immunoassay strip is the same as that in Example 2, and the test results are as Figure 10 shown.

[0121] It can be seen from the experimental results that when the sample pad 1-1 is treated with S14, S16, and S21, as well as in the control group (Control), the generated ECL signal and the T / C stability are not good. The possible reason is that the sample pad 1-1 treated with these surfactants cannot well redissolve and release the ECL signal antibody, which affects the binding of the biomarker to the ECL signal antibody, and does not sufficiently block the redundant sites on the glass fiber and the NC membrane, thus resulting in poor stability of the detection results. In contrast, the sample pad 1-1 treated with S9 and S19 shows good stability, and when LH is 1 mIU / mL and 5 mIU / mL, the ECL signals are not very different, but the T / C of S19 is higher than that of S9. Therefore, in this study, S19 is preferably used to treat the sample pad 1-1.

[0122] (6) Optimize the surfactant concentration in the treatment solution of the sample pad 1-1

[0123] 1. The LH concentration to be measured is 1 mIU / mL and 5 mIU / mL, the camera white balance is 2800K, the camera exposure time is 166 ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6 μL, the capture antibody concentrations of both the T line and the C line are 0.25 mg / mL, the immunoreaction time is 3.5 min, and the sample loading volume is 30 μL.

[0124] 2. Set up the experimental group: Set different concentrations of S19 (0, 0.1%, 0.25%, 0.5%, 0.75%, and 1%) for the optimization experiment.

[0125] 3. The detection process of the dry-type electrochemiluminescence lateral flow immunoassay strip is the same as that in Example 2, and the test results are as Figure 11 shown.

[0126] It can be seen from the experimental results that when the concentration of S19 reaches 0.5%, 0.75% and 1%, and the concentration of LH is 1 mIU / mL and 5 mIU / mL, the ECL signals and T / C on the T line and C line are both low and the stability is extremely poor. The results of 7 repeated detections show that there is a probability of inhibiting the generation of ECL signals, which in turn leads to poor stability. The possible reason for this phenomenon is that treating the sample pad 1-1 with too high a concentration of S19 will cause electrical conduction obstruction and hinder the normal generation of ECL signals. In addition, when the concentration of S19 is 0, the stability is also not good, probably because the concentration of the surfactant is too low, resulting in poor blocking effect of the excess sites on the glass fiber and NC membrane or affecting the immune binding of the biomarker and the ECL signal antibody. In contrast, when the concentration of S19 is 0.1% and 0.25%, the ECL signal and T / C stability are excellent, but the T / C of 0.25% is higher than that of 0.1%. Therefore, it is preferred that the concentration of S19 in the treatment solution of the sample pad 1-1 is 0.25%.

[0127] (7) Preferred type of NC membrane for the detection pad 1-3

[0128] 1. The concentration of LH to be measured is 1 mIU / mL, the white balance of the camera is 2800K, the exposure time of the camera is 166 ms, the driving voltage is 11V, the mixed solution of the ECL signal antibody is 6 μL, the capture antibody concentration of the T line and C line is 0.25 mg / mL, the immunoreaction time is 3.5 min, and the sample loading volume is 30 μL.

[0129] 2. Set up the experimental group: Select five types of NC membranes (HPM140 (Shenzhen Shenwei Industry Co., Ltd.), CN95 (Sartorius), CN140 (Sartorius), 10547149 (Whatman), CN120 (Hangzhou Blues Trading Co., Ltd.)) for the optimization experiment.

[0130] 3. The detection process of the dry-type electrochemiluminescence lateral flow immunoassay strip is the same as that in Example 2, and the test results are as Figure 12 shown.

[0131] It can be seen from the experimental results that both the ECL signal and the T / C stability based on the NC membrane of the CN95 model are very poor. The possible reason for this phenomenon is that the pore size of the NC membrane of this model is large and the flow rate is too fast, resulting in insufficient time for the formation of "T-line ECL signal antibody - biomarker - T-line capture antibody", thus causing a low ECL signal and poor stability, and further making the T / C stability very poor. The ECL signal and T / C based on the NC membranes of the HPM140 and CN120 models are relatively high, but the stability is not good. The possible reason for this phenomenon is that the flow rate of the sample solution on the NC membrane is relatively slow, allowing sufficient time for the reaction of "T-line ECL signal antibody - biomarker - T-line capture antibody". However, it may be due to the unstable production process during the manufacturer's production of the NC membrane, resulting in unstable quality of the produced NC membrane, and further making the detection results unstable. In contrast, the ECL signal and T / C based on the NC membranes of the CN140 and 10547149 models are relatively high and the stability is excellent. The possible reason for this phenomenon is that the flow rate of the sample solution on the NC membrane is relatively slow, allowing sufficient time for the formation of "T-line ECL signal antibody - biomarker - T-line capture antibody", and the production process of the NC membrane by the manufacturer is stable, resulting in stable quality of the produced NC membrane. Given that the ECL signal and T / C of the CN140 model NC membrane are both higher than those of the 10547149 model, this study finally preferably selects the CN140 model NC membrane purchased from Sartorius as the material for detection pads 1 - 3.

[0132] (8) Optimize the pH value of the PBS for the T-line capture antibody

[0133] 1. The concentration of the LH to be measured is 1 mIU / mL, the camera white balance is 2800K, the camera exposure time is 166 ms, the driving voltage is 11V, the volume of the ECL signal antibody mixed solution is 6 μL, the concentration of the T-line and C-line capture antibodies is 0.25 mg / mL, the immunoreaction time is 3.5 min, and the sample loading volume is 30 μL.

[0134] 2. Set up the experimental groups: Set up PBS with different pH values (6.0, 7.0, 8.0, 9.0, and 10.0) for the optimization experiment.

[0135] 3. The detection process of the dry electrochemical luminescence lateral flow immunoassay strip is the same as that in Example 2, and the test results are as Figure 13 shown.

[0136] It can be seen from the experimental results that when the pH value is 8.0, the T / C is significantly higher than those at 6.0, 7.0, 9.0, and 10.0, and the standard deviation is the smallest, indicating the best stability. This may be because when the pH value is 8.0, it is close to the isoelectric point of the T-line capture antibody, making the antibody in a state of easy precipitation. The actual immobilization amount, immobilization firmness, and activity on the NC membrane are relatively good, so the detection results are better. Therefore, in this study, the pH value of the PBS for the T-line capture antibody is preferably 8.0.

[0137] (9) Optimize the pH value of the PBS for the C-line capture antibody

[0138] 1. The concentration of the LH to be measured is 1 mIU / mL, the camera white balance is 2800K, the camera exposure time is 166 ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6 μL, the concentrations of the T-line and C-line capture antibodies are 0.25 mg / mL, the immunoreaction time is 3.5 min, and the sample loading volume is 30 μL.

[0139] 2. Set up experimental groups: Set up PBS with different pH values (6.0, 7.0, 8.0, 9.0, and 10.0).

[0140] 3. The detection process of the dry electrochemical luminescence lateral flow immunoassay strip is the same as that in Example 2, and the test results are as Figure 14 shown.

[0141] It can be seen from the experimental results that when the pH values are 8.0, 9.0, and 10.0, the ECL signal and the stability of T / C are poor. This may be because when the pH value is greater than 8.0, the buffer environment is not conducive to the combination of the C-line capture antibody and the binding sites on the NC membrane, thereby affecting the result stability. When the pH values are 6.0 and 7.0, the ECL signal and the stability of T / C are excellent. However, the T / C at pH 7.0 is significantly higher. This may be because when the pH value is 7.0, it is close to the isoelectric point of the C-line capture antibody, making the antibody in a state of easy precipitation. The actual immobilization amount, immobilization firmness, and activity on the NC membrane are the best. Therefore, in this study, the pH value of the PBS for the C-line capture antibody is preferably 7.0.

[0142] (10) Optimize the concentration of the T-line capture antibody

[0143] 1. The concentration of the LH to be measured is 1 mIU / mL, the camera white balance is 2800K, the camera exposure time is 166 ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6 μL, the concentration of the T-line capture antibody is to be determined, the concentration of the C-line capture antibody is 0.25 mg / mL, the immunoreaction time is 3.5 min, and the sample loading volume is 30 μL.

[0144] 2. Set up the experimental group: Perform optimization experiments with different concentrations of the T-line capture antibody (0.1, 0.25, 0.5, 0.75, and 1 mg / mL).

[0145] 3. The detection process of the dry-type electrochemiluminescence lateral flow immunoassay strip is the same as that in Example 2, and the test results are as Figure 15 shown.

[0146] It can be seen from the experimental results that when the concentration of the T-line capture antibody is lower than 0.5 mg / mL, T / C gradually increases with the increase in the concentration of the T-line capture antibody. However, when the concentration of the T-line capture antibody is higher than 0.5 mg / mL, T / C gradually decreases with the increase in the concentration of the T-line capture antibody. The possible reason for this phenomenon is that the excessive T-line capture antibody causes the ECL signal antibody to be retained in the C-line area, increasing the C-line ECL signal, and thus leading to a decrease in T / C. Based on this study, the preferred concentration of the T-line capture antibody is 0.5 mg / mL.

[0147] (11) Optimize the camera white balance

[0148] 1. The concentration of LH to be measured is 1 mIU / mL, the camera white balance is to be determined, the camera exposure time is 166 ms, the driving voltage is 11 V, the ECL signal antibody mixed solution is 6 μL, the concentration of the T-line capture antibody is 0.5 mg / mL, the concentration of the C-line capture antibody is 0.25 mg / mL, the immunoreaction time is 3.5 min, and the sample loading volume is 30 μL.

[0149] 2. Set up the experimental group: Perform optimization experiments with different camera white balances (2800, 2900, 3000, 3100, and 3200 K).

[0150] 3. The detection process of the dry-type electrochemiluminescence lateral flow immunoassay strip is the same as that in Example 2, and the test results are as Figure 16 shown.

[0151] It can be seen from the experimental results that when the camera white balance reaches 2900 K, T / C reaches the highest. In the range of camera white balance from 2800 to 3200 K, the ratio of the signal T / C to the background T / C (SBR) gradually decreases. To obtain a higher T / C and SBR, a camera white balance of 2900 K is preferred.

[0152] (12) Optimize the camera exposure time

[0153] 1. The concentration of LH to be measured is 1 mIU / mL, the camera white balance is 2900 K, the camera exposure time is to be determined, the driving voltage is 11 V, the ECL signal antibody mixed solution is 6 μL, the concentration of the T-line capture antibody is 0.5 mg / mL, the concentration of the C-line capture antibody is 0.25 mg / mL, the immunoreaction time is 3.5 min, and the sample loading volume is 30 μL.

[0154] 2. Set up the experimental group: Conduct optimization experiments with different camera exposure times (50, 166, 250, 350, and 450 ms).

[0155] 3. The detection process of the dry-type electrochemiluminescence lateral flow immunoassay strip is the same as that in Example 2, and the test results are as Figure 17 shown.

[0156] It can be seen from the experimental results that when the exposure time is between 50 - 166 ms, T / C gradually increases; when it is between 166 - 450 ms, T / C shows a gradually decreasing trend. Within the exposure time range of 50 - 450 ms, SBR gradually decreases. To obtain a higher T / C and SBR, the preferred camera exposure time is 166 ms.

[0157] (13) Preferred driving voltage

[0158] 1. The concentration of LH to be measured is 1 mIU / mL, the camera white balance is 2900 K, the camera exposure time is 166 ms, the driving voltage is to be determined, the volume of the ECL signal antibody mixed solution is 6 μL, the concentration of the capture antibody on the T line is 0.5 mg / mL, the concentration of the capture antibody on the C line is 0.25 mg / mL, the immunoreaction time is 3.5 min, and the sample loading volume is 30 μL.

[0159] 2. Set up the experimental group: Conduct optimization experiments with different driving voltages (8, 9, 10, 11, 12, and 13 V).

[0160] 3. The detection process of the dry-type electrochemiluminescence lateral flow immunoassay strip is the same as that in Example 2, and the test results are as Figure 18 shown.

[0161] It can be seen from the experimental results that when the driving voltage is lower than 11 V, T / C increases with the increase of the driving voltage. This is because the driving voltage in this range has not fully caused the ECL reaction of the ECL signal antibody, so the ECL signal and T / C increase with the increase of the driving voltage. However, when the driving voltage is higher than 11 V, T / C decreases with the increase of the driving voltage. This may be due to the fact that too high a driving voltage causes the ECL signal to be generated too quickly and quenched too quickly, resulting in the analysis device being unable to fully capture the ECL signal, and thus leading to a decrease in T / C. Therefore, the preferred driving voltage is 11 V.

[0162] (14) Preferred immunoreaction time

[0163] 1. The LH concentration to be measured is 1 mIU / mL, the camera white balance is 2900 K, the camera exposure time is 166 ms, the driving voltage is 11 V, the ECL signal antibody mixed solution is 6 μL, the T-line capture antibody concentration is 0.5 mg / mL and the C-line capture antibody concentration is 0.25 mg / mL. The immunoreaction time is to be determined, and the sample loading volume is 30 μL.

[0164] 2. Set up the experimental groups: Conduct optimization experiments by setting different immunoreaction times (1.5, 3.5, 5.5, 7.5, and 9.5 min).

[0165] 3. The detection process of the dry-type electrochemiluminescence lateral flow immunoassay strip is the same as that in Example 2, and the test results are as Figure 19 shown.

[0166] It can be seen from the experimental results that when the immunoreaction time is less than 3.5 min, the ECL signal and T / C increase with the increase of the immunoreaction time. However, when the immunoreaction time is higher than 3.5 min, the ECL signal and T / C basically reach a plateau. The possible reason for this phenomenon is that the formation of the "T-line ECL signal antibody - biomarker - T-line capture antibody" immunosandwich complex is basically saturated. Therefore, the preferred immunoreaction time is 3.5 min.

[0167] (15) Preferred sample loading volume

[0168] 1. The LH concentration to be measured is 1 mIU / mL, the camera white balance is 2900 K, the camera exposure time is 166 ms, the driving voltage is 11 V, the ECL signal antibody mixed solution is 6 μL, the T-line capture antibody concentration is 0.5 mg / mL and the C-line capture antibody concentration is 0.25 mg / mL. The immunoreaction time is 3.5 min, and the sample loading volume is to be determined.

[0169] 2. Set up the experimental groups: Conduct optimization experiments by setting different sample loading volumes (20, 25, 30, 35, and 40 μL).

[0170] 3. The detection process of the dry-type electrochemiluminescence lateral flow immunoassay strip is the same as that in Example 2, and the test results are as Figure 20 shown.

[0171] It can be seen from the experimental results that when the sample loading volume is less than 35 μL, the ECL signal and T / C increase with the increase of the sample loading volume. However, when the sample loading volume is higher than 35 μL, the ECL signal and T / C basically reach a plateau. The possible reason for this phenomenon is that the formation of the "T-line ECL signal antibody - biomarker - T-line capture antibody" immunosandwich complex has reached saturation. Therefore, the preferred sample loading volume in this study is 35 μL.

[0172] (16) Preferred sample dilution ratio

[0173] 1. The original concentrations of the LH samples to be measured are 1, 10, and 50 mIU / mL, the camera white balance is 2900K, the camera exposure time is 166 ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6 μL, the concentration of the capture antibody on the T line is 0.5 mg / mL, the concentration of the capture antibody on the C line is 0.25 mg / mL, the immunoreaction time is 3.5 min, and the sample loading volume is 35 μL.

[0174] 2. Set up the experimental groups: Set the volume ratios of the sample to PBS as 1:6, 1:9, and 1:12 for the optimization experiment.

[0175] 3. The detection process of the dry-type electrochemiluminescence lateral flow immunoassay strip is the same as that in Example 2, and the test results are as Figure 21 shown.

[0176] It can be seen from the experimental results that when the volume ratio of the sample to PBS is 1:9, the slope of the obtained fitting curve is the highest, reaching 2.145, and R 2 is 0.9987. In addition, the fitting curve when the volume ratio of the sample to PBS is 1:9 has a better effect than when the volume ratios are 1:6 and 1:12. The possible reason for this phenomenon is that at 1:6, the sample matrix affects the electrical conduction, thereby affecting the generation of the ECL signal, making the T / C low when the LH sample is at high concentrations (10 and 50 mIU / mL); at 1:12, the low-concentration biomarker cannot make the total amount of the "T-line ECL signal antibody - biomarker - T-line capture antibody" complex reach a relatively saturated state on the detection pads 1-3. Therefore, the preferred dilution ratio of the LH sample is 1:9.

[0177] Example 4

[0178] Using the optimized conditions explored in Example 3, the dry-type electrochemiluminescence lateral flow immunoassay strip of Example 1 is used for LH detection.

[0179] 1. The camera white balance is 2900K, the camera exposure time is 166 ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6 μL, the concentration of the capture antibody on the T line is 0.5 mg / mL, the concentration of the capture antibody on the C line is 0.25 mg / mL, the immunoreaction time is 3.5 min, and the sample loading volume is 35 μL.

[0180] 2. Set up the experimental groups: Set several different LH concentration values (0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 11, and 22 mIU / mL), and LH with a concentration of 0.01 mIU / mL is used for blank control detection.

[0181] 3. The detection process of the dry-type electrochemiluminescence lateral flow immunoassay strip is the same as that in Example 2, and the test results are as Figure 22 shown.

[0182] It can be seen from the experimental results that when the LH concentration is in the range of 0.05 - 22 mIU / mL, T / C increases with the increase of the LH concentration. There is a good linear relationship between T / C (denoted as Y) and the logarithm of the LH concentration (denoted as X), and the linear equation is Y = 2.127X + 2.832 (R 2 = 0.9985, n = 5), and the detection limit is estimated to be 0.0496 mIU / mL.

[0183] The calculation method of the detection limit is: Y L = Y b + 3S b , where Y b represents the average T / C value in the blank control, and S b represents the standard deviation in the blank control (5 repeated experiments). Using the obtained Y L value to calculate the corresponding LH concentration is the detection limit.

[0184] Example 5

[0185] An actual sample detection experiment of the dry-type electrochemiluminescence lateral flow immunoassay strip was carried out under the optimized conditions explored in Example 3.

[0186] 1. The camera white balance is 2900K, the camera exposure time is 166 ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6 μL, the T-line capture antibody concentration is 0.5 mg / mL, the C-line capture antibody concentration is 0.25 mg / mL, the immunoreaction time is 3.5 min, and the sample addition volume is 35 μL.

[0187] 2. Set up the experimental group: Dilute the collected urine samples by 10 times to obtain three diluted samples with LH concentrations of 0.11, 1.21, and 6.96 mIU / mL respectively for actual sample detection.

[0188] 3. The detection process of the dry-type electrochemiluminescence lateral flow immunoassay strip is the same as that in Example 2, and the test results are shown in Table 1.

[0189] Table 1

[0190]

[0191] As can be seen from Table 1, the test strip detection results are 0.0104, 1.093, and 6.996 mIU / mL respectively, the recovery rates are 93.69%, 91.08%, and 100.22% respectively, and the relative standard deviations are 8.51%, 9.78%, and 10.42% respectively. Therefore, the test strip of the present utility model can achieve good detection of LH in actual urine samples.

[0192] Example 6

[0193] Perform the storage stability experiment of the dry electrochemical luminescence lateral flow immunoassay test strip under the optimized conditions explored in Example 3.

[0194] 1. The camera white balance is 2900K, the camera exposure time is 166 ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6 μL, the T-line capture antibody concentration is 0.5 mg / mL, and the C-line capture antibody concentration is 0.25 mg / mL. The immunoreaction time is 3.5 min, and the sample loading volume is 35 μL.

[0195] 2. Set up the experimental group: Store the prepared dry electrochemical luminescence lateral flow immunoassay test strip in an oven at 37 °C, and then perform detections at intervals of 0, 3, 3, 3, 5, 5, and 5 days.

[0196] 3. The detection process of the dry electrochemical luminescence lateral flow immunoassay test strip is the same as that in Example 2, and the test results are as Figure 23 shown.

[0197] It can be seen from the experimental results that in an environment of 37 °C, the dry electrochemical luminescence lateral flow immunoassay test strip can be stored for 19 days. According to the Arrhenius equation, 19 days of acceleration at 37 °C is equivalent to storing at 4 °C for about two and a half years with good storage stability.

[0198] The above embodiments are the preferred embodiments of the present utility model, but the embodiments of the present utility model are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present utility model shall be equivalent replacement methods and are all included in the protection scope of the present utility model.

Claims

1. A dry electrochemiluminescent lateral flow immunoassay test strip, characterized in that: It includes a lateral flow immunoassay strip, an electrode pad and a connection pad; The lateral flow immunoassay strip is composed of a sample pad, a conjugate pad, a detection pad with a backing NC membrane and an absorption pad stacked on a transparent adhesive plate; the detection pad is provided with a T line and a C line; The electrode pad includes an integrated closed bipolar electrode and a driving electrode; the integrated closed bipolar electrode includes a closed bipolar electrode detection anode and a quality control anode and a shared cathode of the two anodes; The lateral flow immunoassay test strip is inverted on the electrode pad; the NC membrane of the detection pad faces downward and the backing faces upward; the T line and C line on the detection pad are in contact with the detection anode and the quality control anode on the electrode pad respectively; the binding pad is close to one end of the quality control anode and is in contact with the non-electrode area of ​​the electrode pad; the sample pad is close to the binding pad and is in contact with the non-electrode area of ​​the electrode pad; the absorption pad is close to one end of the detection anode and is in contact with the negative driving electrode corresponding to the detection anode.

2. The dry electrochemiluminescent lateral flow immunoassay test strip according to claim 1, characterized in that: The connection pad is located on the shared cathode, on the hydrophobic bottom plate between the shared cathode and the positive driving electrode, and is in contact with a portion of the positive driving electrode.

3. The dry electrochemiluminescent lateral flow immunoassay test strip according to claim 1, characterized in that: The driving electrodes include a negative driving electrode corresponding to the anode and a positive driving electrode corresponding to the shared cathode; the two anodes and the corresponding negative driving electrodes as well as the shared cathode and the corresponding positive driving electrode are naturally separated by a hydrophobic bottom plate.

4. The dry electrochemiluminescent lateral flow immunoassay test strip according to claim 1, characterized in that: The electrode pad is provided with double-sided tape between the detection anode, the shared cathode and the driving electrode for fixing part of the absorption pad and part of the detection pad, and double-sided tape is provided on one side of the quality control anode and the shared cathode for fixing part of the sample pad and part of the binding pad.

5. A dry electrochemiluminescent lateral flow immunoassay kit, characterized in that: A dry electrochemiluminescent lateral flow immunoassay test strip comprising any one of claims 1 to 4.

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