Three-dimensional bipolar electrochemiluminescence lateral flow immunization test strip and application thereof in immunization detection

Abstract

The application discloses a three-dimensional bipolar ECL lateral flow immunochromatographic test strip and application thereof in immunodetection, and relates to the technical field of immunodetection. The three-dimensional bipolar ECL lateral flow immunochromatographic test strip comprises a three-dimensional bipolar electrode sheet, a lateral flow immunochromatographic test strip and a connecting pad; the three-dimensional bipolar electrode sheet is arranged below, the lateral flow immunochromatographic test strip and the connecting pad are arranged above; the three-dimensional bipolar electrode sheet is designed in a layered mode and comprises an electrode sheet layer one and an electrode sheet layer two; the electrode sheet layer two is arranged below, and the electrode sheet layer one is arranged above; the electrode sheet layer two comprises a positive driving electrode; the positive driving electrode comprises a positive driving electrode head end, a positive driving electrode tail end and connecting wires thereof. The three-dimensional bipolar electrode sheet is designed for the first time, the electrode sheet layer one and the electrode sheet layer two are combined through back glue with adhesion, the shortcomings of a traditional bipolar ECL electrode sheet are broken, and the three-dimensional bipolar ECL lateral flow immunochromatographic test strip is more suitable for the demand of on-site rapid detection.

Description

Technical Field

[0001] This invention belongs to the field of electrochemiluminescence (ECL) sensing technology, specifically relating to a three-dimensional bipolar ECL lateral flow immunoassay strip and its application in immunoassay. Background Technology

[0002] Currently, common methods for detecting biomarkers include enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography (CLIA), and ECL (electrochemical chromatography-mass spectrometry). Colloidal gold immunochromatography is widely used in the market, but it is only suitable for qualitative or semi-quantitative detection and cannot perform accurate quantitative detection. Other detection methods are generally complex to operate, time-consuming, require sophisticated instruments, and are costly.

[0003] Electroluminescent luminescent (ECL) technology generates light signals by exciting luminescent materials through electrochemical reactions. It boasts advantages such as high sensitivity, low background noise, and ease of temporal / spatial control, and has been widely applied in protein immunoassay, gene diagnostics, and small molecule detection. The core component of traditional ECL chips is typically an electrode sheet, whose performance directly affects the detection limit, signal-to-noise ratio, and stability. Traditional ECL electrode sheets often suffer from the following drawbacks: First, they often employ a two-dimensional planar design, resulting in a low effective surface area, which limits the loading of luminescent materials, restricts signal intensity, and reduces sensitivity. Second, existing electrode sheet materials have limited functionality and poor compatibility, making it difficult to simultaneously meet the requirements of high conductivity and strong luminescent material immobilization capabilities. Third, traditional electrode sheet fabrication processes (such as electron beam lithography and template methods) are complex and costly, hindering large-scale production.

[0004] Existing ECL sensor housings (sometimes called casings) are typically short and flat, lacking aesthetic appeal and thus failing to attract consumers. Furthermore, existing ECL sensor housings are usually large and costly, hindering widespread application. The reagent addition wells (sample wells, buffer solution wells) are not aligned with the ECL observation window (i.e., the sample pad, connection pad, and detection pad are not aligned), increasing the difficulty of housing production and testing operations, and adding complexity to ECL sensor assembly. This also makes it difficult to mass-produce ECL test strips quickly. Summary of the Invention

[0005] The purpose of this invention is to design a three-dimensional bipolar ECL lateral flow immunoassay strip and its application in immunoassay. A large-format three-dimensional bipolar electrode sheet, a large-format lateral flow immunoassay strip, and a large-format connecting pad are assembled into a large-format three-dimensional bipolar ECL lateral flow immunoassay strip. This large-format ECL test strip is continuously cut in a certain manner using an inductive strip cutter to quickly prepare independent, individual ECL test strips, and dry ECL technology is used to quantitatively detect biomarkers.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A three-dimensional bipolar ECL lateral flow immunoassay strip includes a three-dimensional bipolar electrode sheet, a lateral flow immunoassay strip, and a connecting pad; the three-dimensional bipolar electrode sheet is located at the bottom, and the lateral flow immunoassay strip and the connecting pad are located at the top.

[0008] The three-dimensional bipolar electrode sheet is elongated and has a layered design, including electrode sheet layer one and electrode sheet layer two; electrode sheet layer two is on the bottom and electrode sheet layer one is on the top, and they are bonded together by their adhesive backing.

[0009] The second electrode sheet includes a positive driving electrode head end, a positive driving electrode tail end, and their connecting wires.

[0010] The electrode layer one includes a negative driving electrode and an integrated bipolar electrode;

[0011] The negative driving electrode is positioned to correspond to the first end of the positive driving electrode of electrode layer two.

[0012] The integrated bipolar electrode includes a detection anode, a quality control anode, and a shared cathode, with one end of the detection anode close to the negative driving electrode. The detection anode and the quality control anode are connected in series and are located on the same straight line as the shared cathode. This design can not only effectively prevent the reaction solutions from flowing together to form an open bipolar electrode, but also facilitate the mass production of ECL test strips.

[0013] The shared cathode and the tail end of the positive driving electrode are physically spaced apart and are covered by a connecting pad for electrical connection.

[0014] The lateral flow immunoassay strip includes a sample pad, a conjugate pad, a detection pad, and an absorbent pad; the lateral flow immunoassay strip and the connecting pad are arranged in a straight line.

[0015] The binding pad was dried to fix the ECL signaling antibody;

[0016] The detection pad includes a T-line and a C-line; the T-line is coated with T-line capture antibody, and the C-line is coated with C-line capture antibody.

[0017] The T-line and C-line correspond to the detection anode and the control anode, respectively; the sample pad is located between the shared cathode and the control anode, and the binding pad is located between the sample pad and the detection pad; the absorbent pad is close to the detection anode and in contact with part of the negative drive electrode.

[0018] During preparation, the sample pad, binding pad, detection pad, and absorbent pad are sequentially stacked on the adhesive-backed carrier, and then inverted onto the electrode sheet layer one to form the above-mentioned correspondence.

[0019] The preferred carrier is a transparent PET sheet.

[0020] During preparation, the lateral flow immunoassay strip, connecting pad and three-dimensional bipolar electrode sheet can all be made into large-scale versions first, and after they are glued together, they can be cut into individual three-dimensional bipolar ECL lateral flow immunoassay strips.

[0021] The electrode sheet is bonded with hydrophobic double-sided adhesive to fix the lateral flow immunoassay strip and the connecting pad;

[0022] Preferably, double-sided tape is adhered between the negative driving electrode and the detection anode to fix part of the absorbent pad and part of the detection pad; double-sided tape is adhered between the quality control anode and the shared cathode to fix part of the sample application pad, the conjugate pad, and part of the detection pad; and double-sided tape is adhered to the non-electrode area near the top of the shared cathode to fix the connecting pad. The lateral flow immunoassay strip and the connecting pad are distributed in a straight line.

[0023] Electrode layer one and electrode layer two are preferably made of transparent PET sheet, and are prepared by screen printing conductive ink on transparent PET sheet; the detection pad material is preferably NC film; the absorbent pad and connecting pad material is preferably absorbent paper; the sample pad and bonding pad material is preferably glass fiber; the casing is prepared by 3D printer.

[0024] The conjugate pad preparation process is as follows: glass fibers are cut into the required size using a laser cutter, then the conjugate pad treatment solution is dropped onto the glass fibers, and then the treated glass fibers are placed in a 37°C oven and baked for 1 hour; the ECL signal antibody mixture is uniformly sprayed onto the glass fibers using a gold spraying film scribing instrument, and then the glass fibers are placed in a 37°C oven and baked for 1 hour to dry.

[0025] The sample pad preparation process is as follows: the glass fiber is cut into the required size using a laser cutter, the sample pad treatment solution is dripped onto the glass fiber, and then the glass fiber is placed in a 37°C oven and baked for 1 hour.

[0026] The detection pad preparation process is as follows: T-line capture antibody solution and C-line capture antibody solution are applied onto the NC membrane using a gold sputtering membrane scribing instrument, and then the NC membrane is placed in a 37°C oven for 2 hours to dry.

[0027] The absorbent pad and connecting pad are made of absorbent filter paper cut by a laser cutting machine;

[0028] Both the sample pad treatment solution and the conjugate pad treatment solution were prepared by dissolving 0.05% polyvinylpyrrolidone (PVP), 0.025% casein and 0.25% Tween-20 in phosphate-buffered saline (PBS).

[0029] The ECL signaling antibody mixture is prepared by mixing an intramolecular self-enhancing ECL signaling antibody conjugated with a T-line labeled antibody (referred to as T-line ECL signaling antibody), an intramolecular self-enhancing ECL signaling antibody conjugated with a C-line labeled antibody (referred to as C-line ECL signaling antibody), and a labeled antibody treatment solution; the labeled antibody treatment solution is prepared by dissolving 5 mg / mL trehalose, 0.05% PVP, 0.025% casein, 0.03% proclin-300, and 0.1% Tetronic 1307 (referred to as S9) surfactant in PBS buffer.

[0030] The T-line capture antibody solution is prepared by dissolving 15 mg / mL sucrose, 0.1% S9, and 0.15–0.35 mg / mL T-line capture antibody in PBS solution; the C-line capture antibody solution is prepared by dissolving 5 mg / mL sucrose, 5 mg / mL trehalose, 0.1% S9, and 0.25 mg / mL C-line capture antibody in PBS solution.

[0031] The preparation process of the T-line ECL signaling antibody and C-line ECL signaling antibody solution is described in CN 118688451A.

[0032] A three-dimensional bipolar ECL lateral flow immunosensor (3D-BP-ECL-LFIS) includes the aforementioned three-dimensional bipolar ECL lateral flow immunosensor strip;

[0033] The 3D-BP-ECL-LFIS also includes a casing;

[0034] The casing is elongated and includes a top cover and a bottom plate. The top cover has an observation window, a sample application hole, and a buffer solution addition hole, all of which are located on the same straight line. The bottom plate has an electrode contact area and four sets of protrusions for fixing the three-dimensional bipolar ECL lateral flow immunoassay strip.

[0035] Furthermore, the three-dimensional bipolar ECL lateral flow immunoassay strip is adhered to the base plate via the adhesive backing of the electrode sheet layer two;

[0036] Furthermore, the electrode contact area corresponds to the head end of the negative driving electrode and the positive driving electrode; the observation window corresponds to the T line and the C line; the sample application well corresponds to the sample pad; and the buffer addition well corresponds to the connection pad.

[0037] The three-dimensional bipolar ECL lateral flow immunoassay strip or sensor of the present invention can be used for the immunoassay detection of a variety of biomarkers;

[0038] Depending on the antibody, the biomarker is one of luteinizing hormone (LH), cardiac troponin I, C-reactive protein, peptidin, serum amyloid, or Alzheimer's disease-associated neuron linein.

[0039] Specifically, the immune detection includes the following steps:

[0040] (1) Add a sample solution containing the biomarker to be tested to the sample pad and perform an immune reaction;

[0041] In the above process, the sample droplets flow to the conjugate pad via capillary action and bind to the ECL signaling antibody to form a complex (T-line ECL signaling antibody - biomarker). Subsequently, this complex continues to flow to the detection pad and binds to the T-line capture antibody on the detection pad, forming a "T-line ECL signaling antibody - biomarker - T-line capture antibody" immune complex. Simultaneously, the C-line signaling antibody is captured and bound by the C-line capture antibody on the detection pad to form a "C-line ECL signaling antibody - C-line capture antibody" immune complex.

[0042] Furthermore, the volume (V) of the sample solution s The volume is 30–50 μL, preferably 40 μL;

[0043] Furthermore, the time of the immune response (t) i The cooking time is 1.5–5.5 min, preferably 3.5 min;

[0044] (2) Add buffer solution to the sample pad to rinse the residue on the test pad onto the absorbent pad; at the same time, add buffer solution to the connection pad to connect the circuit;

[0045] The buffer solution has a pH value of 6.0 to 10.0 and can be PBS buffer, disodium bicarbonate-citric acid buffer (GASDP), Tris-HCl, glycine-NaOH buffer (GSH), or borax-NaOH buffer (BSH).

[0046] The volume of the buffer solution used is 30–50 μL;

[0047] Preferably, the amount of buffer solution used in the sample pad (V) b The value is 40 μL;

[0048] The rinsing time is several minutes, preferably 3 minutes;

[0049] (3) Place the three-dimensional bipolar ECL lateral flow immunoassay strip or sensor processed in steps (1) and (2) into an ECL point detection device (as disclosed in application number: 2024210642259) for detection, and set the camera white balance (W). b), camera exposure time (t) E ) and driving voltage (E tot ECL immune response was then performed. During the reaction, the COMS camera captured the reaction area throughout the process, collecting ECL signals on the T and C lines. The luminescence videos on the T and C lines were analyzed and processed through a mobile APP, and the corresponding luminescence signal values ​​were displayed. Finally, the ratio of the luminescence signal value on the T line to the luminescence signal value on the C line (i.e., T / C) was used for quantitative analysis of the biomarker to be tested.

[0050] Furthermore, the W b The K value is 2800–3200K, with 2900K being preferred.

[0051] Furthermore, the t E The timeframe is 50–450 ms, with 166 ms being the preferred value;

[0052] Furthermore, the E tot The voltage range is 8–18V, with 16V being preferred.

[0053] The present invention has the following advantages and effects compared with the prior art:

[0054] 1. This invention is the first to design a 3D-BP-ECL-LFIS sensor, which is elongated and strip-shaped. This greatly overcomes the shortcomings of existing bipolar ECL lateral flow immunosensors, such as complicated preparation and high cost.

[0055] 2. This invention is the first to design a three-dimensional bipolar electrode sheet, which includes an electrode sheet layer one and an electrode sheet layer two, bonded together by an adhesive backing. This overcomes the shortcomings of traditional bipolar ECL electrode sheets (such as wider electrode sheets and lack of aesthetics), making it more suitable for the needs of rapid on-site testing.

[0056] 3. This invention is the first to adopt a manufacturing mode of large-format three-dimensional bipolar electrode sheet, large-format lateral flow immunoassay strip and large-format connecting pad. The large-format three-dimensional bipolar electrode sheet is used to attach the large-format lateral flow immunoassay strip and the large-format connecting pad with hydrophobic double-sided adhesive to make a large-format three-dimensional bipolar ECL lateral flow immunoassay strip.

[0057] 4. In this invention, the detection anode and control anode on the three-dimensional bipolar electrode sheet are arranged in series and aligned with the shared cathode in a straight line. Correspondingly, the sample pad, conjugate pad, detection pad, absorbent pad, and connecting pad are also arranged in a straight line, while the buffer addition hole, sample application hole, and observation window on the cartridge are also arranged in a straight line. This design facilitates the mass production of three-dimensional bipolar ECL lateral flow immunoassay strips, simplifies the manufacturing process, and eliminates batch errors in strip preparation.

[0058] 5. The linear integrated bipolar electrode design proposed for the first time in this invention can cleverly solve the problem of easy cross-contamination between the report channel and the support channel reaction solution of existing bipolar ECL electrode sheets; at the same time, it shortens the width of the casing, reduces the size and weight of the casing, and thus reduces production costs.

[0059] 6. This invention patent is the first to use an inductive cutting method to prepare three-dimensional bipolar ECL lateral flow immunoassay strips, which improves processing efficiency, processing accuracy, and production consistency and reliability.

[0060] 7. The 3D-BP-ECL-LFIS 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. Attached Figure Description

[0061] Figure 1 This is a schematic diagram of the overall structure of the three-dimensional bipolar ECL lateral flow immunoassay strip of the present invention.

[0062] Among them, 1-1-three-dimensional bipolar electrode sheet, 1-2-lateral flow immunoassay strip, 1-3-connecting pad.

[0063] Figure 2 This is an exploded schematic diagram of the three-dimensional bipolar ECL lateral flow immunoassay strip of the present invention;

[0064] Figure 3 This is an exploded view of the three-dimensional bipolar electrode sheet of the present invention;

[0065] Figure 4 This is an overall schematic diagram of the lateral flow immunoassay strip of the present invention;

[0066] Among them, 1-1-1-Electrode sheet one, 1-1-1-1-Negative driving electrode, 1-1-1-2-Integrated bipolar electrode, 1-1-1-2-1-Detection anode, 1-1-1-2-2-Quality control anode, 1-1-1-2-3 Shared cathode, 1-1-1-3-1-Hydrophobic double-sided adhesive, 1-1-1-3-2-Hydrophobic double-sided adhesive, 1-1-1-3-3-Hydrophobic double-sided adhesive, 1-1-2-Electrode sheet two, 1-1-2-1-Positive driving electrode head end, 1-1-2-2-Positive driving electrode tail end, 1-1-2-3-Connecting wire, 1-2-1-Sample pad, 1-2-2-Binding pad, 1-2-3-Detection pad, 1-2-4-Absorbent pad, 1-2-5-Carrier with adhesive backing.

[0067] Figure 5 This is a schematic diagram showing the disassembled casing.

[0068] Among them, 2-1-top cover, 2-2-bottom plate, 2-1-1-observation window, 2-1-2-sample addition well, 2-1-3-buffer solution addition well, and 2-2-1-electrode contact area.

[0069] Figure 6 For T / C and W b The relationship diagram.

[0070] Figure 7 For T / C and t E The relationship diagram.

[0071] Figure 8 For T / C and E tot The relationship diagram.

[0072] Figure 9 The T / C and T-line capture antibody concentration ([CAb) t The relationship diagram of ]).

[0073] Figure 10 T / C and signal antibody volume (V) t1-c1 Relationship diagram.

[0074] Figure 11 This is a graph showing the relationship between T / C and ti.

[0075] Figure 12 For T / C and V s The relationship diagram.

[0076] Figure 13 For T / C and V b The relationship diagram.

[0077] Figure 14 The dynamic analysis curves are shown when the volume ratio of T-line signal antibody, C-line signal antibody, and signal antibody treatment solution is 9:9:2 (the inset is a linear fitting curve of the data).

[0078] Figure 15 The dynamic analysis curves are shown when the volume ratio of T-line signal antibody, C-line signal antibody, and signal antibody treatment solution is 6:3:1 (the inset is a linear fitting curve of the data).

[0079] Figure 16 This is a graph showing the actual sample detection and evaluation.

[0080] Figure 17 This is a graph used to evaluate the storage stability when testing LH.

[0081] Figure 18 This is a schematic diagram of the overall 3D-BP-ECL-LFIS.

[0082] Among them, 1-Three-dimensional bipolar ECL lateral flow immunoassay strip, 2-Clubbing clip.

[0083] Figure 19 This is a schematic diagram of the overall layout of the large-format three-dimensional bipolar ECL lateral flow immunoassay strip.

[0084] Figure 20 This is a schematic diagram of the overall structure of a large-format three-dimensional bipolar electrode sheet.

[0085] Figure 21 This is a schematic diagram of the overall layout of the large-format lateral flow immunoassay strip.

[0086] Figure 22 This is a schematic diagram of the overall design of the large-format connecting pad.

[0087] Figure 23 This is a schematic diagram of the entire large electrode sheet layer one.

[0088] Figure 24 This is a schematic diagram of the entire second layer of the large electrode sheet.

[0089] Figure 25 This is a schematic diagram showing the breakdown of a large-format lateral flow immunoassay strip.

[0090] Among them, A-large version with adhesive backing carrier, B-large version absorbent pad, C-large version detection pad, D-large version binding pad, and E-large version sample pad. Detailed Implementation

[0091] 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.

[0092] Example 1

[0093] A three-dimensional bipolar ECL lateral flow immunoassay strip 1 ( Figure 1 , Figure 2 The device includes a three-dimensional bipolar electrode 1-1, a lateral flow immunoassay strip 1-2, and a connecting pad 1-3; the three-dimensional bipolar electrode 1-1 is located at the bottom, and the lateral flow immunoassay strip 1-2 and the connecting pad 1-3 are located at the top.

[0094] The three-dimensional bipolar electrode sheet 1-1 ( Figure 3 It is long and strip-shaped, and adopts a layered design, including electrode sheet layer 1-1-1 and electrode sheet layer 1-1-2; the electrode sheet layer 1-1-2 is at the bottom and the electrode sheet layer 1-1-1 is at the top, and they are bonded together by their adhesive backing.

[0095] The electrode layer 1-1-2 includes a positive driving electrode head end 1-1-2-1, a positive driving electrode tail end 1-1-2-2 and its connecting wires 1-1-2-3;

[0096] The electrode sheet 1-1-1 includes a negative driving electrode 1-1-1-1 and an integrated bipolar electrode 1-1-1-2;

[0097] The negative driving electrode 1-1-1-1 corresponds to the positive driving electrode head 1-1-2-1.

[0098] The integrated bipolar electrode 1-1-1-2 includes a detection anode 1-1-1-2-1, a quality control anode 1-1-1-2-2, and a shared cathode 1-1-1-2-3. One end of the detection anode 1-1-1-2-1 is close to the negative driving electrode 1-1-1-1. The detection anode 1-1-1-2-1 and the quality control anode 1-1-1-2-2 are connected in series and are aligned with the shared cathode 1-1-1-2-3. This design not only effectively prevents the reaction solutions from interconnecting to form an open bipolar electrode, but also facilitates the mass production of ECL test strips.

[0099] The shared cathode 1-1-1-2-3 and the positive driving electrode tail end 1-1-2-2 are physically spaced apart, and they are covered by the connecting pad 1-3 for electrical connection.

[0100] The connecting pad 1-3 is located away from the quality control anode 1-1-1-2-2 and the detection anode 1-1-1-2-1;

[0101] The lateral flow immunoassay strips 1-2 ( Figure 4 It includes sample pad 1-2-1, conjugate pad 1-2-2, detection pad 1-2-3, absorbent pad 1-2-4, and carrier with adhesive backing 1-2-5;

[0102] The binding pad 1-2-2 is used to dry and fix ECL signaling antibodies;

[0103] The detection pad 1-2-3 includes a T line and a C line; the T line is coated with T line capture antibody, and the C line is coated with C line capture antibody;

[0104] The sample pad 1-2-1, conjugate pad 1-2-2, detection pad 1-2-3, and absorbent pad 1-2-4 are sequentially stacked on the adhesive-backed carrier 1-2-5, and then inverted onto the three-dimensional electrode sheet 1-1-1. The T-line and C-line correspond to the detection anode 1-1-1-2-1 and the quality control anode 1-1-1-2-2. The sample pad 1-2-1 is located between the shared cathode 1-1-1-2-3 and the quality control anode 1-1-1-2-2, and the conjugate pad 1-2-2 is located between the sample pad 1-2-1 and the detection pad 1-2-3. The absorbent pad 1-2-4 is close to the detection anode 1-1-1-2-1 and in contact with part of the negative driving electrode 1-1-1-1.

[0105] The adhesive-backed carrier 1-2-5 is preferably a transparent PET sheet;

[0106] The three-dimensional bipolar electrode sheet 1-1, the lateral flow immunoassay strip 1-2, and the connecting pad 1-3 can all be made into large-scale versions first. After they are glued together, they can be cut into individual three-dimensional bipolar ECL lateral flow immunoassay strips 1 by an induction strip cutter.

[0107] The electrode sheet layer 1-1-1 is bonded with hydrophobic double-sided adhesive 1-1-1-3 to fix the lateral flow immunoassay strip 1-2 and the connecting pad 1-3;

[0108] Preferably, double-sided tape 1-1-1-3-1 is adhered between the negative driving electrode 1-1-1-1 and the detection anode 1-1-1-2-1 to fix part of the absorbent pad 1-2-4 and part of the detection pad 1-2-3; double-sided tape 1-1-1-3-2 is adhered between the quality control anode 1-1-1-2-2 and the shared cathode 1-1-1-2-3 to fix part of the sample pad 1-2-1, the conjugate pad 1-2-2, and part of the detection pad 1-2-3; and double-sided tape 1-1-1-3-3 is adhered to the non-electrode area near the top of the shared cathode 1-1-1-2-3 to fix the connecting pad 1-3. The lateral flow immunoassay strip 1-2 and the connecting pad 1-3 are distributed in a straight line.

[0109] The 3D-BP-ECL-LFIS also includes a casing 2;

[0110] The aforementioned card 2 ( Figure 5 The strip is elongated and includes a top cover 2-1 and a bottom plate 2-2. The top cover 2-1 is provided with an observation window 2-1-1, a sample application hole 2-1-2, and a buffer addition hole 2-1-3, all of which are located on the same straight line. The bottom plate 2-2 is provided with an electrode contact area 2-2-1 and four sets of protrusions for fixing the three-dimensional bipolar ECL lateral flow immunoassay strip 1. The three-dimensional bipolar ECL lateral flow immunoassay strip 1 and the casing 2 form a 3D-BP-ECL-LFIS.

[0111] Example 2

[0112] The 3D-BP-ECL-LFIS detection process of this invention is as follows:

[0113] First, 40 μL of sample solution containing the biomarker to be tested is added to the sample pad and allowed to stand for 3.5 min. The sample solution flows through capillary action to the binding pad and binds to the ECL signaling antibody to form a complex (T-line ECL signaling antibody-LH). Subsequently, this complex continues to flow to the detection pad and binds to the T-line capture antibody on the detection pad, forming a “T-line ECL signaling antibody-LH-T-line capture antibody” immune complex. At the same time, the C-line signaling antibody is captured and bound by the C-line capture antibody on the detection pad to form a “C-line ECL signaling antibody-mouse IgG” immune complex.

[0114] Subsequently, 40 μL of PBS solution was added to the sample pad to rinse the residue on the test pad onto the absorbent pad for 3 minutes; at the same time, 30 μL of PBS solution was added to the connection pad to connect the circuit.

[0115] Next, the prepared 3D-BP-ECL-LFIS was placed in an ECL instantaneous detection device (disclosed in application number: 2024210642259) for detection at 2900 kW. b 166ms t E and 16VE tot ECL immune response was performed under the following conditions; during the reaction, the COMS camera captured the reaction area throughout the process, collecting ECL signals on the T line and C line. The luminescence video on the T line and C line was analyzed and processed through a mobile APP, and the corresponding luminescence signal values ​​were displayed. Finally, the ratio of the luminescence signal value on the T line to the luminescence signal value on the C line (i.e., T / C) was used for quantitative analysis of the biomarker to be tested.

[0116] The following test will be conducted using a sample solution containing 1 mIU / mL LH and the 3D-BP-ECL-LFIS of this invention as an example to measure W. b The effect on the luminous signal value of 3D-BP-ECL-LFIS.

[0117] Set up the experimental group: Select five Ws b Optimization experiments were conducted using numerical values ​​(2800, 2900, 3000, 3100, 3200K).

[0118] Test results are as follows Figure 6 As shown in the figure, the experimental results indicate that the range of 2800–3200 kW is [missing information]. b Within the range, when W b When the signal reaches 2900K, the corresponding T / C reaches its highest value; simultaneously, the ratio of signal T / C to background T / C (SBR) is between 2800 and 3200KW. b The value gradually decreases within the range. To obtain higher T / C and SBR, W... b The preferred value is 2900K.

[0119] Example 3

[0120] Important factors affecting T / C in Example 2 (t) E E tot [CAb] t ]、V t1-c1 t i V s and V b Optimize the selection.

[0121] (1) Preferred t E

[0122] 1. The concentration of LH to be tested is 1 mIU / mL, W b For 2900K, t E Pending, E tot 16V, [CAb] t The concentration was 0.25 mg / mL, V t1-c1 It is 12 μL / cm, t i For 3.5 minutes, V s 40 μL, V b The value is 40 μL.

[0123] 2. Set up experimental groups: Select five t groups. E The optimal values ​​(50, 166, 250, 350, 450ms) were selected through an optimization experiment.

[0124] 3. The 3D-BP-ECL-LFIS detection process is as described in Example 2, and the test results are as follows: Figure 7 As shown.

[0125] The experimental results show that t E Within the range of 50–450 ms, when t E When 166ms is reached, T / C reaches its maximum value, while t E The SBR gradually decreases within the range of 50–450 ms. To obtain higher T / C and SBR, t E The preferred value is 166ms.

[0126] (2) Preferred E tot

[0127] 1. The concentration of LH to be tested is 1 mIU / mL, W b For 2900K, t E For 166ms, E tot Pending, [CAb] t The concentration was 0.25 mg / mL, V t1-c1 It is 12 μL / cm, t i For 3.5 minutes, V s 40 μL, Vb The value is 40 μL.

[0128] 2. Set up experimental groups: Select five E tot Optimization experiments were conducted using numerical values ​​(8, 10, 12, 14, 15, 16, 17, 18V).

[0129] 3. The 3D-BP-ECL-LFIS detection process is as described in Example 2, and the test results are as follows: Figure 8 As shown.

[0130] The experimental results show that E tot Within the range of 8–18V, when E tot Below 16V, T / C increases with E tot Increase and increase; when E tot Above 16V, T / C increases with E tot It increases and then decreases. Therefore, E tot The preferred voltage is 16V.

[0131] (3) Preferred [CAb] t ]

[0132] 1. The concentration of LH to be tested is 1 mIU / mL, W b For 2900K, t E For 166ms, E tot 16V, [CAb] t [To be determined, V] t1-c1 It is 12 μL / cm, t i For 3.5 minutes, V s 40 μL, V b The value is 40 μL.

[0133] 2. Set up experimental groups: Select five [CAb] groups. t The optimal values ​​(150, 200, 250, 300, 350 μg / mL) were used in the optimization experiment.

[0134] 3. The 3D-BP-ECL-LFIS detection process is as described in Example 2, and the test results are as follows: Figure 9 As shown.

[0135] The experimental results show that when [CAb] t When the concentration is below 250 μg / mL, the T / C ratio increases with [CAb]. t [CAb] increases as it increases, but when [CAb] increases as it increases, [CAb] increases as it increases. t When the concentration is above 250 μg / mL, the T / C ratio increases with [CAb]. t The increase and decrease in [CAb] is likely due to the stacking of excess protein molecules on the detection pad, which hinders electron transfer between the luminescent reagent and the anode of the integrated bipolar electrode. Therefore, [CAb]t The preferred concentration is 250 μg / mL.

[0136] (4) Preferred V t1-c1

[0137] 1. The concentration of LH to be tested is 1 mIU / mL, W b For 2900K, t E For 166ms, E tot 16V, [CAb] t The concentration was 0.25 mg / mL, V t1-c1 Pending, t i For 3.5 minutes, V s 40 μL, V b The value is 40 μL.

[0138] 2. Set up experimental groups: Select five V groups. t1-c1 Optimization experiments were conducted using numerical values ​​(8, 10, 12, 14, 16 μL).

[0139] 3. The 3D-BP-ECL-LFIS detection process is as described in Example 2, and the test results are as follows: Figure 10 As shown.

[0140] The experimental results show that there was no significant difference in T / C within the range of 8–16 μL. When V t1-c1 When the volume changes from 8 μL to 12 μL, the ECL signal intensity on the T and C lines varies with V. t1-c1 Increase significantly; when V t1-c1 When the volume was changed from 12 μL to 16 μL, the ECL signal slowly increased. This phenomenon may be due to the gradual saturation of the added ECL signaling antibody. Therefore, V t1-c1 The preferred value is 12 μL.

[0141] (5) Preferred t i

[0142] 1. The concentration of LH to be tested is 1 mIU / mL, W b For 2900K, t E For 166ms, E tot 16V, [CAb] t The concentration was 0.25 mg / mL, V t1-c1 It is 12 μL / cm, t i Pending, V s 40 μL, V b The value is 40 μL.

[0143] 2. Set up experimental groups: Select five t groups. i The optimal values ​​(1.5, 2.5, 3.5, 4.5, 5.5 min) were selected through an optimization experiment.

[0144] 3. The 3D-BP-ECL-LFIS detection process is as described in Example 2, and the test results are as follows: Figure 11 As shown.

[0145] The experimental results show that, within the range of 1.5–3.5 min, the ECL signal intensity and T / C ratio on the T line change with t. i Increases as t increases; i After 5.5 minutes of further change, the ECL signal intensity and T / C tended to stabilize. This phenomenon may be because the sandwich immune complex reaches saturation after the reaction, and the number of molecules formed tends to stabilize, reaching a plateau phase. Therefore, t i The preferred time is 3.5 min.

[0146] (6) Preferred V s

[0147] 1. The concentration of LH to be tested is 1 mIU / mL, W b For 2900K, t E For 166ms, E tot 16V, [CAb] t The concentration was 0.25 mg / mL, V t1-c1 It is 12 μL / cm, t i For 3.5 minutes, V s Pending, V b The value is 40 μL.

[0148] 2. Set up experimental groups: Select five V groups. s Optimization experiments were conducted using numerical values ​​(30, 35, 40, 45, 50 μL).

[0149] 3. The 3D-BP-ECL-LFIS detection process is as described in Example 2, and the test results are as follows: Figure 12 As shown.

[0150] The experimental results show that when V s When the volume changes from 30 μL to 40 μL, the ECL signal intensity and T / C on the T line change with V. s Increases as V increases; s When the volume was further reduced to 50 μL, the ECL signal intensity and T / C tended to stabilize. This phenomenon may be because the sandwich immune complex reached saturation after the reaction, and the number of molecules formed tended to stabilize, reaching a plateau phase. Therefore, V s The preferred value is 40 μL.

[0151] (7) Preferred V b

[0152] 1. The concentration of LH to be tested is 1 mIU / mL, W b For 2900K, tE For 166ms, E tot 16V, [CAb] t The concentration was 0.25 mg / mL, V t1-c1 It is 12 μL / cm, t i For 3.5 minutes, V s 40 μL, V b To be determined.

[0153] 2. Set up experimental groups: Select five V groups. b The optimal values ​​(35, 40, 45, 50, 55 μL) were selected through an optimization experiment.

[0154] 3. The 3D-BP-ECL-LFIS detection process is as described in Example 2, and the test results are as follows: Figure 13 As shown.

[0155] The experimental results show that V b As the volume gradually changes from 35 μL to 55 μL, the T / C gradually decreases, while the SRB initially increases and then gradually decreases; when the volume... b The SRB reaches its maximum value at a concentration of 40 μL. A possible reason for this phenomenon is the gradual decrease in ECL signal strength on the C line. To obtain higher T / C and SBR, V... b The preferred value is 40 μL.

[0156] Example 4

[0157] Using the preferred conditions explored in Example 3, LH detection was performed using the 3D-BP-ECL-LFIS from Example 1.

[0158] 1. W b For 2900K, t E For 166ms, E tot 16V, [CAb] t The concentration was 0.25 mg / mL, V t1-c1 It is 12 μL / cm, t i For 3.5 minutes, V s 40 μL, V b The volume of the solution was 40 μL; the volume ratio of the T-line signal antibody, C-line signal antibody, and signal antibody was 9:9:2.

[0159] 2. Set up experimental groups: Set up several different LH concentration values ​​(0.01, 0.1, 0.5, 1, 2.5, 5, 10 and 24 mIU / mL), with 0 mIU / mL LH as a blank control.

[0160] 3. The 3D-BP-ECL-LFIS testing process is the same as in Example 2, and the test results are as follows: Figure 14 As shown.

[0161] The experimental results show that within the range of 0.01–24 mIU / mL, T / C increases with increasing LH concentration; T / C (represented by Y) has a good linear relationship with the logarithm of LH concentration (represented by X). The fitted linear equation is Y = 1.173X + 2.782(R²). 2 =0.9987, n=6), the limit of detection is estimated to be 0.0070 mIU / mL. The limit of detection is calculated as follows: Y L =Y b +3S b , where Y b This represents the mean T / C, S of the blank control group. b The standard deviation of the blank control group is represented by the obtained Y. L Substituting into the linear equation yields the LH concentration (i.e., the detection limit).

[0162] Example 5

[0163] Using the preferred conditions explored in Example 3, LH detection was performed using the 3D-BP-ECL-LFIS from Example 1.

[0164] 1. W b For 2900K, t E For 166ms, E tot 16V, [CAb] t The concentration was 0.25 mg / mL, V t1-c1 It is 12 μL / cm, t i For 3.5 minutes, V s 40 μL, V b The volume of the solution was 40 μL; the volume ratio of the T-line signal antibody, C-line signal antibody, and signal antibody was 6:3:1.

[0165] 2. Set up experimental groups: Set up several different LH concentration values ​​(0.01, 0.1, 0.5, 1, 2.5, 5, 10 and 24 mIU / mL), with 0 mIU / mL LH as a blank control.

[0166] 3. The 3D-BP-ECL-LFIS testing process is the same as in Example 2, and the test results are as follows: Figure 15 As shown.

[0167] The experimental results show that within the range of 0.01–24 mIU / mL, T / C increases with increasing LH concentration; T / C (represented by Y) has a good linear relationship with the logarithm of LH concentration (represented by X). The fitted linear equation is Y = 1.915X + 4.308(R²). 2 =0.9963, n=6), the detection limit is estimated to be 0.0074 mIU / mL, and the detection limit is calculated in the same way as above.

[0168] Example 6

[0169] Using the preferred conditions explored in Example 3, actual sample testing experiments were conducted using the 3D-BP-ECL-LFIS from Example 1.

[0170] 1. W b For 2900K, t E For 166ms, E tot 16V, [CAb] t The concentration was 0.25 mg / mL, V t1-c1 It is 12 μL / cm, t i For 3.5 minutes, V s 40 μL, V b The value is 40 μL.

[0171] 2. Experimental group setup: Human urine samples from groups A, B, C, and D were diluted 10-fold with PBS. ELISA kits were used to detect the actual samples, and 3D-PB-ECL-LFIS was also used to detect the same actual samples. Finally, the detection results of the ELISA method and the 3D-PB-ECL-LFIS method were compared.

[0172] 3. The 3D-BP-ECL-LFIS testing process is the same as in Example 2, and the test results are as follows: Figure 16 As shown.

[0173] Linear fitting was performed using the average LH concentration from six 3D-PB-ECL-LFIS assays as the ordinate and the LH concentration from two ELISA assays as the abscissa. When the volume ratio of the T-line signal antibody, C-line signal antibody, and signal antibody in the treatment solution was 9:9:2, the linear fitting equation was Y = 1.0394X + 0.13734(R²). 2 =0.9986); When the volume ratio of the T-line signal antibody, C-line signal antibody, and signal antibody treatment solution is 6:3:1, the linear fitting equation is Y = 0.9587X + 0.95868(R² = 0.9986); 2 =0.9985). The experimental results show that the 3D-PB-ECL-LFIS method has a high correlation with the ELISA method, indicating that the 3D-PB-ECL-LFIS method of the present invention has good reliability in detecting LH in actual samples.

[0174] Example 7

[0175] Storage stability experiments were conducted using the preferred conditions explored in Example 3 and the 3D-BP-ECL-LFIS from Example 1.

[0176] 1. W bFor 2900K, t E For 166ms, E tot 16V, [CAb] t The concentration was 0.25 mg / mL, V t1-c1 It is 12 μL / cm, t i For 3.5 minutes, V s 40 μL, V b The value is 40 μL.

[0177] 2. Setting up the experimental group: 3D-BP-ECL-LFIS prepared with T-line signal antibody, C-line signal antibody and signal antibody treatment liquid volume ratios of 9:9:2 and 6:3:1 respectively was vacuum sealed and placed in a 37℃ oven; then T / C was measured every 4 days for seven weeks.

[0178] 3. The 3D-BP-ECL-LFIS testing process is the same as in Example 2, and the test results are as follows: Figure 17 As shown.

[0179] The experimental results show that when the volume ratio of T-line signal antibody, C-line signal antibody, and signal antibody treatment solution is 9:9:2, 3D-BP-ECL-LFIS can be stored at 37°C for 28 days. According to the Arrhenius equation, 28 days of accelerated testing at 37°C is equivalent to four years of storage at 4°C. When the volume ratio of T-line signal antibody, C-line signal antibody, and signal antibody treatment solution is 6:3:1, 3D-BP-ECL-LFIS can be stored at 37°C for 24 days. According to the Arrhenius equation, 24 days of accelerated testing at 37°C is equivalent to three years and six months of storage at 4°C. These results indicate that 3D-BP-ECL-LFIS has good storage stability. The longer storage period is achieved with a volume ratio of 9:9:2, which may be related to the higher ECL signal intensity on the C-line. This also demonstrates that the volume ratio of T-line signal antibody, C-line signal antibody, and signal antibody treatment solution can affect the storage stability of 3D-BP-ECL-LFIS.

[0180] Example 8

[0181] A 3D-BP-ECL-LFIS, the composition and structure of which are as follows: Figure 18 As shown, it includes a three-dimensional bipolar ECL lateral flow immunoassay strip 1 and a cartridge 2;

[0182] The independent, single three-dimensional bipolar ECL lateral flow immunoassay strip 1 is composed of a large-format three-dimensional bipolar ECL lateral flow immunoassay strip ( Figure 19 It is prepared by cutting using an induction strip cutter (model HGS220S, Hangzhou Fenghang Technology Co., Ltd.);

[0183] The large-format three-dimensional bipolar ECL lateral flow immunoassay strip includes a large-format three-dimensional bipolar electrode sheet ( Figure 20 Large-format lateral flow immunoassay strips ( Figure 21 ) and large board connecting pad ( Figure 22 );

[0184] The large-format three-dimensional bipolar electrode sheet includes a large-format electrode sheet layer one ( Figure 23 ) and large electrode sheet layer two ( Figure 24 The large electrode layer one and the large electrode layer two each consist of 25 horizontally arranged electrode layers one and two, which are aligned and bonded together by the adhesive backing of the large electrode layer one.

[0185] The large-format lateral flow immunoassay strip consists of a large-format adhesive carrier (A), a large-format absorbent pad (B), a large-format detection pad (C), a large-format conjugation pad (D), and a large-format sample pad (E). Figure 25 The test strip is constructed from 25 horizontally arranged independent lateral flow immunoassay strips. A large three-dimensional bipolar electrode sheet is bonded to the large lateral flow immunoassay strip via double-sided adhesive on the electrode sheet layer 1. The large connecting pad is attached to the shared cathode and positive driving electrode tail end on the electrode sheet layer 1 via double-sided adhesive on the electrode sheet layer 1, and is arranged parallel to the large lateral flow immunoassay strip. Through the above process, the required large three-dimensional bipolar ECL lateral flow immunoassay strip is obtained for cutting with an inductive strip cutter, thereby producing an independent, single three-dimensional bipolar ECL lateral flow immunoassay strip 1.

[0186] 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. A three-dimensional bipolar ECL lateral flow immunoassay strip, characterized in that... It includes a three-dimensional bipolar electrode sheet, a lateral flow immunoassay strip, and a connecting pad; the three-dimensional bipolar electrode sheet is at the bottom, and the lateral flow immunoassay strip and the connecting pad are at the top; The three-dimensional bipolar electrode sheet adopts a layered design, including electrode sheet layer one and electrode sheet layer two; electrode sheet layer two is at the bottom and electrode sheet layer one is at the top. The second electrode layer includes a positive driving electrode; the positive driving electrode includes a positive driving electrode head end, a positive driving electrode tail end, and a connecting wire thereof. The electrode layer one includes a negative driving electrode and an integrated bipolar electrode; the integrated bipolar electrode includes a detection anode, a quality control anode and a shared cathode in sequence, wherein one end of the detection anode is close to the negative driving electrode; The negative driving electrode is positioned to correspond to the first end of the positive driving electrode of electrode layer two. The shared cathode and the positive driving electrode are spaced apart and are covered by a connecting pad.

2. The three-dimensional bipolar ECL lateral flow immunoassay strip according to claim 1, characterized in that: In the integrated bipolar electrode, the detection anode, the quality control anode, and the shared cathode are located in a straight line.

3. The three-dimensional bipolar ECL lateral flow immunoassay strip according to claim 1, characterized in that: The lateral flow immunoassay strip includes a sample pad, a conjugate pad, a detection pad, and an absorbent pad; the lateral flow immunoassay strip and the connecting pad are located in a straight line.

4. The three-dimensional bipolar ECL lateral flow immunoassay strip according to claim 3, characterized in that: The detection pad includes a T-line and a C-line.

5. The three-dimensional bipolar ECL lateral flow immunoassay strip according to claim 4, characterized in that: The T-line and C-line correspond to the detection anode and the quality control anode, respectively; the sample pad is located between the shared cathode and the quality control anode, and the binding pad is located between the sample pad and the detection pad; the absorbent pad is close to the detection anode and in contact with part of the negative driving electrode.

6. The three-dimensional bipolar ECL lateral flow immunoassay strip according to claim 5, characterized in that: During preparation, the sample pad, binding pad, detection pad and absorbent pad are stacked sequentially on the adhesive-backed carrier, and then upside down on the electrode sheet layer one.

7. The three-dimensional bipolar ECL lateral flow immunoassay strip according to claim 6, characterized in that: During preparation, the lateral flow immunoassay strip, connecting pad, and three-dimensional bipolar electrode sheet are first made into large-scale models, then bonded together and cut into individual three-dimensional bipolar ECL lateral flow immunoassay strips.

8. The three-dimensional bipolar ECL lateral flow immunoassay strip according to claim 1, characterized in that: The electrode sheet is bonded with hydrophobic double-sided adhesive to fix the lateral flow immunoassay strip and the connecting pad.

9. The three-dimensional bipolar ECL lateral flow immunoassay strip according to claim 3, characterized in that: The aforementioned binding pad is used to dry and fix ECL signaling antibodies.

10. The three-dimensional bipolar ECL lateral flow immunoassay strip according to claim 4, characterized in that: The T-line packet is captured by T-line antibodies, and the C-line packet is captured by C-line antibodies.

11. A three-dimensional bipolar ECL lateral flow immunosensor, characterized in that... Includes the three-dimensional bipolar ECL lateral flow immunoassay strip as described in any one of claims 1 to 10.

12. The three-dimensional bipolar ECL lateral flow immunosensor according to claim 11, characterized in that... It also includes jamming.

13. The three-dimensional bipolar ECL lateral flow immunosensor according to claim 12, characterized in that: The casing includes a top cover and a bottom plate. The top cover has an observation window, a sample application hole, and a buffer solution addition hole, all of which are located in a straight line. The bottom plate has an electrode contact area and four sets of protrusions for fixing the three-dimensional bipolar ECL lateral flow immunoassay strip.

14. The application of the three-dimensional bipolar ECL lateral flow immunoassay strip according to any one of claims 1 to 10 and / or the three-dimensional bipolar ECL lateral flow immunoassay sensor according to any one of claims 11 to 13 in the immunoassay detection of multiple biomarkers.

15. The application according to claim 14, 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 linein.

16. The application according to claim 14, characterized in that... Includes the following steps: (1) Add a sample solution containing the biomarker to be tested to the sample pad and perform an immune reaction; (2) Add buffer solution to the sample pad to rinse the residue on the test pad onto the absorbent pad; at the same time, add buffer solution to the connection pad to connect the circuit; (3) The three-dimensional bipolar ECL lateral flow immunoassay strip or sensor processed in steps (1) and (2) is placed into the ECL instant detection device for detection. After setting the camera white balance, camera exposure time and driving voltage, the ECL immunoassay reaction is performed. Finally, the ratio of the luminescence signal value on the T line to the luminescence signal value on the C line is used for quantitative analysis of the biomarker to be tested.

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