Nucleic acid-based algal toxin on-site rapid quantitative detection paper chip device and detection method thereof

Abstract

The invention belongs to the technical field of small molecule detection, and relates to a nucleic acid-based algal toxin on-site rapid quantitative detection paper chip device and a detection method thereof. A reaction area of the paper chip device is coated with an aptamer c-DNA and a fluorescent group labeled-DNA, and a reading area of the paper chip device is coated with a capture nucleic acid probe d-DNA; the nucleic acid sequence of the aptamer c-DNA is as shown in SEQ ID NO.1, and the 5'end of the c-DNA is marked with biotin; the nucleic acid sequence of the capture nucleic acid probe is shown as SEQ ID NO.1, the nucleic acid sequence of the capture nucleic acid probe is shown as SEQ ID NO.2, the 5'end of the capture nucleic acid probe is marked with an FAM fluorophore, the nucleic acid sequence of the capture nucleic acid probe d-DNA is shown as SEQ ID NO.3, and the 5 'end of the capture nucleic acid probe d-DNA is marked with biotin. The whole detection process does not exceed 10 minutes, the cost is low, and rapid on-site quantitative detection of algal toxin in water is achieved.

Description

Technical Field

[0001] This invention belongs to the field of small molecule detection technology, specifically relating to a paper chip device for rapid on-site quantitative detection of algal toxins in water and its application. Background Technology

[0002] In recent years, eutrophication of water bodies has led to frequent cyanobacterial blooms, accompanied by a significant increase in toxin levels, exacerbating water quality deterioration. Among various toxins, microcystin-releasing agents (MC-LRs) are among the most common and potent. They can alter the activity of related enzymes in organisms, leading to the accumulation of phosphorylated proteins in the liver, physiological dysfunction, cell necrosis, multiple organ failure, and even death. In light of this, the World Health Organization (WHO) has classified MC-LRs as neurotoxins, and the International Agency for Research on Cancer (IARC) has classified them as Group 2B (probably carcinogenic to humans), setting a limit of 1 μg / L for their concentration in drinking water. -1 Therefore, developing a simple, highly sensitive, accurate, and rapid on-site detection method to achieve real-time monitoring of MC-LR in water is of significant practical importance.

[0003] Currently, various techniques have been developed for the detection of microcystin-releasing agents (MC-LR) in water, including chromatography, spectroscopic analysis, electrochemical analysis, and biosensor technology. While these methods offer high accuracy and excellent sensitivity, they generally suffer from the following limitations: they require expensive and complex instruments, involve cumbersome sample pretreatment or concentration processes, and are time-consuming and costly. Crucially, they are difficult to implement for rapid, on-site detection of MC-LR in water.

[0004] Paper-based microarrays have been widely used in food, pharmaceutical, and environmental sample testing in recent years due to their low cost, diverse functions, excellent portability, good biocompatibility, and environmental friendliness. Furthermore, paper-based microarrays can be combined with fluorescence detection methods to construct simpler, more intuitive, and faster detection systems. The principle involves a competitive reaction between the analyte and complementary DNA carrying a fluorescent group in the reaction zone. This complementary DNA detaches and migrates through the detection channel to the signal readout area, where it is specifically recognized and bound by immobilized DNA. The amount of captured complementary DNA is positively correlated with the concentration of the analyte; the analyte can be quantified by detecting the fluorescence intensity of the complementary DNA. Based on these characteristics, paper-based microarrays can be designed as miniaturized, portable on-site detection platforms.

[0005] Currently, based on competitive enzyme-linked immunosorbent assay (ELISA), cCommercially available MC-LR detection kits based on the ELISA principle already exist, but they suffer from drawbacks such as a negative correlation between color signal output and MC-LR concentration, leading to potential result confusion, and insufficient stability of protein-based test strips. Paper-based chips based on the principle of nucleic acid-specific recognition, by setting the reaction area and signal reading area as two independent functional regions, achieve a positive correlation between fluorescence signal output and MC-LR concentration. This can overcome the shortcomings of commercially available kits and effectively enable rapid, sensitive, and convenient on-site detection of large numbers of samples. However, this method faces the following technical challenges in practical applications: 1. Paper materials exhibit non-specific adsorption of competitive nucleic acids, affecting the accuracy of quantification; 2. The complex matrix composition of environmental samples can easily interfere with nucleic acid recognition reactions, reducing the reliability of detection; 3. The separation design of independent functional areas requires precise control of the solution migration path. The paper chip structure design and detection process optimization are necessary to ensure that the reaction solution flows completely into the signal reading area. Summary of the Invention

[0006] This invention provides a paper-based chip device for rapid on-site quantitative detection of algal toxins in water and its application. The reaction region surface of the paper-based chip device is coated with a highly specific aptamer (…). c -DNA), and binds to an excess of complementary DNA with a fluorescent group (ƒ-DNA), so that c DNA and ƒ The ratio of DNA to 1:1. Given... c The binding affinity of -DNA to MC-LR is greater than its binding affinity to ƒ-DNA. When the target molecule MC-LR flows through the reaction zone, a competitive reaction occurs, displacing ƒ-DNA. After elution, the displaced ƒ-DNA flows through the detection channel into the signal readout region, where it is captured by the nucleic acid probe. d - Rapid DNA identification and quantitative analysis via fluorescence detection, with a positive correlation between fluorescence signal and algal toxin concentration, ensures accurate quantification and compensates for the shortcomings of commercially available test kits.

[0007] The present invention adopts the following technical solution: This invention provides a paper-based chip device for on-site, real-time, quantitative detection of algal toxins in water, wherein the reaction region of the paper-based chip device is coated with an aptamer. c DNA and fluorescently labeled ƒ -DNA, the readout region of the paper-based chip device contains captured nucleic acid probes. d -DNA; The aptamer c The nucleic acid sequence of the DNA is shown in SEQ ID NO.1. c - The 5' end of the DNA is labeled with biotin; ƒThe nucleic acid sequence of the DNA is shown in SEQ ID NO.2, and the 5' end is labeled with a FAM fluorescent group; the capture nucleic acid probe d The nucleic acid sequence of the DNA is shown in SEQ ID NO.3. d - The 5' end of the DNA is labeled with biotin.

[0008] In the above technical solution, the aptamer further... c - The concentration of DNA is 10 μmol / L to 10 mmol / L; d - The concentration of DNA is 10 μmol / L to 10 mmol / L.

[0009] In the above technical solution, the method for coating the reaction region and the reading region further includes: Chitosan solution was added to both the reaction zone and the signal reading zone. After drying at room temperature, glutaraldehyde solution was added and incubated at room temperature for 120–150 min. After washing and drying, streptavidin solution was added and incubated for 10–20 min. After washing and drying, BSA solution was added for blocking. After reacting for 10–20 min, the mixture was washed with Tween-20 and PBS buffer and dried at room temperature. c-DNA solution was added dropwise to the reaction zone and incubated for 10–20 min. After washing and drying, f-DNA solution was added and reacted for 10–20 min. After washing and drying, the mixture was stored for later use. Chitosan solution was added dropwise to the signal reading zone. d -DNA solution, incubate for 10-20 min, wash and dry, store at 4℃ for later use.

[0010] In the above technical solution, the concentration of the chitosan solution is 0.24~0.26 mg / mL, the volume fraction of the glutaraldehyde solution is 2.4~2.6%, the concentration of the streptavidin solution is 0.9~1.1 mg / mL, the volume fraction of the BSA solution is 0.4~0.6%, the volume fraction of Tween-20 is 0.04~0.06%, and the pH of the PBS buffer is 7.4~7.6.

[0011] In the above technical solution, further, the paper chip device has two separate circular areas on the same plane of the filter paper, namely a reaction chip area and a colorimetric chip area; the filter paper has a fold line, which is located between the reaction area and the signal reading area, allowing the filter paper to be folded in half. When the filter paper is folded along the fold line, the reaction area and the signal reading area are on different planes; two rectangular channels are provided between the reaction area and the signal reading area, wherein the first rectangular channel is coplanar with and connected to the reaction area, but separate from the signal reading area; the second rectangular channel is located directly above the first rectangular channel and is connected to the signal reading area; when the reaction area and the signal reading area are on the same plane, the second rectangular channel covers the first rectangular channel directly above it, and the first rectangular channel is completely located within the area of ​​the second rectangular channel; when folded along the fold line so that the reaction area and the signal reading area are on different planes, the second rectangular channel connected to the signal reading area is separated from the reaction area and the first rectangular channel, and the first rectangular channel and the second rectangular channel are on different planes respectively.

[0012] In the above technical solution, the reaction zone, signal reading zone, first rectangular channel and second rectangular channel are all hydrophilic zones; except for the reaction zone, signal reading zone and two rectangular channels, the areas are all painted with wax, and molten liquid wax is injected into the mold to form hydrophobic zones to achieve functional division of the areas; the molten wax is injected into the mold, and the paper chip is demolded after cooling to room temperature.

[0013] This invention also provides a method for on-site, real-time quantitative detection of algal toxins in water using a paper-based chip device. The method is based on the principle of nucleic acid specific recognition and competitive reaction. The paper-based chip device is prepared using a wax drawing method combined with nucleic acid modification. Algal toxins in the sample compete with complementary DNA (ƒ-DNA) carrying fluorescent groups for binding to the binding sites of aptamer c-DNA coated on the surface of the reaction zone. Since the binding force of the aptamer c-DNA to MC-LR is greater than that to ƒ-DNA, when the algal toxin target molecule passes through the reaction zone, the ƒ-DNA is replaced through a competitive reaction. The replaced f-DNA molecules, after elution, flow into the signal reading zone, where they specifically bind to and are rapidly identified by the capture nucleic acid probe d-DNA coated on the surface of the signal reading zone. The fluorescence intensity of the signal reading zone is detected using a fluorescence detector or a handheld fluorescence instrument. The fluorescence intensity is directly proportional to the algal toxin content, achieving on-site, real-time quantitative detection of algal toxins in water and overcoming the shortcomings of existing detection kits.

[0014] The present invention provides a method for detecting algal toxins, the method using the aforementioned paper chip device, comprising the following steps: (1) Fold the paper chip device along the fold line so that the reaction area faces up and the signal reading area faces down. Take the test solution and add it dropwise to the reaction area. The reaction is carried out at room temperature. The test solution includes a blank solution without algal toxins, a standard solution containing algal toxins, and an actual water sample that has been pretreated. (2) After the reaction is complete, add washing solution to the reaction area and repeatedly aspirate and aspirate with a pipette to fully dissolve the displaced ƒ-DNA in the washing solution; then unfold the paper chip device and lay it flat so that the second layer of rectangular channels covers the first layer of rectangular channels. With the help of capillary force, the washing solution migrates to the signal reading area through the second layer of rectangular channels in sequence. (3) Fold the reaction area back to back under the signal reading area. After the washing solution completely wets the signal reading area, ƒ-DNA is specifically recognized and enriched by d-DNA. After a period of reaction, insert one side of the paper chip signal reading area into a fluorometer or handheld fluorometer and record the fluorescence intensity of each signal reading area. Among them, the fluorescence intensity of the blank solution without algal toxin is close to the background signal, and the fluorescence intensity of the sample containing algal toxin shows a linear response, that is, the fluorescence intensity is proportional to the algal toxin content. (4) Using the concentration of algal toxin in the standard solution containing algal toxin as the abscissa and the corresponding fluorescence intensity as the ordinate, a working curve is plotted and fitted to obtain the standard curve equation. Based on the equation and the fluorescence intensity data of the sample to be tested, the content of algal toxin in the solution to be tested is calculated.

[0015] In the above technical solution, the reaction time in step (1) is 5~10 min, the pretreatment is membrane filtration, and the pore size of the membrane is 0.45 μm; The washing solution in step (2) is a PBS washing solution containing 0.05% Tween-20 and pH=7.4; The specific recognition reaction time in step (3) is 1~5 min; The signal acquisition device in step (4) is a handheld fluorometer, which performs photoelectric signal conversion through Sepu3010 and uses Sepu3010 software to display and record the fluorescence intensity data of the signal reading area.

[0016] In the above technical solution, the coating method between the reaction region and the signal reading region is further as follows: An aptamer with a concentration of 10 μmol / L to 10 mmol / L ( c The reaction region is coated with ƒ-DNA, and the concentration of fluorescently labeled complementary DNA (ƒ-DNA) is 10 μmol / L; the capture nucleic acid probe (ƒ-DNA) is added at a concentration of 10 μmol / L to 10 mmol / L. d -DNA) is coated on the signal reading region. Compared with liquid chromatography-mass spectrometry, the method of this invention does not require complex sample pretreatment processes and expensive instruments, and has the advantages of being simple and rapid. Compared with traditional enzyme-linked immunosorbent assay (ELISA), it uses low-cost paper chips instead of expensive 96-well plates, significantly reducing detection costs. Compared with commercial test strips, by separating the reaction area and the signal reading area and optimizing the ratio between color signal output and analyte concentration, it can more accurately and clearly reflect the true content of algal toxins in the sample and avoid result confusion.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention uses wax painting technology combined with biomodification methods to prepare paper chip devices. Based on the principle of nucleic acid specific recognition, it utilizes the interaction between algal toxins in the sample and the reaction zone. ƒ -DNA pairs c Detection is achieved through competitive binding to DNA binding sites. This is due to the aptamer... c -DNA has a higher affinity for MC-LR than ƒ -DNA, when algal toxins flow through the reaction zone, a competitive reaction causes fluorescent groups to... f -DNA is replaced; after one elution step, the replaced DNA is... ƒ -DNA detection region capture nucleic acid probe d - Rapid DNA identification and quantitative analysis via fluorescence detection show a positive correlation between fluorescence signal intensity and algal toxin concentration, resulting in clear and unambiguous results. The entire detection process takes no more than 10 minutes, and the cost per test is less than 1 RMB, offering advantages such as low cost and high stability.

[0018] In this method, the reaction area and the signal reading area are set separately, resulting in low background signal and high signal-to-noise ratio, which significantly improves the detection sensitivity. The detection limit can reach 0.1 μg / L, which is lower than the limit of algal toxins in drinking water set by the World Health Organization (1 μg / L).

[0019] Compared with traditional algal toxin detection methods, this invention overcomes technical bottlenecks such as cumbersome sample pretreatment, high detection costs, long detection time, and reliance on complex instruments, and realizes rapid on-site quantitative detection of algal toxins.

[0020] This method, after binding to the captured DNA in the reaction zone, blocks the unreacted carboxyl and amino groups on the surface of the reaction zone, effectively inhibiting their activity. c -Non-specific adsorption of DNA significantly improves the accuracy of detection results.

[0021] This method uses paper materials as the detection element, which is environmentally friendly and portable, and the detection method is simple to operate and easy to implement.

[0022] After optimization of experimental conditions, the linear detection range of this method is 0.01~10. 4 μg / mL, covering 6 orders of magnitude, with a wide dynamic detection range.

[0023] This invention enables the parallel detection of multiple target molecules by dividing the chip into different functional regions.

[0024] This method enables the quantitative analysis of algal toxins in water by constructing a standard curve. Attached Figure Description

[0025] The other objectives and technical advantages of the present invention will become clearer through the detailed description of the technical solution of the present invention in conjunction with the accompanying drawings, which will help to fully understand the technical features and innovations of the present invention.

[0026] Figure 1 is a schematic diagram of the principle of the on-site rapid quantitative detection method for algal toxins in water based on paper chips according to Embodiment 1 of the present invention.

[0027] Figure 2 is a flowchart of the use of the paper chip device according to Embodiment 1 of the present invention. In the figure, 1 is the reaction area, 2 is the signal reading area, 3 is the first layer rectangular channel, 4 is the second layer rectangular channel, and 5 is the fold line.

[0028] Figure 3 shows the algal toxin standard curve obtained by the algal toxin detection method of the present invention. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments, but this does not limit the present invention in any way.

[0030] To further illustrate the present invention, it is described in detail below with reference to embodiments and application examples. It should be understood that the listed embodiments and application examples are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0031] Table 1 Nucleic acid sequences used in this invention

[0032] Example 1: Fabrication of paper chip devices: The fabrication of paper-based chip devices includes the following steps: a. As shown in Figure 2, the paper chip device is fabricated based on Whatman No.1 filter paper. Two separate circular areas are located on the same plane: a reaction area 1 and a signal reading area 2. A fold line 5 is provided on the filter paper between the reaction area and the signal reading area. After folding along this line, the reaction area and the signal reading area are on different planes. Two rectangular channels are provided between the reaction area and the signal reading area: the first rectangular channel is coplanar with and connected to the reaction area, but separate from the signal reading area; the second rectangular channel is connected to the signal reading area. When the filter paper is laid flat, the second rectangular channel is directly above and covers the area of ​​the first rectangular channel; when the filter paper is folded, the second rectangular channel connected to the signal reading area is separated from both the reaction area and the first rectangular channel. The reaction area and the signal reading area are both 5 mm in diameter and white; both rectangular channels are 2 mm wide and 3 mm long, also white; the remaining areas of the filter paper, except for the reaction area, the signal reading area, and the two rectangular channels, are black. When the two rectangular channels overlap, liquid from the reaction area flows into the signal reading area through capillary action. Chip usage process as follows Figure 1 As shown.

[0033] b. A paper chip is prepared using a 3D printed mold. The mold includes a reaction area, a signal reading area, a first layer of rectangular channels, and a second layer of rectangular channels. The interior of the circular area and the two rectangular channels are white hydrophilic regions, while the exterior of the circular area is a black hydrophobic region. Molten wax is injected into the mold, and the paper chip is demolded after cooling to room temperature.

[0034] c. To fabricate a paper chip, first, fix the reaction zone... c -DNA (nucleic acid sequence shown in Table 1), with a concentration of 10 μmol / L. The specific steps are as follows: First, add 0.25 mg / mL chitosan solution to the reaction zone, dry at room temperature, then add 2.5% glutaraldehyde solution and incubate at room temperature for 2 h; subsequently, wash thoroughly with PBS containing 0.05% Tween-20, pH=7.4, and dry at room temperature; next, add 1 mg / mL streptavidin solution, incubate for 10-20 min, then wash and dry; then add 0.5% BSA solution for blocking, react for 10 min, then wash and dry at room temperature; finally, add dropwise... c Incubate the f-DNA solution for 10 min, wash and dry, and finally add 10 μmol / L f-DNA, then wash three times with PBS solution.

[0035] d. Fix in the reaction zone d-DNA (nucleic acid sequence shown in Table 1), with a concentration of 10 μmol / L. Incubate for 10–20 min, wash and dry, and store at 4°C for later use.

[0036] Example 2: Quantitative detection of algal toxins in river water: a. Place the paper chip prepared in Example 1 horizontally, fold the reaction area and signal reading area back to back so that the reaction area faces upward. Take a river water sample, allow it to settle overnight, and filter the supernatant. Transfer 10 μL of the filtered sample and add it to the chip reaction area, reacting at room temperature for 8 min. The test solution includes a blank solution, a standard solution containing algal toxin standards, and a pretreated actual water sample (the actual water sample is filtered through a 0.45 μm filter membrane).

[0037] b. After the reaction is complete, add 40 μL of 1×PBS buffer solution to the reaction area and repeatedly pipette to ensure that the displaced ƒ-DNA is fully dissolved in the buffer solution. At this point, the solution is evenly distributed in the first layer of rectangular channels and the reaction area. Then, unfold the paper chip and lay it flat so that the reaction area and the signal reading area are on the same plane. With the help of capillary action of the paper chip, the solution migrates to the signal reading area through the second layer of rectangular channels.

[0038] c. Fold the reaction zones back-to-back under the signal readout zone. The displaced ƒ-DNA will be captured by the nucleic acid probe on the signal readout zone. d - DNA rapid identification: After 2 min of reaction, the fluorescence intensity in each signal reading region was recorded. The fluorescence intensity of the control group with added blank solution tended to the background signal, while the fluorescence intensity of the group with added algal toxin was linear, that is, the fluorescence intensity was directly proportional to the content of algal toxin. d. The fluorescence intensity of the signal reading area was measured using a handheld fluorometer. The photoelectric signal was converted using a Sepu3010 fluorometer, and the signal was displayed and recorded using Sepu3010 software. The fluorescence intensity data refers to the fluorescence intensity data of the signal reading area, with the concentration of algal toxins in the standard solution as the x-axis. The concentration range of the standard solution is 0.01~10. 4 μg / mL, with the corresponding fluorescence intensity data as the ordinate, such as Figure 3 As shown, the working curve was plotted and fitted to obtain the standard curve equation. The content of algal toxins in the sample solution was calculated from the standard curve equation and the fluorescence intensity data of the sample.

Claims

1. A paper-based chip device for on-site, real-time quantitative detection of algal toxins in water, characterized in that, The reaction region of the paper chip device is coated with an aptamer. c -DNA and fluorescently labeled ƒ-DNA, the read region of the paper chip device is covered with capture nucleic acid probes. d -DNA; The aptamer c The nucleic acid sequence of the DNA is shown in SEQ ID NO.

1. c The 5' end of the ƒ-DNA is labeled with biotin; the nucleic acid sequence of the ƒ-DNA is shown in SEQ ID NO.2, and the 5' end is labeled with a FAM fluorescent group; the nucleic acid sequence of the capture nucleic acid probe d-DNA is shown in SEQ ID NO.

3. d - The 5' end of the DNA is labeled with biotin.

2. The paper chip device according to claim 1, characterized in that, The aptamer c - The concentration of DNA is 10 μmol / L to 10 mmol / L; d - The concentration of DNA is 10 μmol / L to 10 mmol / L.

3. The paper chip device according to claim 1, characterized in that, The method for coating the reaction region and the reading region includes: Chitosan solution was added to both the reaction zone and the signal reading zone, and after drying at room temperature, glutaraldehyde solution was added. The mixture was incubated at room temperature for 120–150 min, washed, and dried. Streptavidin solution was added, and the mixture was incubated for 10–20 min, washed, and dried. BSA solution was added for blocking, and the reaction was allowed to proceed for 10–20 min. The mixture was then washed with Tween-20 and PBS buffer, dried at room temperature, and then added dropwise to the reaction zone. c -DNA solution, incubate for 10-20 min, wash and dry, then add ƒ-DNA solution, react for 10-20 min, wash and dry again, store for later use (protect from light), and add dropwise to the signal reading region. d -DNA solution, incubate for 10-20 min, wash and dry, store at 4 ℃ for later use.

4. The paper chip device according to claim 3, characterized in that, The chitosan solution has a concentration of 0.24~0.26 mg / mL, the glutaraldehyde solution has a volume fraction of 2.4~2.6%, the streptavidin solution has a concentration of 0.9~1.1 mg / mL, the BSA solution has a volume fraction of 0.4~0.6%, the Tween-20 solution has a volume fraction of 0.04~0.06%, and the PBS buffer has a pH of 7.4~7.

6.

5. The paper chip device according to claim 1, characterized in that, The paper chip device has two separate circular areas on the same plane of the filter paper, namely the reaction chip area (1) and the colorimetric chip area (2); the filter paper has a fold line (5), which is located between the reaction area (1) and the signal reading area (2) so that the filter paper can be folded in half. When the filter paper is folded along the fold line (5), the reaction area (1) and the signal reading area (2) are on different planes; there are two layers of rectangular channels between the reaction area (1) and the signal reading area (2), wherein the first layer of rectangular channels (3) is coplanar with and connected to the reaction area (1), and separate from the signal reading area (2); the second layer of rectangular channels (4) is located in the first layer of rectangular channels (3) and connected to the reaction area (1), and separate from the signal reading area (2); The first rectangular channel (3) is directly above the signal reading area (2); when the reaction area (1) and the signal reading area (2) are on the same plane, the second rectangular channel (4) covers the first rectangular channel (3) directly above it, and the first rectangular channel (3) is completely located within the area of ​​the second rectangular channel (4); when the reaction area (1) and the signal reading area (2) are on different planes by folding along the fold line (5), the second rectangular channel (4) connected to the signal reading area (2) is separated from the reaction area (1) and the first rectangular channel (3), and the first rectangular channel and the second rectangular channel are on different planes respectively.

6. The paper-based chip device according to claim 5, characterized in that, The reaction zone (1), signal reading zone (2), first rectangular channel (3) and second rectangular channel (4) are all hydrophilic zones; except for the reaction zone (1), signal reading zone (2) and the two rectangular channels, the area is painted with wax, and molten liquid wax is injected into the mold to form a hydrophobic zone; the molten wax is injected into the mold, and the paper chip is demolded after cooling to room temperature.

7. A method for detecting algal toxins, characterized in that, The method uses the paper chip device according to any one of claims 1-6 and includes the following steps: (1) Fold the paper chip device along the fold line so that the reaction area faces up and the signal reading area faces down. Take the test solution and add it dropwise to the reaction area. The reaction is carried out at room temperature. The test solution includes a blank solution without algal toxins, a standard solution containing algal toxins, and an actual water sample that has been pretreated. (2) After the reaction is complete, add washing solution to the reaction area and repeatedly aspirate and aspirate with a pipette to fully dissolve the displaced ƒ-DNA in the washing solution; then unfold the paper chip device and lay it flat so that the second layer of rectangular channels covers the first layer of rectangular channels. With the help of capillary force, the washing solution migrates to the signal reading area through the second layer of rectangular channels in sequence. (3) Fold the reaction zone back-to-back under the signal reading zone. After the washing solution has completely soaked the signal reading zone, ƒ -DNA is d - DNA-specific recognition and enrichment; after a period of reaction, insert one side of the signal reading area of ​​the paper chip into a fluorometer or handheld fluorometer and record the fluorescence intensity of each signal reading area; among them, the fluorescence intensity of the blank solution without algal toxins is close to the background signal, and the fluorescence intensity of the sample containing algal toxins shows a linear response, that is, within the linear range, the fluorescence intensity is proportional to the algal toxin content. (4) Using the concentration of algal toxin in the standard solution containing algal toxin as the abscissa and the corresponding fluorescence intensity as the ordinate, a working curve is plotted and fitted to obtain the standard curve equation. Based on the equation and the fluorescence intensity data of the sample to be tested, the content of algal toxin in the solution to be tested is calculated.

8. The method according to claim 7, characterized in that, The reaction time in step (1) is 5~10 min, and the pretreatment is a membrane filtration process with a pore size of 0.45 μm. The washing solution in step (2) is a PBS washing solution containing 0.05% Tween-20 and pH=7.4; The specific recognition reaction time in step (3) is 1~5 min; The signal acquisition device in step (4) is a handheld fluorometer, which performs photoelectric signal conversion through Sepu3010 and uses Sepu3010 software to display and record the fluorescence intensity data of the signal reading area.

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