Multi-channel dry-type electrochemical luminescence lateral flow detection device

Abstract

The utility model discloses a multi-channel dry-type electrochemical luminescence lateral flow detection device. The detection device comprises an electrode plate and a lateral flow fiber chip, the electrode plate comprises a negative driving electrode, a positive driving electrode and at least one bipolar electrode group; the bipolar electrode group comprises at least two bipolar electrodes which are distributed in series; the bipolar electrode is an integrated bipolar electrode, the anode of the bipolar electrode is provided with two branches, and the cathode of the negative driving electrode or the cathode of the adjacent bipolar electrode is connected with the two branches through a detection sheet in the lateral flow fiber chip. According to the utility model, a novel array closed bipolar electrode structure is adopted, and the number of cathodes of the bipolar electrodes needing to be pretreated and an area needing to be added with a solution are reduced through a method of connecting a plurality of integrated bipolar electrodes in series and sharing a pair of driving electrodes, so that the number of times of adding the solution is reduced, and the complexity of operation is reduced.

Description

Technical Field

[0001] The utility model belongs to the field of microfluidic in vitro diagnosis, and specifically relates to a multi-channel dry electrochemical luminescence lateral flow detection device. Background Art

[0002] In vitro diagnostics refers to products and services that obtain clinical diagnostic information by performing in vitro tests on human sample solutions (blood, body fluids, tissues, etc.) to determine diseases or body functions. In vitro diagnostic technology is developing rapidly, with tests based on gene, protein and cell levels emerging in an endless stream, and developing in a simpler, faster and more diversified direction.

[0003] Electrochemiluminescence technology has become an important in vitro diagnostic technology due to its high sensitivity and wide dynamic range. At present, electrochemiluminescence can be simply divided into three-electrode electrochemiluminescence and bipolar electrode electrochemiluminescence. Bipolar electrode electrochemiluminescence includes two basic configurations: closed and open. In recent years, closed bipolar electrode electrochemiluminescence has developed rapidly, from single detection to high-throughput or multiplex detection to meet the needs of multi-faceted detection. Previously reported closed bipolar electrode electrochemiluminescence usually has good analytical performance, but they still face some problems. For example, the common closed bipolar electrode electrochemiluminescence detection flux is often low and the electrode preparation is cumbersome; the common array closed bipolar electrode electrochemiluminescence requires multiple additions of related reagents and is large in volume; the common shared cathode closed bipolar electrode electrochemiluminescence electrode is more modified and lacks accurate quality control methods. Summary of the invention

[0004] The purpose of the utility model is to provide a multi-channel dry electrochemical luminescence lateral flow detection device to solve the following defects of the prior art:

[0005] 1. In the common array closed bipolar electrode electrochemiluminescence detection, multiple pairs of closed bipolar electrodes need to be used in parallel, and each bipolar electrode cathode or anode needs to be pretreated and reagents added, which makes the manual operation process more cumbersome and affects the detection stability.

[0006] The utility model adopts a novel array closed bipolar electrode structure, and reduces the number of bipolar electrode cathodes that need to be pretreated and the area that needs to add solution by constructing multiple integrated bipolar electrodes in series and sharing a pair of driving electrodes. This reduces the number of electrodes and the number of times to add reagents in the common array closed bipolar electrode electrochemical luminescence detection by nearly half.

[0007] 2. The existing electrochemiluminescence detection method of sharing a bipolar electrode cathode is to perform high-throughput detection by arranging multiple anodes in parallel on an integrated bipolar electrode. This structure results in the same potential difference between each branch bipolar electrode anode and the shared cathode, so a certain sliding resistor needs to be added to perform multi-color electrochemiluminescence detection.

[0008] The array closed bipolar electrode structure adopted by the utility model has a plurality of bipolar electrodes connected in series. As far as the driving voltage is concerned, the potential difference between the cathode and the anode of the bipolar electrodes arranged in series can be changed by changing the size and material of the bipolar electrodes.

[0009] 3. The existing multi-channel closed bipolar electrochemiluminescence detection method is to arrange multiple anodes in parallel on an integrated bipolar electrode and divide the branch anode into multiple ones by wax screen printing technology. When this method is used for multivariate detection, there is a difference in the potential difference between each bipolar electrode anode and the shared cathode, and the reduction reaction efficiency on the shared cathode is limited.

[0010] The utility model adopts a closed bipolar electrode array screen-printed on a hydrophobic material for electrochemiluminescence detection. On the one hand, the cumbersome process of multiple printing and electrode modification is reduced by screen-printing electrodes on a hydrophobic material and using a lateral flow fiber chip. On the other hand, the use of multiple shared cathodes evenly distributes the redox reactions on multiple bipolar electrode anodes and shared cathodes in the existing multi-channel closed bipolar electrochemiluminescence detection method to multiple integrated bipolar electrodes in series, thereby improving the efficiency of each pair of redox reactions and thus improving the efficiency of electrochemiluminescence.

[0011] 4. Existing closed bipolar electrode electrochemiluminescence detection usually has only one quality control zone, which leads to large detection errors.

[0012] The detection method of the utility model has two luminous areas in the same detection channel, one is the detection line (T line) and the other is the quality control line (C line). The ratio of the electrochemical luminescence intensity on the two (i.e., T / C) is used to quantitatively detect the target, which greatly reduces the detection error.

[0013] The purpose of the utility model is achieved through the following technical solutions:

[0014] A multi-channel dry electrochemical luminescence lateral flow detection device, comprising an electrode sheet and a lateral flow fiber chip;

[0015] The electrode sheet is composed of a conductive material on a hydrophobic material, and includes a negative driving electrode, a positive driving electrode, and at least one bipolar electrode group;

[0016] The bipolar electrode group comprises at least two bipolar electrodes connected in series;

[0017] The bisexual electrode is an integrated bisexual electrode, the anode of the bisexual electrode has two branches, and the cathode of the negative driving electrode or the adjacent bisexual electrode is connected to the two branches through the detection sheet in the lateral flow fiber chip; preferably, the distance from the cathode of the negative driving electrode or the adjacent bisexual electrode to the two branches is equal;

[0018] The cathode of the bipolar electrode is connected to the positive driving electrode via a connecting piece in the lateral flow fiber chip;

[0019] When there are two or more bipolar electrode groups, the bipolar electrode groups are connected in parallel between the driving electrodes; multiple bipolar electrode groups connected in parallel can increase the detection flux;

[0020] The lateral flow fiber chip comprises a sample adding sheet, a binding sheet, a detection sheet, an absorption sheet, a transparent adhesive board and a connecting sheet; the detection sheet, the binding sheet, the sample adding sheet and the absorption sheet are sequentially assembled on the transparent adhesive board to form a lateral flow detection channel; a plurality of the lateral flow detection channels and a single connecting sheet are assembled on a single transparent adhesive board;

[0021] The detection sheet covers the anode of the bipolar electrode and the negative driving electrode or the cathode of the adjacent bipolar electrode;

[0022] The detection sheet is a fiber material with T-line capture probes and C-line capture probes modified on the surface, and the fiber material is preferably a nitrocellulose membrane (NC membrane);

[0023] Furthermore, the positions and sizes of the T line and C line on the detection sheet are consistent with the two branches of the bipolar electrode anode; the C line on the detection sheet is closer to the binding sheet;

[0024] The connecting piece covers the cathode and the positive driving electrode of the bipolar electrode.

[0025] The sample sheet is a fiber material treated by a sample sheet treatment liquid, and the fiber material is preferably glass fiber;

[0026] The binding sheet is a fiber material treated with a binding sheet treatment solution and dried to modify the electrochemiluminescent probe, and the fiber material is preferably glass fiber;

[0027] The fiber material of the absorbent sheet is preferably absorbent paper;

[0028] The fiber material of the connecting sheet is preferably glass fiber;

[0029] The transparent adhesive plate is preferably PET plastic with adhesive backing;

[0030] The sample-adding sheet treatment solution and the binding sheet treatment solution both contain phosphate buffered saline (PBS) containing 0.05% polyvinyl pyrrolidone, 0.025% casein and 0.025% surfactant S9;

[0031] The electrode sheet is made by printing conductive carbon ink on a hydrophobic PET plate with adhesive backing by screen printing.

[0032] The detection device further comprises a housing;

[0033] The housing comprises an upper cover and a lower cover;

[0034] The upper cover is provided with an observation window and an electrode contact area; the lower surface of the upper cover is provided with a plurality of protrusions for fixing the lateral flow fiber chip;

[0035] The upper surface of the lower cover is provided with a fixing area for fixing the electrode sheet and the lateral flow fiber chip, and the side surface of the lower cover is provided with a plurality of liquid adding holes for adding sample solution or buffer solution.

[0036] The multi-channel dry electrochemiluminescent lateral flow detection device can be used for high-throughput detection such as nucleic acid hybridization and protein immunoassay (for different detections, only the electrochemiluminescent probes and capture probes used are different). Taking nucleic acid high-throughput detection as an example, the detection steps are as follows:

[0037] (1) Design and preparation of electrochemiluminescent probes and capture probes

[0038] For different targets, select their characteristic fragments as target sequences (DNAT);

[0039] Design T-line electrochemiluminescent probe sequences and capture probe sequences according to the target sequence, and design universal C-line electrochemiluminescent probe sequences and capture probe sequences;

[0040] The above four sequences need to meet the following conditions:

[0041] The 5′ end of the T-line electrochemiluminescent probe sequence and the 3′ end of DNAT are complementary for 5 to 20 bases;

[0042] The 3′ end of the T-line capture probe sequence and the 5′ end of DNAT are complementary for 5 to 20 bases;

[0043] The C-line electrochemiluminescent probe sequence and the capture probe sequence are completely base complementary;

[0044] The 5′ end of the T-line and C-line electrochemiluminescent probe sequences were modified with an amino group;

[0045] The 5′ end of the T-line and C-line capture probe sequences were modified with biotin;

[0046] The electrochemiluminescent probe preparation method adopts a published method (patent number: ZL202310708705.8), the difference being that the protein is replaced by a nucleic acid sequence.

[0047] (2) Sensing interface preparation

[0048] The C-line electrochemiluminescent probe and the T-line electrochemiluminescent probe are mixed in equal volumes to prepare the required electrochemiluminescent probe, and are sprayed on the surface of the binding sheet by a gold spray film sprayer. When different targets are detected, only the T-line electrochemiluminescent probe needs to be changed;

[0049] The aforementioned T-line and C-line capture probes were pre-mixed with streptavidin in a certain ratio for 30 minutes, and a gold-sprayed film scribing instrument was used to scribing the T-line and C-line of the detection sheet, respectively. When detecting different targets, only the T-line capture probe needs to be changed;

[0050] The shell, the electrode sheet and the lateral flow fiber chip are assembled in sequence to form a multi-channel dry electrochemiluminescence lateral flow detection device.

[0051] (3) Detection process

[0052] The sample solution is dripped onto the sample adding sheet (or the liquid adding hole corresponding to the shell) of the detection channel. The sample solution flows from the sample adding sheet to the binding sheet. The DNAT in the sample solution and the T-line electrochemiluminescent probe in the binding sheet form a complex. The complex further flows with the solution to the detection sheet and the T-line capture probe to form a three-nucleic acid chain hybridization complex. The C-line electrochemiluminescent probe in the binding sheet flows with the solution to the detection sheet and the C-line capture probe to form a two-nucleic acid chain hybridization complex.

[0053] After a period of incubation, a certain volume of washing buffer is added to the sample adding piece (corresponding liquid adding hole of the shell) to promote the formation of the complex on the one hand and wash away the unbound electrochemiluminescent probe on the other hand;

[0054] After flushing, the multi-channel dry electrochemiluminescence lateral flow detection device is placed in a portable electrochemiluminescence detector for detection, and the nucleic acid concentration in each channel is quantitatively analyzed using T / C;

[0055] The incubation time is preferably 3 min, and the volume of the washing buffer is preferably 10 μL;

[0056] Furthermore, the washing buffer is PBS.

[0057] The portable electrochemical luminescence detector is a device that controls a COMS camera to take pictures and analyze luminescent images through a mobile phone App.

[0058] Compared with the prior art, the utility model has the following advantages and effects:

[0059] 1. The utility model adopts a novel array closed bipolar electrode structure, which reduces the number of bipolar electrode cathodes that need to be pretreated and the area where solution needs to be added by connecting multiple integrated bipolar electrodes in series and sharing a pair of driving electrodes, thereby reducing the number of times the solution is added and reducing the complexity of the operation.

[0060] 2. The utility model adopts an array closed bipolar electrode structure with multiple integrated bipolar electrodes connected in series, which can accurately control the driving voltage on different bipolar electrodes.

[0061] 3. The utility model adopts the method of screen printing an array of closed bipolar electrodes on a hydrophobic material for electrochemical luminescence detection, which separates the hydrophilic region and the hydrophobic region into two layers, which not only reduces the difficulty of production, but also improves the storage stability.

[0062] 4. The utility model reduces the difficulty of manufacturing the sensing interface by preparing the sensing interface on the NC film.

[0063] 5. The utility model sets two luminous areas in the same detection channel and performs quantitative detection through T / C to improve the accuracy of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 Schematic diagram of a multi-channel dry electrochemiluminescence lateral flow detection device.

[0065] Figure 2 Schematic diagram of the electrode sheet of the multi-channel dry electrochemiluminescence lateral flow detection device.

[0066] Figure 3 Schematic diagram of the lateral flow fiber chip of the multi-channel dry electrochemiluminescence lateral flow detection device.

[0067] Figure 4 This is a schematic diagram of the lateral flow detection channel structure of a multi-channel dry electrochemiluminescence lateral flow detection device.

[0068] Figure 5 This is a schematic diagram of the outer shell of a multi-channel dry electrochemiluminescence lateral flow detection device.

[0069] Figure 6 This is a schematic diagram of the upper surface of the upper cover of the multi-channel dry electrochemiluminescence lateral flow detection device.

[0070] Figure 7 This is a schematic diagram of the lower surface of the upper cover of a multi-channel dry electrochemiluminescence lateral flow detection device.

[0071] Figure 8 Schematic diagram of the lower cover of the multi-channel dry electrochemiluminescence lateral flow detection device.

[0072] Fig. 9 Schematic diagram of the assembly of a multi-channel dry electrochemiluminescence lateral flow detection device.

[0073] Fig.10 This is the detection principle diagram of the multi-channel dry electrochemiluminescence lateral flow detection device.

[0074] Fig.11 This is a diagram showing the results of detecting different concentrations of Escherichia coli CRISPR / Cas 12a products using a multi-channel dry electrochemiluminescence lateral flow detection device.

[0075] Fig.12 This is a diagram showing the results of detecting Escherichia coli CRISPR / Cas12a products and PCR products using a multi-channel dry electrochemiluminescence lateral flow detection device.

[0076] Fig.13 This is a schematic diagram of the structure of the four-element detection electrode sheet of the multi-channel dry electrochemiluminescence lateral flow detection device. DETAILED DESCRIPTION

[0077] The present invention is further described in detail below in conjunction with embodiments and drawings, but the implementation manner of the present invention is not limited thereto.

[0078] Example 1

[0079] A multi-channel dry electrochemical luminescence lateral flow detection device, such as Figure 1 As shown, it includes an electrode sheet 1, a lateral flow fiber chip 2 and a housing 3.

[0080] Its structure and preparation process are as follows:

[0081] 1. Use drawing software Adobe Illustrator CS6 to design the electrode sheet structure, and use screen printing technology to prepare the electrode sheet 1.

[0082] The structure of the prepared electrode sheet 1 is as follows Figure 2 As shown, the electrode sheet 1 is formed by screen printing conductive carbon ink on a PET plastic plate 1-1, and includes a negative driving electrode 1-2, a positive driving electrode 1-3, and two integrated bipolar electrodes 1-4-1, 1-4-2;

[0083] The integrated bipolar electrode 1-4 has three branches, one branch is the bipolar electrode cathode 1-4-1-1, 1-4-2-1, and the other two branches are the bipolar electrode anode 1-4-1-2, 1-4-1-3 and 1-4-2-2, 1-4-2-3;

[0084] Two integrated bipolar electrodes 1-4-1 and 1-4-2 are connected in series through the detection sheet 2-5 in the lateral flow fiber chip 2, wherein the two anodes 1-4-1-2, 1-4-1-3 of the integrated bipolar electrode 1-4-1 and the cathode 1-4-2-1 of the adjacent integrated bipolar electrode 1-4-2 are located in the corresponding area 1-5 of the detection sheet 2-5 on the electrode sheet 1, and the two anodes 1-4-1-2, 1-4-1-3 are axially symmetrically distributed about the corresponding cathode 1-4-1-1;

[0085] One end 1-2-1 of the negative driving electrode 1-2 is used to connect to an external circuit, and the other end 1-2-2 is connected to the integrated bipolar electrode anodes 1-4-2-2, 1-4-2-3 through the detection sheet 2-5 in the lateral flow fiber chip 2, and is located in the area 1-6 corresponding to the detection sheet 2-5 on the electrode sheet 1 and is located on the symmetry axis of the two anodes 1-4-2-2, 1-4-2-3 of the integrated bipolar electrode;

[0086] One end 1-3-1 of the positive driving electrode 1-3 is used to connect to an external circuit, and the other end 1-3-2 is connected to the integrated bipolar electrode cathode 1-4-1-1 through the connecting piece 2-2, and is located in the area 1-7 corresponding to the connecting piece 2-2 on the electrode sheet 1, and each branch 1-3-2 is equidistant from the corresponding integrated bipolar electrode cathode 1-4-1-1;

[0087] 2. Use the drawing software Adobe Illustrator CS6 to design the structure of each part of the lateral flow fiber chip, cut it with a laser cutting machine, modify it with a gold spray film scribing instrument, and further assemble it to obtain the lateral flow fiber chip.

[0088] Lateral flow fiber chip 2 Figure 3 As shown, it includes a transparent adhesive plate 2-1, a connecting sheet 2-2, and a lateral flow detection channel 2-3;

[0089] The detection sheet 2-3-3, the binding sheet 2-3-2, the sample adding sheet 2-3-1, and the absorption sheet 2-3-4 are assembled on the transparent adhesive plate 2-4 in sequence to form a lateral flow detection channel ( Figure 4 );

[0090] The backing side of the detection sheet 2-3-3 is attached to the transparent adhesive plate 2-4, the binding sheet 2-3-2 is stacked on the detection sheet 2-3-3, and the overlapping length is 2mm; the sample sheet 2-3-1 is stacked on the binding sheet 2-3-2, and the overlapping length is 2mm; the absorption sheet 2-3-4 is stacked on the detection sheet 2-3-3, and the overlapping length is 4mm;

[0091] Sample 2-3-1 is a glass fiber treated with a sample treatment solution, with a length of 6 mm and a width of 3 mm;

[0092] The binding sheet 2-3-2 is a glass fiber treated with a binding sheet treatment solution and dried to modify an electrochemiluminescent probe, with a length of 6 mm and a width of 3 mm;

[0093] The detection sheet 2-3-3 is an NC membrane with T lines 2-3-3-2 and C lines 2-3-3-1, and the surface of the membrane is modified with T line capture probes and C line capture probes, respectively. The detection sheet 2-3-3 is 20 mm long and 3 mm wide.

[0094] The material of the absorbent sheet 2-3-4 is absorbent paper, with a length of 19 mm and a width of 4 mm (i.e., the overlapping length with the detection sheet);

[0095] The material of the connecting piece 2-2 is glass fiber treated with a connecting piece treatment liquid, with a length of 28 mm and a width of 3 mm;

[0096] The transparent adhesive plates 2-1 and 2-4 are PET plastic with adhesive backing; the transparent adhesive plate 2-4 is 28 mm long and 3 mm wide, and is used to assemble the lateral flow detection channel; the transparent adhesive plate 2-1 is 70 mm long and 15 mm wide, and is used to fix the lateral flow detection channel and the connecting piece;

[0097] Two lateral flow detection channels 2-3 and a single connecting piece 2-2 are fixed on a single transparent adhesive plate 2-1;

[0098] The shell 3 structure is designed using the drawing software Solidworks 2020 and printed using a 3D printer. The structure includes an upper cover 3-1 and a lower cover 3-2 ( Figure 5 ).

[0099] The upper surface 3-1-1 of the upper cover 3-1 is provided with observation windows, which are T-line observation window 3-1-1-1 and C-line observation window 3-1-1-2 of lateral flow detection channel 1, and T-line observation window 3-1-1-3 and C-line observation window 3-1-1-4 of lateral flow detection channel 2; the lower surface 3-1-2 of the upper cover 3-1 is provided with protrusions 3-1-2-1, 3-1-2-2 ( Figure 6 and Figure 7 );

[0100] Lower cover 3-2( Figure 8 ) is provided with a liquid adding hole 3-2-1; an electrode sheet 1 fixing area 3-2-2; a lateral flow fiber chip 2 fixing area 3-2-3 and an electrode contact area 3-2-4;

[0101] 4. Assemble the electrode sheet 1, the lateral flow fiber chip 2 and the housing 3 to form a multi-channel dry electrochemical luminescence lateral flow detection device. The schematic diagram of the assembly process is shown in FIG. Fig. 9 shown.

[0102] First, stick the electrode sheet 1 to the electrode sheet 1 fixing area 3-2-2 of the lower cover 3-2; then, put the assembled lateral flow fiber chip 2 upside down on the electrode sheet 1 and in the lateral flow fiber chip 2 fixing area 3-2-3; finally, fasten the upper cover 3-1 and the lower cover 3-2 to complete the assembly of the multi-channel dry electrochemical luminescence lateral flow detection device.

[0103] Example 2

[0104] The multi-channel dry electrochemiluminescent lateral flow detection device in Example 1 is used to detect Escherichia coli CRISPR / Cas 12a products, and the detection process is as follows:

[0105] 1. Design of nucleic acid sequences required for detecting E. coli CRISPR / Cas 12a products

[0106] Primers were designed based on the rfbE gene of Escherichia coli;

[0107] Upstream primer: 5′-gggttaactgttatgttgtactgcttc-3′;

[0108] Downstream primer: 5′-aatccacgttgagtccagacattc-3′;

[0109] E. coli crRNA: 5′-taatttctactaagtgtagatccaaccgtcattgacaggaa-3′;

[0110] E. coli CRISPR / Cas 12a product capture probe sequence: 5′-ttctttttcttatacatttact-3′;

[0111] E. coli CRISPR / Cas 12a product signal probe sequence: 5'-tacagacctgagttgcacctaa-3';

[0112] C-line signal probe sequence: 5′-ggcacaaacacgcacctc-3′;

[0113] C-line capture probe sequence: 5′-gaggtgcgtgtttgtgcc-3′;

[0114] 2. Preparation of sensing interface

[0115] The sensing interface preparation process is as follows:

[0116] The cells were treated with a binding agent (PBS containing 0.05% PVP, 0.025% casein and 0.025% S9). Figure 4The binding sheet 2-3-2 in the embodiment is dried at 37°C for 30 min; the electrochemical luminescence signal probe is modified on the surface of the binding sheet 2-3-2 by using a gold spray film scribing instrument, and then dried at 37°C for 120 min;

[0117] The capture probe was modified on the surface of the detection piece 2-3-3 by using a gold spray film streaking instrument and dried at 37°C for 120 min;

[0118] All modifications in lateral flow assay channels one and two were identical.

[0119] The sensing principle of the detection piece 2-3-3 is as follows Fig.10 As shown, after the solution containing the target sequence enters the binding plate 2-3-2, the target sequence and the signal probe sequence undergo base complementary pairing to form a complex (signal sequence / target sequence); the complex further flows to the detection plate 2-3-3 and combines with the capture probe to form a new complex (signal probe / target sequence / capture probe); the signal probe emits light after power is turned on.

[0120] 3. Sample preparation

[0121] The E. coli DNA was extracted by the magnetic bead method. 200 μL of the sample to be tested was added to a centrifuge tube, and 300 μL of lysis solution, 100 μL of isopropanol, and 15 μL of magnetic beads were added in sequence, and the sample was lysed at 56°C. After magnetic separation, 350 μL of washing solution 1 was added. After magnetic separation again, 350 μL of washing solution 2 was added. After magnetic separation again, 50 μL of elution solution was added. After magnetic separation again, the E. coli DNA sample solution was obtained.

[0122] The above lysate, magnetic beads, washing solution 1, washing solution 2 and elution solution are all from commercial kits;

[0123] Escherichia coli DNA was amplified by polymerase chain reaction (PCR). The amplification solution included: 5 μL of Escherichia coli DNA sample solution, 5 μL of 10×Ex Taq Buffer, 4 μL of dNTP, 0.24 μL of Taq enzyme, 1 μL of upstream primer (10 μM) and 1 μL of downstream primer (10 μM); the reaction program was 95°C for 2 min, and 30 cycles (95°C for 15 s, 60°C for 30 s).

[0124] The above 10x Ex Taq Buffer, dNTPs, and Taq enzyme were all from commercial kits;

[0125] The target sequence was obtained by CRISPR / Cas 12a cis-cleavage reaction. The reaction solution included: NEBuffer r2.1Reaction Buffer (10×) 2μL, PCR reaction solution 2μL, 2μM crRNA 2μL, 1mM EnGen Lba Cas12a (Cpf1) 2μL, and Nuclease-free water 12μL; the reaction procedure was 45°C 3min, 95°C 3min; the obtained cleavage product solution was diluted to the required concentration.

[0126] 4. Sample loading and detection

[0127] The obtained cleavage product solution was diluted 100 times; 10 μL of the diluent, the undiluted cleavage product solution and PBS were added to the liquid adding hole in Example 1 respectively; after waiting for 3 minutes, 10 μL of the washing buffer was added for washing;

[0128] After flushing, the multi-channel dry electrochemiluminescence lateral flow detection device was placed in a portable electrochemiluminescence detector, a driving voltage of 17V was applied for detection, and the nucleic acid concentration in each channel was quantitatively analyzed using T / C. The results were as follows: Fig.11 As shown in the figure, it can be seen that the device of the utility model can perform multi-channel quantitative detection.

[0129] Example 3

[0130] The multi-channel dry electrochemiluminescent lateral flow detection device in Example 1 is used to simultaneously detect Escherichia coli CRISPR / Cas 12a and PCR amplification products, and the detection process is as follows:

[0131] 1. Design the required nucleic acid sequence

[0132] The nucleic acid sequence required for detection of E. coli CRISPR / Cas 12a products is the same as that in Example 2.

[0133] The sequences required for E. coli PCR amplification are as follows:

[0134] Upstream primer: 5′-tgtccacacgatgccaatg-3′;

[0135] Downstream primer: 5′-ctgaggatcttggttggcg-3′;

[0136] The sequence of the E. coli PCR amplification product (target sequence) obtained by amplification with the upstream primer and the downstream primer is as follows: 5′-cgccaaccaagatcctcagctatagggtgcttttgatatttttccgagtacattggcatcgtgtggaca-3′;

[0137] Capture probe sequence of E. coli PCR amplification product: 5′-caaaagcaccctatagct-3′;

[0138] Signal probe sequence of E. coli PCR amplification product: 5′-atgtactcggaaaaatat-3′;

[0139] The C-line signal probe sequence and the C-line capture probe sequence are the same as those in Example 2.

[0140] 2. Preparation of sensing interface

[0141] The preparation process of the sensing interface is similar to that of Example 2, except that the Escherichia coli CRISPR / Cas 12a product capture probe sequence, the Escherichia coli CRISPR / Cas 12a product signal probe sequence, the C-line signal probe sequence, and the C-line capture probe sequence are modified in the lateral flow detection channel one; and the Escherichia coli PCR amplification product capture probe sequence, the Escherichia coli PCR amplification product signal probe sequence, the C-line signal probe sequence, and the C-line capture probe sequence are modified in the lateral flow detection channel two.

[0142] 3. Sample preparation

[0143] The preparation process of E. coli CRISPR / Cas 12a product was the same as that in Example 2;

[0144] The preparation process of E. coli PCR amplification product is as follows:

[0145] The E. coli DNA was extracted using the bacterial genomic DNA rapid extraction kit. Then, 2 μL of 10× PCR buffer (containing Mg) was added to the PCR tube. 2+ ), 1μL dNTP (2.5mM), 1.6μL upstream and downstream primers (2.5μM), 0.16μL ExTaq DNA polymerase (5U / μL), 1.6μL Escherichia coli DNA and 12.24μL ultrapure water. After mixing, transfer the solution to the convection tube, centrifuge again and add 2μL silicone oil to complete the preparation of the reaction solution in the convection tube. Then, place the convection tube in a convection PCR instrument and heat it at a constant temperature for several minutes for PCR amplification. After the PCR is completed, the amplified product is aspirated and diluted to the corresponding multiple by adding ultrapure water. The obtained amplified product dilution is melted by heating at high temperature 98℃ for 4min, and then the product after high temperature heating is quickly transferred to ice, and the obtained single-stranded amplified product is stored at -20℃ for subsequent detection.

[0146] 4. Sample loading and detection

[0147] The sample loading and detection process is similar to Example 2. Take 10 μL of E. coli CRISPR / Cas 12a product and 10 μL of 10-fold diluted E. coli PCR amplification product and add them to lateral flow detection channel 1 and channel 2 respectively. Then, wait for 3 minutes and add 10 μL of washing buffer for washing;

[0148] After flushing, the multi-channel dry electrochemiluminescence lateral flow detection device was placed in a portable electrochemiluminescence detector and a driving voltage of 17V was applied for detection. The nucleic acid concentration in each channel was quantitatively analyzed using T / C. The results were as follows: Fig.12 As shown in the figure, it can be seen that the device of the utility model can simultaneously perform multi-channel quantitative detection of different targets.

[0149] Example 4

[0150] Another embodiment of the structure of the electrode sheet in a multi-channel dry electrochemiluminescence lateral flow detection device.

[0151] The electrode sheet structure was designed using the drawing software Adobe Illustrator CS6.

[0152] The structure of the designed electrode sheet 4 is as follows Fig.13 As shown, it includes a negative driving electrode 4-1, a positive driving electrode 4-2, and six integrated bipolar electrodes 4-3, 4-4, 4-5, 4-6, 4-7 and 4-8;

[0153] Each integrated bipolar electrode structure is the same as that in Example 1, and has three branches, one branch is a bipolar electrode cathode, and the other two branches are bipolar electrode anodes;

[0154] The first row has three integrated bipolar electrodes 4-3, 4-5, and 4-7 connected in series, and their series connection and arrangement are similar to those in Example 1; the second row has three integrated bipolar electrodes 4-4, 4-6, and 4-8 with the same structure as the first row; the electrodes in the first row and the second row are arranged in parallel;

[0155] One end 4-1-1 of the negative driving electrode is used to connect to an external circuit, and the other end branches 4-1-2 and 4-1-3 are respectively connected to the anodes of the first and second rows of integrated bipolar electrodes 4-3 and 4-4. The connection and arrangement of each branch and the anode of the integrated bipolar electrode are similar, and the distance from each branch to the corresponding anode of the integrated bipolar electrode is equal.

[0156] One end 4-2-1 of the positive driving electrode is used to connect to an external circuit, and the other end branches 4-2-2 and 4-2-3 are respectively connected to the cathodes of the first and second rows of integrated bipolar electrodes 4-7 and 4-8. The connection method and arrangement method of each branch and the cathode of the integrated bipolar electrode are similar, and the distance from each branch to the corresponding cathode of the integrated bipolar electrode is equal.

[0157] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principle of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A multi-channel dry electrochemiluminescence lateral flow detection device, characterized in that: It includes an electrode sheet and a lateral flow fiber chip; The electrode sheet includes a negative driving electrode, a positive driving electrode, and at least one group of bipolar electrodes; The bipolar electrode group comprises at least two bipolar electrodes connected in series; The bipolar electrode is an integrated bipolar electrode, the anode of the bipolar electrode has two branches, and the negative driving electrode or the cathode of the adjacent bipolar electrode is connected to the two branches through the detection sheet in the lateral flow fiber chip.

2. The detection device according to claim 1, characterized in that: The distances between the cathode of the negative driving electrode or the adjacent bipolar electrode and the two branches of the anode of the bipolar electrode are equal.

3. The detection device according to claim 1, characterized in that: The cathode of the bipolar electrode is connected to the positive driving electrode via a connecting piece in the lateral flow fiber chip.

4. The detection device according to claim 1, characterized in that: When there are two or more bipolar electrode groups, the bipolar electrode groups are distributed in parallel between the driving electrodes.

5. The detection device according to claim 1, characterized in that: The lateral flow fiber chip comprises a sample adding sheet, a binding sheet, a detection sheet, an absorption sheet, a transparent adhesive plate and a connecting sheet; the detection sheet, the binding sheet, the sample adding sheet and the absorption sheet are sequentially assembled on the transparent adhesive plate to form a lateral flow detection channel.

6. The detection device according to claim 5, characterized in that: Several of the lateral flow detection channels and a single connecting piece are assembled on a single transparent adhesive plate.

7. The detection device according to claim 5, characterized in that: The surface of the detection sheet is modified with T-line capture probes and C-line capture probes.

8. The detection device according to claim 7, characterized in that: The positions and sizes of the T line and the C line on the detection sheet are consistent with the two branches of the bipolar electrode anode; the C line is closer to the binding sheet.

9. The detection device according to claim 1, characterized in that: The detection device comprises a shell.

10. The detection device according to claim 9, characterized in that: The shell comprises an upper cover and a lower cover.

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