A closed-loop bipolar electrochemiluminescence lateral current gene detection method

Abstract

This application discloses a closed-loop bipolar electrochemiluminescence lateral flow gene detection method, which includes the following steps: designing a C-region capture probe and a signal probe, as well as a T-region capture probe and a signal probe, based on the target DNA sequence; coating a signal probe coupled with a terpyridine-ruthenium-polylysine complex onto the hydrophilic channel A of the test strip binding sheet; modifying and fixing the C-region capture probe and the T-region capture probe respectively in the corresponding regions by streaking a mixed solution containing the capture probe; sequentially adding the test sample solution and a buffer solution to the hydrophilic channel A of the test strip loading sheet; simultaneously adding a buffer solution to the hydrophilic channel B to connect the circuit and trigger the electrochemiluminescence reaction; and collecting the electrochemiluminescence signal to achieve detection. The detection method of this invention uses a self-enhanced electrochemiluminescence probe as a signal probe and modifies it on the binding sheet to amplify the electrochemiluminescence signal, thereby achieving ultrasensitive quantitative detection of T-DNA.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic detection, specifically relating to a closed-loop bipolar electrochemiluminescence lateral flow gene detection method. Background Technology

[0002] Microfluidics is a technology that allows for the precise control and manipulation of microscale fluids, enabling automation and high efficiency throughout the entire analytical process. Due to its advantages such as low reagent consumption, high sensitivity, and low analytical cost, it has become a research hotspot in the field of point-of-care testing (POCT) in recent years. Currently, based on the combination of microfluidics with various detection technologies, a variety of rapid and convenient methods for detecting infectious diseases have been developed, demonstrating great application potential in the field of in vitro diagnostics.

[0003] Genes are the basic units of heredity, consisting of DNA or RNA sequences that carry genetic information and are passed on to the next generation through replication. Gene testing refers to the technique of examining genes through blood or other bodily fluids. In many infectious diseases, pathogen quantification has proven to be a useful prognostic indicator and an important reference for monitoring treatment response. Currently, some commonly used gene testing methods include electrochemiluminescence immunoassay, electrochemical methods, fluorescence analysis, and colorimetric analysis.

[0004] Electrochemiluminescence is a chemiluminescence phenomenon excited by electrochemistry. As early as 1991, Bard et al. used Ru(bpy)3... 2+ Electrochemiluminescence has been applied to immunoassays and DNA analysis. With the continuous advancement of research, electrochemiluminescence, with its advantages of low background, high sensitivity, wide detection range, and good controllability, has developed into a powerful analytical technique widely used in the field of biosensing. It has made substantial progress in basic research, environmental monitoring, clinical diagnosis, immunoassay, and food safety.

[0005] Lateral flow assays can be used to detect the presence of target analytes in samples (urine, saliva, sweat, serum, plasma, whole blood, etc.), offering the advantage of providing quantitative, semi-quantitative, or qualitative results within minutes without requiring specific and expensive equipment. This method is low-cost, easy to use, has a short testing time, and can be stored stably, making it ideal for medical diagnostics, home testing, point-of-care testing (POCT), or laboratory applications. Summary of the Invention

[0006] The purpose of this invention is to provide a closed-loop bipolar electrochemiluminescence lateral flow gene detection method. This method solves the problems of complex operation, high cost, and long time consumption in current nucleic acid detection processes, and can achieve rapid quantification of pathogens as well as detection with high specificity and high sensitivity.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A closed-loop bipolar electrochemiluminescence lateral flow gene detection method is based on lateral flow gene detection strips and / or detection kits;

[0009] The lateral flow gene detection reagent strip includes an electrode and two hydrophilic channels A and B, with the electrode partially overlapping the two hydrophilic channels.

[0010] The hydrophilic channels A and B are distributed on the sample application plate, binding plate, detection plate, and absorption plate;

[0011] The sample dispensing plate is used to add the sample solution to be tested and to allow the sample solution to flow to the detection plate;

[0012] The binding sheet is used to dry the signal probe labeled with an electrochemiluminescent substance. One end of it is connected to the detection sheet, and the other end is connected to the sample application sheet.

[0013] The electrode is located on the electrode plate;

[0014] The electrode includes an integrated bipolar electrode and a pair of driving electrodes (positive and negative); the integrated bipolar electrode includes a cathode and at least two anodes connected in parallel; wherein, the integrated bipolar electrode anode closer to the sample plate is called the quality control anode, and the integrated bipolar electrode anode closer to the absorption plate is called the detection anode;

[0015] During sample addition and testing, the anode of the integrated dual-polarity electrode and the corresponding negative drive electrode are located in the reporting channel, while the cathode of the integrated dual-polarity electrode and the corresponding anode drive electrode are located in the support channel.

[0016] The detection strip overlaps with the integrated bipolar electrode; the one that overlaps with the anode of the integrated bipolar electrode is called hydrophilic channel A, and the other hydrophilic channel is called hydrophilic channel B.

[0017] The portion of the hydrophilic channel A that overlaps with the control anode is called the control zone (C zone), and the portion of the hydrophilic channel A that overlaps with the detection anode is called the detection zone (T zone). The C zone and the T zone are used to modify and fix the capture probes of the control zone and the detection zone, respectively, for monitoring the feasibility of the test strip and the concentration of biomarkers.

[0018] The test strip is made of nitrocellulose membrane.

[0019] A base plate can be mounted under the sample loading plate, binding plate, detection plate, electrode plate, and absorption plate.

[0020] The lateral flow gene detection kit contains the aforementioned detection reagent strips;

[0021] The test kit also includes an upper cover, a transparent cover, and a lower cover.

[0022] The reagent strip is fixed on the lower cover, and the transparent cover plate covers the binding plate, detection plate, electrode plate and absorption plate (partially covered) of the reagent strip;

[0023] The top cover covers the transparent cover plate;

[0024] The top cover is provided with a sample addition hole, a buffer addition hole, an observation window, and an electrode contact area;

[0025] The sample application well and buffer addition well correspond to hydrophilic channel A and hydrophilic channel B on the sample application strip, respectively;

[0026] The observation window corresponds to the quality control area and the detection area located in the hydrophilic channel A on the test piece;

[0027] The electrode contact area corresponds to the driving electrode.

[0028] The closed-loop bipolar electrochemiluminescence lateral current gene detection method includes the following steps:

[0029] (1) Design probe DNA sequences

[0030] Design C-region capture probes and signal probes, as well as T-region capture probes and signal probes, based on the target DNA (T-DNA) sequence;

[0031] The C-region capture probe has its 5' end modified with biotin;

[0032] The C-region signal probe is modified with an amino group at its 5' end;

[0033] The T-region capture probe has its 3' end modified with biotin;

[0034] The T-region signal probe is modified with an amino group at its 5' end;

[0035] The 5' end of the T-DNA is complementary to the 3' end of the T-region capture probe;

[0036] The 3' end of the T-DNA is complementary to the 5' end of the T-region signal probe;

[0037] The C-region capture probe sequence is completely complementary to the C-region signal probe sequence.

[0038] The number of complementary base pairs between the T-DNA and the T-region signal probe and capture probe is preferably 18 bp;

[0039] (2) Pretreatment and modification of test strips

[0040] Add the treatment solution to the hydrophilic channel A of the test strip and dry it. The treatment solution is used to improve the hydrophilicity of the test strip and increase the absorption rate of the test sample solution.

[0041] A treatment solution was added to the hydrophilic channel A of the test strip binding tablet and dried. Then, a signal probe coupled with a terpyridine ruthenium-polylysine complex was drop-coated onto the hydrophilic channel A of the test strip binding tablet and vacuum dried at 25℃-37℃. The addition of the treatment solution was to improve the stability of the signal probe, so that the signal probe could be released better and faster, and to eliminate non-specific adsorption.

[0042] The test strips of the test reagent strips are soaked in surfactant solution and dried at 37°C under constant temperature and humidity. The mixed solution containing the capture probes is used to modify and fix the capture probes in the corresponding areas of the C region and the T region by scribing with a film scribing instrument. The modification width is preferably 2 mm. The pretreatment with surfactant solution is to enable the test sample solution to diffuse evenly on the test strip and make the luminescence effect more stable.

[0043] Add the treatment solution to the hydrophilic channel A of the absorbent strip of the test reagent and dry it. The purpose of adding the treatment solution is to improve the fluidity of the liquid.

[0044] The sample preparation solution is a buffer solution (pH 7.4) containing 1% SDS, 0.5% PVP, and 0.5% BSA;

[0045] The conjugate treatment solution is a buffer solution (pH 7.4) containing 0.5% gelatin, 2.5% trehalose, 1% BSA, 0.1% Triton X-100, and 0.1% Proclin 300 preservatives; the amount of the modified signal probe on the conjugate is 6 μL;

[0046] The surfactant solution of the detection strip is a mixed solution containing 0.5% S9 (Tetronic 1307) and 0.9% NaCl; the mixed solution containing the capture probe is a mixed solution containing 5% sucrose, 5% trehalose, 0.5 mg / mL streptavidin and 50 μM capture probe.

[0047] The absorbent tablet treatment solution is a buffer solution containing 0.5% Tween.

[0048] (3) Sample testing

[0049] Add the test sample solution to the hydrophilic channel A of the test strip. After waiting for several minutes, add buffer solution to the hydrophilic channel A on the test strip. This washes away any unbound signal probes in the detection area and fills the hydrophilic channel A with solution. At the same time, add buffer solution to the hydrophilic channel B to connect the circuit. Then connect the positive and negative electrodes of the test strip to the power supply and apply an appropriate voltage to trigger the electrochemiluminescence reaction.

[0050] Electrochemiluminescence signals are acquired in the form of images and videos, and further analyzed by the program to achieve qualitative and quantitative detection of pathogen nucleic acids;

[0051] The driving voltage of the closed-loop bipolar electrochemiluminescence is preferably 8.5V; the hybridization reaction time is preferably 3min; the buffer solution is phosphate-buffered saline (PBS); the electrochemiluminescence lateral flow gene detection method can be used to detect one of the following: nucleic acid, amplification product, CRISPR / Cas cis and trans cleavage products, protein, and small molecule marker.

[0052] The basic principle of this invention is:

[0053] The biotin-modified capture probe and streptavidin were incubated at room temperature. The capture probe was then applied to the corresponding area on the detection strip using a streaking device. The affinity between biotin and streptavidin immobilized the capture probe on the detection strip, improving the immobilization efficiency and stability. The immobilized capture probe then hybridized with T-DNA, resulting in a hybridization complex that was immobilized in the detection area.

[0054] When the test sample solution containing T-DNA is added to the sample application strip of the reagent strip, the solution flows through the binding strip, where the T-DNA hybridizes with the T-region signal probe modified on the binding strip to form a "T-region signal probe-T-DNA" complex. It then flows further to the detection strip, where a "T-region signal probe-T-DNA-T-region capture probe" hybridization complex is formed after a complete hybridization reaction. Simultaneously, the C-region signal probe hybridizes with the C-region capture probe to form a "C-region signal probe-C-region capture probe" hybridization complex. After the reaction is complete, a buffer solution is added to the hydrophilic channel A of the sample application strip to wash away any unbound signal probes in the detection area and to fill the hydrophilic channel A with solution. At the same time, a buffer solution is added to the hydrophilic channel B to connect the circuit. Finally, the positive and negative electrodes of the reagent strip's driving electrode are connected to a power source, and an appropriate voltage is applied to trigger the electrochemiluminescence reaction.

[0055] The self-enhanced electrochemiluminescence probe in the hybridization complex is coupled with a terpyridine ruthenium-polylysine complex, which is enriched with a large number of luminescent groups, effectively amplifying the electrochemiluminescence signal and enabling ultrasensitive quantitative detection of the biomarker T-DNA.

[0056] The present invention has the following advantages and effects compared with the prior art:

[0057] 1. The test strip of the present invention uses a nitrocellulose membrane. Compared with traditional electrochemiluminescence chips, the modification operation is simple, the reaction is fast and sensitive, the detection results are more stable, and false positive results caused by solution residue are avoided to a certain extent.

[0058] 2. In addition to the test strip, the test strip of the present invention can use materials such as cloth, paper, and glass fiber as substrate materials. The materials are simple, readily available, and environmentally friendly, resulting in lower costs and facilitating mass production.

[0059] 3. The electrode pad of the test strip of the present invention is a closed bipolar electrode, with two bipolar anode electrodes symmetrically distributed in parallel. This design not only reduces the area of ​​the electrode and hydrophilic channel, but also reduces the space occupied by the electrode, reduces the consumption of printing materials and reagents, and lowers the cost.

[0060] 4. The electrode pads of the test strip of the present invention have the potential to arrange multiple detection anodes, enabling multi-channel multi-element detection.

[0061] 5. The detection method of the present invention uses a self-enhanced electrochemiluminescence probe as a signal probe and modifies it on the binding plate to amplify the electrochemiluminescence signal, thereby achieving ultrasensitive quantitative detection of T-DNA.

[0062] 6. The detection method of the present invention can be used to detect amplification products, CRISPR / Cas products, proteins, small molecule markers, etc., and the detection methods and detection modes are diversified, with good versatility and practicality.

[0063] 7. The test strips and kits of the present invention are small and portable, do not require large instruments as the basis for detection, and are simple to operate and easy to carry compared with traditional electrochemiluminescence analyzers. They can be used for POCT applications in different scenarios and meet the needs of home-use self-test kits. Attached Figure Description

[0064] Figure 1 This is a schematic diagram showing the structural breakdown of a closed-loop bipolar electrochemiluminescence lateral flow gene detection reagent strip.

[0065] Figure 2 This is a schematic diagram of the overall structure of a closed-loop bipolar electrochemiluminescence lateral flow gene detection reagent strip.

[0066] Figure 3 This is a schematic diagram of the composition and structure of the electrode sheet of a closed-loop bipolar electrochemiluminescence lateral flow gene detection reagent strip.

[0067] Figure 4This is a structural breakdown diagram of a closed-loop bipolar electrochemiluminescence lateral flow gene detection kit.

[0068] Figure 5 This is a schematic diagram of the overall structure of a closed-loop bipolar electrochemiluminescence lateral flow gene detection kit.

[0069] Figure 6 Electrochemiluminescence T / C numerical bars for CRISPR / Cas12a cis-cleavage products of PCR amplification products at different concentrations.

[0070] Figure 7 This is a bar chart showing the electrochemiluminescence T / C ratios of convective PCR products at different dilutions.

[0071] Figure 8 The bar chart shows the electrochemiluminescence T / C values ​​of convection PCR amplification products after 100-fold dilution at different amplification times.

[0072] Wherein: 1-Sample loading strip; 2-Binding strip; 3-Detection strip; 4-Electrode strip; 5-Absorption strip; 6-Base plate; 7-Support channel; 8-Report channel; 9-Bipolar electrode quality control anode; 10-Bipolar electrode detection anode; 11-Drive electrode negative electrode; 12-Drive electrode positive electrode; 13-Bipolar electrode cathode; 14-Top cover; 15-Transparent cover; 16-Closed bipolar electrochemiluminescence lateral flow gene detection reagent strip; 17-Bottom cover; 18-Sample loading hole; 19-Buffer solution addition hole; 20-Observation window; 21-Electrode contact area. Detailed Implementation

[0073] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the implementation of the present invention is not limited thereto.

[0074] Example 1

[0075] The design and fabrication of electrochemiluminescence lateral flow gene detection strips and kits include the following steps:

[0076] (1) Design of reagent strips

[0077] The electrochemiluminescence lateral flow gene detection reagent strip includes electrodes and two hydrophilic channels A and B, such as... Figure 1 and Figure 2 As shown, the electrode partially overlaps with the two hydrophilic channels;

[0078] The hydrophilic channels A and B are distributed on the sample application plate 1, the binding plate 2, the detection plate 3, and the absorption plate 5;

[0079] The sample addition tablet 1 is used to add the sample solution to be tested. It can perform chromatographic filtration of the sample solution to be tested, and quickly absorb the sample solution to be tested and flow to the binding tablet 2.

[0080] The binding plate 2 is used to dry the signal probe labeled with electrochemiluminescent material. One end of it is connected to the detection plate 3 and the other end is connected to the sample loading plate 1, thus creating a good flow channel for the sample to be tested.

[0081] The electrode is located on electrode plate 4;

[0082] The electrode comprises an integrated bipolar electrode and a pair of driving electrodes (negative electrode 11 and positive electrode 12), such as Figure 3 As shown; the integrated bipolar electrode includes a cathode (13) and at least two anodes (9 and 10) connected in parallel; wherein, the integrated bipolar electrode anode closer to the sample loading sheet 1 is called the quality control anode 9, and the integrated bipolar electrode anode closer to the absorption sheet 5 is called the detection anode 10;

[0083] During sample addition and testing, the anode (9 and 10) of the integrated bipolar electrode and the corresponding negative electrode 11 of the driving electrode are located in the reporting channel 8, and the cathode 13 of the integrated bipolar electrode and the corresponding positive electrode 12 of the driving electrode are located in the support channel 7.

[0084] The detection piece 3 overlaps with the integrated bipolar electrode; the one that overlaps with the anode (9 and 10) of the integrated bipolar electrode is called hydrophilic channel A, and the other hydrophilic channel is called hydrophilic channel B.

[0085] The portion of the hydrophilic channel A corresponding to the control anode 9 is called the control region (C region), and the portion of the hydrophilic channel A corresponding to the detection anode 10 is called the detection region (T region). The C region and the T region are used to modify and fix the capture probe of the control region (i.e., the C region capture probe) and the capture probe of the detection region (i.e., the T region capture probe), respectively, for monitoring the feasibility of the test strip and the T-DNA concentration.

[0086] A base plate 6 can be mounted under the sample loading plate 1, binding plate 2, detection plate 3, electrode plate 4, and absorption plate 5.

[0087] (2) Preparation of reagent strips

[0088] The shapes of the hydrophilic channels and electrodes in sample sheet 1, bonding sheet 2, detection sheet 3, electrode sheet 4, and absorption sheet 5 were designed using the drawing software Adobe Illustrator CS6, and based on this, 300-mesh polyester microchannel mesh and electrode mesh were fabricated.

[0089] Sample plate 1, binding plate 2 and absorbent plate 5 are made by PP ink screen printing technology, and hydrophilic channels surrounded by hydrophobic ink are formed by screen printing non-woven fabric; detection plate 3 is made by cutting nitrocellulose membrane into a specific size; electrode plate 4 is made by screen printing hydrophobic fabric with conductive carbon ink.

[0090] (3) Structure of the electrochemiluminescence lateral flow gene detection kit

[0091] The structure of the electrochemiluminescence lateral flow gene detection kit is as follows: Figure 4 and Figure 5 As shown, it includes an upper cover 14, a transparent cover 15, a reagent strip 16, and a lower cover 17.

[0092] The reagent strip 16 is fixed on the lower cover 17, and the transparent cover plate 15 covers the binding piece 2, detection piece 3, electrode piece 4 and absorption piece 5 of the reagent strip 16 (partially covered);

[0093] The upper cover 14 covers the transparent cover plate 15;

[0094] The upper cover 14 is provided with a sample addition hole 18, a buffer addition hole 19, an observation window 20, and an electrode contact area 21;

[0095] The sample application well 18 and buffer addition well 19 correspond to the sample application strip 1 of hydrophilic channel A and hydrophilic channel B, respectively;

[0096] The observation window 20 corresponds to the C and T regions on the detection piece 3 located in the hydrophilic channel A;

[0097] The electrode contact area 21 corresponds to the driving electrodes (negative electrode 11 and positive electrode 12).

[0098] Example 2

[0099] The application of the closed-loop bipolar electrochemiluminescence lateral flow gene detection method of the present invention in the detection of CRISPR / Cas12a cis-cleavage products is as follows:

[0100] (1) DNA extraction

[0101] The bacterial strain to be tested was cultured, and the DNA of Escherichia coli O157:H7 was extracted using the magnetic bead method. The obtained DNA solution was stored at -20℃ for later use.

[0102] (2) Polymerase chain reaction (PCR) amplification

[0103] Take a PCR tube and add 5 μL of DNA solution, 5 μL of Taq buffer (10×), 4 μL of dNTP (2.5 mM), 0.24 μL of Taq enzyme (5 U / μL), and 1 μL each of forward and reverse primers (10 μM). Mix well and place in a standard PCR instrument for amplification. Amplification parameters are set to 95℃ for 2 min for heat denaturation, followed by 35 thermal cycles (95℃, 15 s; 60℃, 30 s). After cycling, the temperature is lowered to 4℃ and maintained for a period of time. The concentration of the obtained PCR amplification product is measured to be 460 μg / μL using a UV spectrophotometer. The amplification product is stored at -20℃ for later use.

[0104] The primer sequences are as follows:

[0105] Upstream primer: 5'-ttctttttcttatacatttact-3' (SEQ ID NO.1);

[0106] Downstream primer: 5'-tacagacctgagttgcacctaa-3' (SEQ ID NO.2).

[0107] (3) CRISPR / Cas12a cis-cutting

[0108] Take a PCR tube and add 2 μL of reaction buffer (10×), 2 μL of crRNA (2 μM), 2 μL of Cas12a (1 mM), and 12 μL of nuclease-free water. Then add 2 μL of amplification product (460, 46, or 4.6 μg / μL), mix well, and incubate at 45°C for 3 min. Then heat to 95°C to inactivate the Cas protein. After 3 min, immediately place on ice to prevent the cleaved product from reforming into a double-stranded structure. Store the solution of the CRISPR / Cas12a cis-cleavage product at -20°C for subsequent detection.

[0109] The sequence of the PCR amplification product is shown in SEQ ID NO.3;

[0110] The crRNA sequence is as follows:

[0111] 5'-taatttctactaagtgtagatccaaccgtcattgacaggaa-3' (SEQ ID NO. 4);

[0112] The CRISPR / Cas12a cis-cleavage product sequence is as follows:

[0113] 5'-aggtatatcggaaggagatgaagttatattgttccaacactgacat-3' (SEQ ID NO. 5).

[0114] (4) Design the DNA sequence required for gene detection reagent strips based on the CRISPR / Cas12a cis-cut product sequence, including C region signal probe S1, T region signal probe S2, C region capture probe CP1 and T region capture probe CP2.

[0115] The designed DNA sequence is as follows:

[0116] S1: 5'-NH2-(CH2)6-ggcacaaacacgcacctc-3' (SEQ ID NO.6);

[0117] CP1: 5'-Biotin-gaggtgcgtgtttgtgcc-3' (SEQ ID NO.7);

[0118] S2: 5'-NH2-(CH2)6-tacagacctgagttgcacctaa-3' (SEQ ID NO.8);

[0119] CP2: 5'-ttctttttcttatacatttact-Biotin-3' (SEQ ID NO. 9).

[0120] (5) Pretreatment and modification of test strips

[0121] Treatment solution was added to the hydrophilic channel A of sample patch 1 of the test strip for pretreatment and drying. Treatment solution was added to the hydrophilic channel A of binding patch 2 of the test strip for pretreatment and drying. Then, 6 μL of signal probe solution (a mixed solution of signal probe S1 in region C and signal probe S2 in region T) was added to the hydrophilic channel A of the pretreated binding patch 2 and placed in a vacuum dryer at 37°C for about 30 min. The signal probe was coupled with a terpyridine ruthenium-polylysine complex (refer to Chinese patent application 2023107087058). The test patch 3 of the test strip was treated by soaking in a surfactant solution and dried at 37°C under constant temperature and humidity. The mixed solution containing the capture probe was used to modify and fix the capture probe CP1 in region C and the capture probe CP2 in region T in the corresponding areas by scribing with a scribing apparatus. The modification width was preferably 2 mm. Treatment solution was added to the hydrophilic channel A of absorber patch 5 of the test strip for pretreatment and drying.

[0122] The sample loading tablet 1 is prepared in PBS (pH 7.4) containing 1% SDS, 0.5% PVP, and 0.5% BSA; the binding tablet 2 is prepared in PBS (pH 7.4) containing 0.5% gelatin, 2.5% trehalose, 1% BSA, 0.1% Triton X-100, and 0.1% Proclin 300 preservatives, and the amount of the modified signal probe on the binding tablet 2 is 6 μL; the surfactant solution of the detection tablet 3 is a mixed solution containing 0.5% S9 (Tetronic 1307) and 0.9% NaCl; the mixed solution containing the capture probe is a mixed solution of 5% sucrose, 5% trehalose, 0.5 mg / mL streptavidin, and 50 μM capture probe; the absorption tablet 5 is prepared in PBS containing 0.5% Tween.

[0123] (6) Assembly of the test kit

[0124] like Figure 1As shown, the test strip is based on a single-sided adhesive base plate 6 and is assembled by sequentially stacking an absorption sheet 5, an electrode sheet 4, a detection sheet 3, a binding sheet 2, and a sample application sheet 1.

[0125] like Figure 4 , 5 As shown, the assembled reagent strip 16 is placed in the lower cover 17 of the outer shell, and the transparent cover 15 covers the reagent strip 16 to fix the reagent strip and provide a stable detection environment. The upper cover 14 is pressed to complete the assembly of the closed bipolar electrochemiluminescence lateral flow gene detection kit.

[0126] (7) Electrochemiluminescence detection

[0127] 20 μL of CRISPR / Cas12a cis-cleavage product solution was added to sample plate 1 through sample well 18. After the solution flowed to and filled the detection plate 3, the hybridization reaction was allowed to proceed for 3 minutes. Then, 30 μL of PBS was added to sample plate 1 through sample well 18 to wash away unbound signal probes in the detection area and to fill the reporter channel 8 with solution. Simultaneously, 20 μL of PBS was added to the support channel 7 through buffer addition well 19 to connect the circuit. Then, the driving electrode negative electrode 11 and positive electrode 12 of electrode plate 5 were connected to a DC power supply via alligator clip wires, and an 8.5V voltage was applied to trigger the electrochemiluminescence reaction.

[0128] (8) Data Acquisition and Processing

[0129] The luminescence images were acquired and analyzed using an instrument (patent application number 202210518683.4). The obtained data was then imported into Origin software for further analysis and processing. The relationship between the T / C ratio (the ratio of the luminescence intensity in the T region to the luminescence intensity in the C region) and the concentration of the PCR amplification product was obtained (each data point was calculated using 5 replicate experiments).

[0130] Test results are as follows Figure 6 As shown, it can be seen that as the concentration of PCR amplification products increases, the T / C value gradually increases, and the T / C values ​​between different concentrations are significantly different. This indicates that the closed bipolar electrochemiluminescence lateral flow gene detection method of the present invention has the ability to detect CRISPR / Cas12a cis-cleavage products.

[0131] Example 3

[0132] The application of the closed-loop bipolar electrochemiluminescence lateral flow gene detection method of the present invention in the detection of convective PCR products is as follows:

[0133] (1) DNA extraction

[0134] The bacterial strain to be tested was cultured, and DNA was extracted from Escherichia coli O157:H7 using a rapid bacterial genomic DNA extraction kit (Shanghai Sangon Biotech Co., Ltd.). The DNA solution was diluted 100 times with ultrapure water and stored at -20°C for later use.

[0135] (2) Convection PCR amplification

[0136] Take a PCR tube and add 2 μL of 10×PCR buffer (containing Mg) to each tube. 2+ 1 μL of dNTP (2.5 mM), 1.6 μL each of forward and reverse primers (2.5 μM), 0.16 μL of Ex Taq DNA polymerase (5 U / μL), 1.6 μL of DNA solution, and 12.24 μL of ultrapure water were mixed thoroughly. After centrifugation, the solution was transferred to a flow tube, centrifuged again, and 2 μL of silicone oil was added to complete the preparation of the reaction solution in the flow tube. The flow tube was then placed in a flow PCR instrument and heated at a constant temperature for several minutes for PCR amplification. After PCR, the amplification product was aspirated and diluted to the appropriate fold with ultrapure water. The resulting diluted amplification product was heated at 98 °C for 4 min to denature it, and then the heated product was quickly transferred to ice. The resulting single-stranded amplification product was stored at -20 °C for subsequent detection.

[0137] The primer sequences are as follows:

[0138] Upstream primer: 5'-tgtccacacgatgccaatg-3' (SEQ ID NO.10);

[0139] Downstream primer: 5'-ctgaggatcttggttggcg-3' (SEQ ID NO.11).

[0140] The sequence of the convection PCR amplification product is as follows:

[0141] 5'-cgccaaccaagatcctcagctatagggtgcttttgatatttttccgagtacattggcatcgtgtggaca-3'

[0142] (SEQ ID NO.12).

[0143] (3) Design the DNA sequence required for gene detection reagent strips based on the sequence of convection PCR amplification products, including T region signal probe S3 and T region capture probe CP3 (C region signal probe S1 and capture probe CP1 are the same as in Example 2).

[0144] The designed sequences of T-region signal probe S3 and T-region capture probe CP3 are as follows:

[0145] S3: 5'-NH2-(CH2)6-atgtactcggaaaaatat-3' (SEQ ID NO. 13);

[0146] CP3: 5'-caaaagcaccctatagct-Biotin-3' (SEQ ID NO. 14).

[0147] (4) Pretreatment and modification of reagent strips

[0148] The preprocessing and modification process is similar to that in Example 2, and the probes used are as follows: C-region signal probe S1, T-region signal probe S3; C-region capture probe CP1, T-region capture probe CP3.

[0149] (5) The test kit is assembled in the same way as in Example 2.

[0150] (6) Electrochemiluminescence detection is the same as in Example 2.

[0151] (7) Data acquisition and processing are the same as in Example 2.

[0152] Different dilutions of the convective PCR amplification products were analyzed. The convective PCR amplification time was 35 min, and the amplification products were diluted at 10⁻¹⁰ and 10⁻¹⁰ times. 2 10 3 10 4 The corresponding electrochemiluminescence detection results are as follows: Figure 7 As shown, it can be seen that as the dilution factor of the convection PCR amplification product increases, the T / C value gradually decreases, indicating that the closed bipolar electrochemiluminescence lateral flow gene detection method of this invention has the potential to detect convection PCR amplification products.

[0153] The convection PCR amplification products at different amplification times were detected. The convection PCR amplification times were 35 min, 30 min, 25 min, 20 min, 15 min, and 10 min. The obtained amplification products were diluted 100-fold, and the corresponding electrochemiluminescence detection results are shown below. Figure 8 As shown in the figure, the changes in the T / C values ​​reveal two key points: First, within 25 minutes, the number of amplified products gradually increases with increasing amplification time, reaching a plateau after 25 minutes. Second, even with a 10-minute instantaneous amplification time and a 100-fold dilution of the amplified products, the corresponding electrochemiluminescence intensity is significantly higher than the background value. This indicates that the closed-loop bipolar electrochemiluminescence lateral flow gene detection method of this invention has the potential for rapid detection using convective PCR.

[0154] 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 closed-loop bipolar electrochemiluminescence lateral current gene detection method, characterized in that: It is based on lateral flow gene detection strips and / or detection kits; The lateral flow gene detection reagent strip includes an electrode and two hydrophilic channels A and B, with the electrode partially overlapping the two hydrophilic channels; The hydrophilic channels A and B are distributed on the sample application plate, binding plate, detection plate, and absorption plate; The electrode includes an integrated bipolar electrode and a pair of driving electrodes; the integrated bipolar electrode includes a cathode and at least two anodes connected in parallel; wherein, the integrated bipolar electrode anode closer to the sample plate is called the quality control anode, and the integrated bipolar electrode anode closer to the absorption plate is called the detection anode; The electrode is located on an electrode sheet; the electrode sheet is made of conductive carbon ink screen-printed hydrophobic cloth; The detection strip overlaps with the integrated bipolar electrode; the one that overlaps with the anode of the integrated bipolar electrode is called hydrophilic channel A, and the other hydrophilic channel is called hydrophilic channel B. The detection kit contains the lateral flow gene detection strip.

2. The detection method according to claim 1, characterized in that... Includes the following steps: (1) Design probe DNA sequences Design C-region capture probes and signal probes, as well as T-region capture probes and signal probes, based on the target DNA (T-DNA) sequence; (2) Pretreatment and modification of test strips Add the treatment solution to the hydrophilic channel A of the test strip and dry it; Treatment solution was added to the hydrophilic channel A of the test strip binding tablet and dried. Then, a signal probe coupled with a terpyridine ruthenium-polylysine complex was applied and vacuum dried. The test strips of the test kit are soaked in a surfactant solution and dried. The mixed solution containing the capture probes is then used to modify and fix the C-region capture probes and T-region capture probes in the corresponding areas by streaking. Add the treatment solution to the hydrophilic channel A of the absorbent strip of the test reagent strip and then dry it; (3) Sample testing Add the test sample solution to the hydrophilic channel A of the test strip and wait for several minutes. Then add a buffer solution to the hydrophilic channel A on the test strip. At the same time, add a buffer solution to the hydrophilic channel B to connect the circuit. Then connect the positive and negative electrodes of the test strip to the power supply and apply an appropriate voltage to trigger the electrochemiluminescence reaction. Electrochemiluminescence signals are acquired in the form of images and videos, and further analyzed by the program to achieve qualitative and quantitative detection of pathogen nucleic acids; The detection method described herein is a detection method for non-disease diagnosis purposes.

3. The detection method according to claim 2, characterized in that: The C-region capture probe has its 5' end modified with biotin; The C-region signal probe is modified with an amino group at its 5' end; The T-region capture probe has its 3' end modified with biotin; The T-region signal probe is modified with an amino group at its 5' end.

4. The detection method according to claim 2, characterized in that: The 5' end of the T-DNA is complementary to the 3' end of the T-region capture probe; The 3' end of the T-DNA is complementary to the 5' end of the T-region signal probe; The C-region capture probe sequence is completely complementary to the C-region signal probe sequence.

5. The detection method according to claim 2, characterized in that: The sample preparation solution is a buffer solution containing 1% SDS, 0.5% PVP, and 0.5% BSA, with a pH of 7.4; The conjugation solution is a buffer solution containing 0.5% gelatin, 2.5% trehalose, 1% BSA, 0.1% Triton X-100, and 0.1% Proclin 300 preservatives, with a pH of 7.

4.

6. The detection method according to claim 2, characterized in that: The surfactant solution of the detection strip is a mixed solution containing 0.5% S9 and 0.9% NaCl; the mixed solution containing the capture probe is a mixed solution containing 5% sucrose, 5% trehalose, 0.5 mg / mL streptavidin and 50 μM capture probe. The absorbent tablet treatment solution is a buffer solution containing 0.5% Tween.

7. The detection method according to claim 1, characterized in that: In the lateral flow gene detection reagent strip, during sample addition and detection, the anode of the integrated bipolar electrode and the corresponding driving electrode negative electrode are located in the reporting channel, and the cathode of the integrated bipolar electrode and the corresponding driving electrode positive electrode are located in the support channel.

8. The detection method according to claim 1, characterized in that: In the lateral flow gene detection reagent strip, the portion of the hydrophilic channel A that overlaps with the control anode is called the control zone, and the portion of the hydrophilic channel A that overlaps with the detection anode is called the detection zone.

9. The detection method according to claim 1, characterized in that: The test kit also includes an upper cover, a transparent cover, and a lower cover.

10. The detection method according to claim 9, characterized in that: The reagent strip is fixed on the lower cover, and the transparent cover plate covers the binding plate, detection plate, electrode plate and absorption plate of the reagent strip; The top cover is placed over a transparent cover plate.

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