Time-resolved fluorescent quantitative detection microcystin test strip and preparation and detection methods thereof

Abstract

The invention discloses a time-resolved fluorescent quantitative detection microcystin test strip as well as a preparation method and a detection method of the time-resolved fluorescent quantitative detection microcystin test strip. The test strip comprises a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are arranged on a bottom plate. A combination pad of the test strip is coated with a europium-labeled microcystin nucleic acid aptamer nanoprobe, and a nitrocellulose membrane is provided with a detection line and a quality control line. During detection, microcystic toxins in a sample are competitively combined with the probe to influence a T line fluorescence signal, and accurate quantification can be realized by measuring the fluorescence intensity ratio of a T / C line and substituting the fluorescence intensity ratio into a standard curve. The high specificity of the nucleic acid aptamer and the high sensitivity of time-resolved fluorescence are combined, and the probe structure, the reagent formula and the preparation process are optimized, so that the detection has the advantages of high sensitivity, strong specificity, rapidness, simplicity and convenience in operation, good stability and the like, and is suitable for on-site rapid detection of microcystin in a water environment and an algae sample.

Description

Technical Field

[0001] This invention belongs to the field of environmental monitoring and biochemical analysis technology, specifically relating to a time-resolved fluorescence quantitative detection test strip for microcystin, its preparation method, and its detection method. Background Technology

[0002] Microcystins (MCs) are a class of cyclic heptapeptide toxins produced by cyanobacteria. Among them, microcystin-LR (MC-LR) is the most toxic and widely distributed, exhibiting strong hepatotoxicity and potential carcinogenicity, seriously threatening aquatic environmental safety and human health. Therefore, establishing a rapid, sensitive, and accurate method for detecting microcystins is crucial.

[0003] Currently, the main methods for detecting microcystin include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and enzyme-linked immunosorbent assay (ELISA). HPLC and LC-MS have the advantages of high accuracy and specificity, but they require expensive equipment, are complex to operate, and have long detection times, making them unsuitable for rapid on-site detection and large-scale sample screening. ELISA is simple to operate and has a lower cost, but it suffers from problems such as large fluorescence background interference, insufficient sensitivity, and poor stability, making it difficult to meet the needs of detecting low concentrations of microcystin.

[0004] Time-resolved fluorescence analysis is a novel fluorescence detection technique that uses rare earth elements (such as europium and terbium) as fluorescent markers. It takes advantage of the long fluorescence lifetime and large Stokes shift of rare earth elements and eliminates interference from non-specific fluorescence background by setting a delay time. It has advantages such as high sensitivity, strong specificity and good stability.

[0005] Nucleic acid aptamers are single-stranded DNA or RNA oligonucleotides obtained through exponential enrichment ligand system evolution techniques. They bind with high affinity and high specificity to specific target molecules (such as MC-LR). Unlike antibodies, they offer advantages such as simple preparation, high stability, and ease of modification and labeling. The specific binding of a single-stranded oligonucleotide to a target by a nucleic acid aptamer itself does not generate detectable physical or chemical signals. Therefore, nucleic acid aptamers are often used as recognition molecules, combined with nanotechnology. Utilizing the optical and electrical properties of nanomaterials, detection sensors are constructed to convert the specific binding between the aptamer and the target molecule into a quantitatively detectable photoelectric signal. This results in a linear correlation between the target concentration and the fluorescence intensity of the system within a certain range, thereby achieving qualitative and quantitative detection of the target analyte.

[0006] By combining time-resolved fluorescence analysis technology with nucleic acid aptamers, an optimized test strip and method for the time-resolved fluorescence quantitative detection of microcystin based on nucleic acid aptamers have been developed, which has important practical significance for improving the sensitivity, stability and efficiency of detection. Summary of the Invention

[0007] To address the shortcomings of existing microcystin detection methods, such as insufficient sensitivity, poor stability, and complex operation, this invention provides a time-resolved fluorescence quantitative detection test strip for microcystin based on nucleic acid aptamers, along with its preparation and detection method. This test strip features optimized components, a simple and rapid detection method, high sensitivity, high specificity, and good stability, enabling accurate quantitative detection of microcystin. Specifically, this invention utilizes europium (Eu)... 3+ The microcystin-labeled nucleic acid aptamer probe, after optimization of the coupling molar ratio, improves the stability and binding activity of the nucleic acid aptamer. The fluorescence enhancement solution uses a specific ratio of β-diketone, tri-n-octylphosphine oxide, and Triton X-100 to further enhance the stability of the fluorescence signal. The method for time-resolved fluorescence quantitative detection of microcystins using the above test strip includes sample addition reaction, fluorescence enhancement reaction, and fluorescence detection steps. The sample pretreatment steps are simple and suitable for the detection of different types of samples. During detection, the microcystins in the sample compete with the probe for binding, affecting the T-line fluorescence signal. Accurate quantification can be achieved by measuring the fluorescence intensity ratio of the T / C lines and substituting it into a standard curve.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention proposes a time-resolved fluorescence quantitative detection test strip for microcystin, comprising a sample pad, a conjugate pad, a nitrocellulose membrane (NC membrane), and an absorbent pad sequentially arranged on a base plate, wherein the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad are stacked end-to-end sequentially; specifically: The sample pad and the bonding pad are made of glass fiber. The binding pad is sprayed with fluorescently labeled microcystin nucleic acid aptamer probes; The nitrocellulose membrane has a detection line (T line) and a control line (C line) spaced parallel to each other. The detection line has a nucleotide sequence that is complementary to a partial sequence of the microcystin nucleic acid aptamer; the control line has a nucleotide sequence that binds to the terminal sequence of the microcystin nucleic acid aptamer.

[0009] Furthermore, the fluorescently labeled microcystin nucleic acid aptamer probe is obtained by coupling rare earth element-labeled time-resolved nanospheres with microcystin nucleic acid aptamers, wherein the rare earth element is europium.

[0010] Furthermore, the microcystin nucleic acid aptamer sequence was designed as: 5′-GGGGCCATATGCCTACCATCTCTTTTTTTTTTTT-NH2-3′; the detection line aptamer sequence was a partially complementary sequence of the microcystin nucleic acid aptamer, specifically 5′-GAGATGTGCTGCATA-Biotin-3′; and the control line aptamer sequence was a poly A sequence, specifically 5′-Biotin-AAAAAAAAAAAAAAAAA-3′.

[0011] Secondly, the present invention provides a method for preparing the above-mentioned time-resolved fluorescence quantitative detection test strip for microcystin, comprising the following process steps: S1: Glass fiber was selected as the sample pad and conjugate pad material. After being fully soaked in PBS solution, it was dried in an oven and then sealed and stored for later use. S2: The fluorescently labeled microcystin nucleic acid aptamer probe solution was sprayed onto the conjugate pad using a gold spraying machine, dried in an oven, and then sealed and stored for later use. S3: Using a gold spraying machine, the prepared detection line solution and control line solution are sprayed onto a nitrocellulose membrane to obtain the detection line and control line. After drying in an oven, they are sealed and stored for later use. S4: Assembly of test strips: The prepared sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad are sequentially overlapped and assembled on the base plate along the chromatography direction.

[0012] Furthermore, in step S2, the preparation process of the fluorescently labeled microcystin nucleic acid aptamer probe is as follows: a certain amount of carboxylated europium-labeled time-resolved fluorescent microspheres and aminated microcystin nucleic acid aptamers are coupled at 25-30℃. After reacting for 12-16 hours, the mixture is purified by centrifugation, resuspended in 0.01 mol / L PBS resuspension, and stored at 4-5℃.

[0013] Further, in step S3, the preparation process of the detection line solution is as follows: a streptavidin solution of a certain concentration is prepared with PBS solution, a certain volume of biotinylated detection line aptamer solution is added to the streptavidin solution, and the reaction is carried out at 4-5℃ for 2-3 h; after the reaction, the solution is centrifuged in an ultrafiltration centrifuge tube and washed with PBS solution, and finally the solution in the tube is collected and resuspended in PBS solution to the original volume for later use; the preparation process of the quality control line solution is as follows: a streptavidin solution of a certain concentration is prepared with PBS solution, a certain volume of biotinylated quality control line aptamer solution is added to the streptavidin solution, and the reaction is carried out at 4-5℃ for 2-3 h; after the reaction, the solution is centrifuged in an ultrafiltration centrifuge tube and washed with PBS solution, and finally the solution in the tube is collected and resuspended in PBS solution to the original volume for later use.

[0014] Furthermore, in step S4, the overlap is configured as follows: the sample pad overlaps one end of the conjugate pad, the other end of the conjugate pad overlaps one end of the nitrocellulose membrane, and the absorbent pad overlaps the other end of the nitrocellulose membrane, with an overlap length of 2-3 mm between them. The strips are then cut into test strips with a width of 3-4 mm using a strip cutter. The conjugate pad is placed on one side of the detection line on the nitrocellulose membrane, and the absorbent pad is placed on one side of the quality control line on the nitrocellulose membrane.

[0015] Thirdly, the present invention also proposes a detection method for the time-resolved fluorescence quantitative detection strip for microcystin as described above, comprising the following process steps: S1: Prepare microcystin standard solutions with different concentration gradients, using PBS buffer solution as the solvent; S2: Preparation of fluorescence enhancement solution: The fluorescence enhancement solution is an ethanol-water solution containing β-diketone, tri-n-octylphosphine oxide and Triton X-100, wherein the concentration of β-diketone is 0.1-0.3 mol / L and the concentration of tri-n-octylphosphine oxide is 0.05-0.15 mol / L; S3: Microcystin standard solutions of different concentration gradients are added dropwise to the sample pad of the test strip, and fluorescence enhancement solution is added. The mixture is reacted at room temperature for a period of time. The fluorescence intensity ratio of the corresponding detection line and control line is measured using a time-resolved fluorescence detector. A standard curve is plotted with the standard solutions of different concentrations as the abscissa and the fluorescence intensity ratio of the corresponding detection line and control line as the ordinate to obtain the regression equation. S4: The fluorescence intensity ratio of the detection line and the control line of the pretreated sample is obtained by using the process in step S3. Then, the ratio is substituted into the regression equation to calculate the concentration of microcystin in the sample.

[0016] Furthermore, the sample is a water sample or an algae sample. The pretreatment process for the water sample is as follows: take the water sample into a centrifuge tube, centrifuge it, and take the supernatant. The pretreatment process for the algae sample is as follows: take the algae sample, add PBS buffer solution, homogenize it, centrifuge it, take the supernatant, and filter the supernatant through a filter membrane to obtain the sample solution.

[0017] The technical solution of the present invention has the following beneficial effects: 1. Sensitivity: This invention uses europium (Eu) 3+ As a fluorescent marker, combined with a fluorescence enhancement solution, it significantly reduces fluorescence background interference and improves fluorescence signal intensity, with a quantitative detection limit of up to 0.05 ng / mL.

[0018] 2. Specificity: The present invention uses the highly specific microcystin nucleic acid aptamer A2C, which has a cross-reactivity rate of ≤5% with microcystin analogs (such as MC-RR, MC-YR and nodularin), and can accurately distinguish microcystin-LR from other analogs.

[0019] 3. Ease of operation: After optimization of the detection process, the entire detection process can be completed within 10 minutes. The sample pretreatment steps are simple, no complicated instruments and equipment are required, the detection accuracy is ≥85%, and the method reproducibility RSD is ≤10%, which is suitable for rapid on-site detection and large-scale sample screening.

[0020] 4. Excellent stability and reproducibility: By optimizing the probe coupling process, resuspension formulation, and drying and storage conditions of the test strips, the test strips of this invention show no significant change in detection performance (such as the correlation coefficient of the standard curve and the limit of detection) after being stored at 4°C for 6 months. The intra-batch and inter-batch coefficients of variation are both less than 10%, demonstrating excellent stability and reproducibility.

[0021] 5. This invention combines the high specificity of nucleic acid aptamers with the high sensitivity of time-resolved fluorescence, and through optimization of probe structure, reagent formulation and preparation process, the detection has the advantages of high sensitivity, strong specificity, rapid and simple operation and good stability. It is suitable for rapid on-site detection of microcystin in aquatic environment and algal samples. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the structure of the time-resolved fluorescence quantitative detection test strip for microcystin-LR of the present invention; Figure 2 For the interpretation of results of time-resolved fluorescence quantitative detection of microcystin-LR test strips; Figure 3 This is a standard curve for the quantitative detection of microcystin-LR in an embodiment of the present invention. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0024] Example 1: Preparation of test strips

[0025] like Figure 1The present invention provides a time-resolved fluorescence quantitative detection test strip for microcystin, comprising a sample pad, a conjugation pad, a nitrocellulose membrane (NC membrane), and an absorbent pad sequentially arranged on a base plate, wherein the sample pad, conjugation pad, nitrocellulose membrane, and absorbent pad are stacked end-to-end in sequence; wherein: The sample pad and the bonding pad are made of glass fiber. The binding pad is sprayed with fluorescently labeled microcystin nucleic acid aptamer probes; The nitrocellulose membrane has detection lines (T lines) and control lines (C lines) spaced parallel to each other.

[0026] The fluorescently labeled microcystin nucleic acid aptamer probe is obtained by coupling europium-labeled time-resolved nanospheres with microcystin nucleic acid aptamers.

[0027] The europium (Eu) described in this embodiment 3+ Preparation method of labeled microcystin nucleic acid aptamer nanoprobes: (1) Design of microcystin nucleic acid aptamer sequences: The microcystin nucleic acid aptamer A2C: 5′-GGGGCCATATGCCTACCATCTCTTTTTTTTTTTT-NH2-3′; T-line of detection: 5′-GAGATGTGCTGCATA-Biotin-3′; C-line of detection: 5′-Biotin-AAAAAAAAAAAAAAAAA-3′; The working concentration of the microcystin nucleic acid aptamer sequence is 0.1 μmol / L; (2) Preparation of fluorescently labeled nucleic acid aptamer probes: europium (Eu) 3+ Time-resolved nanospheres labeled with microcystin were coupled with microcystin nucleic acid aptamers. The nanospheres underwent carboxylation treatment, and the nucleic acid aptamers underwent amination treatment. The mass ratio of nanospheres to nucleic acid aptamers was 1:5 to 1:8. Specific steps included: taking 20 μL of 100 μmol / L nucleic acid aptamer and 0.1 mg of Eu... 3+ Time-resolved fluorescent microspheres were coupled, with the coupling reaction temperature controlled at 25–30 °C and the reaction time at 12–16 h. After the coupling reaction, the uncoupled microcystin-LR aptamers were removed by centrifugation (8000 r / min, 15 min, 25 °C), and the microcystin-LR aptamers were resuspended in 0.01 mol / L PBS resuspension (0.5% PEG, 1% sucrose, 0.1% Tween-20, 0.02% MgSO4, 0.05% (NH4)2SO4, pH 7.4) and stored at 4 °C. The assembly and preparation process of the time-resolved fluorescence quantitative detection test strip for microcystin: (a) Sample pad and conjugate pad: Glass fiber was selected as the material for the sample pad and conjugate pad. After being fully soaked in 0.001 mol / L PBS solution (0.25% Tween-20, 2% sucrose), it was placed in a 37℃ oven and dried for 2 h. It was then sealed and stored for later use. (b) Spraying with gold: The fluorescently labeled nucleic acid aptamer conjugate solution was sprayed onto the conjugate pad at a spray rate of 2.0 μL / cm using a gold sprayer, placed in a 37°C oven, dried for 1 h, and sealed for later use. (c) Preparation of T and C line solutions: A 1 mg / mL streptavidin solution was prepared using PBS (0.01 mol / L, pH 7.4). A certain volume of 50 μM biotinylated T line aptamer solution was added to 1 mg / mL streptavidin, and the reaction was carried out at 4℃ for 2 h. After the reaction, the solution was centrifuged in an ultrafiltration centrifuge tube (10 KD, Millipore's Amicon Ultra-15) at 6000 r / min for 10 min, washed twice with 0.01 mol / L PBS, and finally the solution in the tube was collected and resuspended in 0.01 mol / L PBS to the original volume for later use. The C line solution was treated in the same way. The prepared T and C line solutions were sprayed onto an NC membrane using a gold spraying apparatus and dried in a 37℃ oven for later use.

[0028] (d) Assembly of the test strip: Assemble the prepared sample pad, conjugate pad, NC membrane, and absorbent pad according to... Figure 1 Assemble and paste the test strips onto the base plate in the order shown, with each part overlapping by approximately 2 mm. Cut the strips into 3 mm wide strips using a strip cutter and package them together with the desiccant in an aluminum foil bag for later use.

[0029] Preparation of the reaction solutions related to this invention: (a) Working solution: 0.01 mol / L PBS solution (0.25% Tween-20, 2% sucrose, pH 7.4); (b) 0.01 mol / L PBS resuspension: (0.5% PEG, 1% sucrose, 0.1% Tween-20, 0.02% MgSO4, 0.05% (NH4)2SO4, pH 7.4); (b) Fluorescence enhancement solution: ethanol-water solution containing β-diketone, tri-n-octylphosphine oxide and Triton X-100, wherein the concentration of β-diketone is 0.1-0.3 mol / L and the concentration of tri-n-octylphosphine oxide is 0.05-0.15 mol / L.

[0030] Technical Principles

[0031] The principle is as follows Figure 2The diagram illustrates the process of spraying fluorescently labeled microcystin nucleic acid aptamer probes onto the conjugation pad and setting a test line and control line on the test strip. The test line is a partially complementary sequence of the MC-LR aptamer, and the control line is a poly A sequence, both immobilized on a nitrocellulose membrane using the streptavidin-biotin system. Sample solution is added to the sample pad; due to chromatography, the solution flows slowly forward. When there is no MC-LR in the sample, the colloidal gold solution labeled with the aptamer is first captured by the complementary sequence of the aptamer immobilized on the test line, resulting in a red T-line and a negative result (e.g., ...). Figure 2 (Illustration). When MC-LR is present in the sample, the colloidal gold solution labeled with the aptamer preferentially binds to MC-LR but not to the complementary sequence of the aptamer at the test line. The T line is less red than the C line or disappears. Because the aptamer has a linker base at the end, it can also bind to the DNA poly A at the control line, making the C line red. The result is weakly positive or positive (e.g., ...). Figure 2 (Illustration). When line C is colorless, it means the test strip is invalid.

[0032] For ease of use, the test strip in this embodiment is further provided with an outer shell, which covers the outside of the test strip; the outer shell includes a base and a cover, and the cover is provided with an observation port and a sample application port; the sample application port opens into the upper part of the sample pad to expose part or all of the sample pad area; the observation port opens into the upper part of the nitrocellulose membrane to expose all the detection lines and control lines.

[0033] Example 2 Detection of Microcystin

[0034] The process requires pretreatment of the samples to be tested. The samples can be water or algae. The pretreatment steps for the sample solution are as follows: For water samples, take 10 mL of water sample into a centrifuge tube, centrifuge at 10000 r / min for 10 min, take the supernatant, filter it through a 0.22 μm filter membrane and set it aside for later use; For algae samples, take 1 g of fresh cyanobacteria sample, add 5 mL of PBS buffer, homogenize with a tissue homogenizer for 2 min, centrifuge at 12000 r / min for 15 min, take the supernatant, filter it through a 0.22 μm filter membrane and set it aside for later use as the sample solution.

[0035] The detection method of the time-resolved fluorescence quantitative detection test strip for microcystin of the present invention includes the following process steps: S1: Prepare microcystin standard solutions with different concentration gradients. The standard is pure microcystin-LR with a purity ≥99%. In this embodiment, the concentration gradient is set to 0.01 ng / mL, 0.05 ng / mL, 0.2 ng / mL, 1 ng / mL, 5 ng / mL, and 20 ng / mL. The solvent is 0.01 mol / L PBS buffer at pH 7.4. S2: Preparation of fluorescence enhancement solution: The fluorescence enhancement solution is an ethanol-water solution containing β-diketone, tri-n-octylphosphine oxide and Triton X-100, wherein the concentration of β-diketone is 0.1-0.3 mol / L and the concentration of tri-n-octylphosphine oxide is 0.05-0.15 mol / L; S3: Take 60 μL of microcystin standard solutions of different concentration gradients and add them to the sample pad of the test strip, and add 10 μL of fluorescence enhancement solution. React at room temperature for 5 min. Use a time-resolved fluorescence detector, set the excitation wavelength to 340 nm, the emission wavelength to 615 nm, the delay time to 400 μs, and the measurement time to 400 μs. Observe and compare the color development of the T and C lines of each group of test strips; record the fluorescence intensity of the corresponding detection line and control line and calculate their ratio; plot the standard solution of different concentrations as the abscissa and the fluorescence intensity ratio of the corresponding detection line and control line as the ordinate to obtain the regression equation. S4: The fluorescence intensity ratio of the detection line and the control line of the pretreated sample is obtained by using the process in step S3. Then, the ratio is substituted into the regression equation to calculate the concentration of microcystin in the sample.

[0036] Results Calculation: A standard curve was plotted with the concentration of microcystin-LR standard as the abscissa (x, ng / mL) and the T / C ratio of fluorescence signal intensity of the T and C lines as the ordinate (y). The standard curve showed a good linear relationship, and the regression equation was y = -1.5769x + 0.1734, with a correlation coefficient R² = 0.987. Substituting the ratio of the T-line fluorescence intensity to the C-line fluorescence intensity detected in the samples into the equation, the microcystin concentration in the water sample was calculated to be 0.08 ng / mL, and the microcystin concentration in the algal sample was 2.15 ng / mL.

[0037] Example 3: Test strip performance verification

[0038] 1. Sensitivity verification: Perform 10 repeated tests on the blank sample and calculate the average fluorescence intensity value. ) and standard deviation (s), in The concentration corresponding to +3s is the detection limit. The concentration corresponding to +10s is the limit of quantitation. The results show that the limit of quantitation is 0.05 ng / mL, indicating that the test strip has high sensitivity.

[0039] 2. Specificity Validation: Microcystin-RR, Microcystin-YR, and nodularin standards at concentrations of 10 ng / mL were tested, and the cross-reactivity rates were calculated. The results showed that the cross-reactivity rates were 3.2%, 2.8%, and 4.5%, respectively, all ≤5%, indicating that the test strip has high specificity.

[0040] 3. Stability verification: The test strips were stored at 4℃ for 6 months, and the detection performance was tested at 0 months, 2 months, 4 months and 6 months. The results showed that the correlation coefficient of the standard curve of the test strips was ≥0.995 for different storage times, and there was no significant change in the limit of detection and limit of quantitation, indicating that the test strips had good stability.

[0041] 4. Precision Validation: Intra-batch and inter-batch repeated testing was performed on standards with concentrations of 0.1 ng / mL, 1 ng / mL, and 10 ng / mL. Within-batch tests were repeated 10 times for each concentration, and inter-batch tests were performed continuously over 5 days. Results showed that the intra-batch coefficient of variation was 2.1%–4.3%, and the inter-batch coefficient of variation was 3.5%–7.2%, both meeting the testing requirements, indicating good precision of the test strips.

[0042] In the specific embodiments of this invention, parameters such as the concentration gradient of the microcystin standard, the labeling rate and concentration of the fluorescently labeled nucleic acid aptamer probe, the coupling molar ratio of fluorescent microspheres to nucleic acid aptamers, the concentration of the enhancer, and the reaction conditions are all optimal parameters obtained through extensive experimental optimization, ensuring the detection performance of the test strip. The sample pretreatment steps have been optimized according to the sample type, making it suitable for the detection of samples with different matrices such as water samples and algal samples, thus expanding the application range of the test strip.

[0043] It should be noted that the scope of protection of this invention is not limited to the specific embodiments described above. For those skilled in the art, appropriate adjustments and modifications can be made to the test strip components and detection method without departing from the principle of this invention, and these adjustments and modifications should also be considered within the scope of protection of this invention. For example, the fluorescent marker can be replaced with terbium (Tb). 3+ Other rare earth elements, such as microcystin nucleic acid aptamers, can be replaced with other oligonucleotide sequences with the same detection efficiency. These replacements are all equivalent replacements of the present invention and fall within the protection scope of the present invention.

[0044] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A time-resolved fluorometric quantitative detection microcystin test strip, characterized in that, The test strip comprises a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad arranged on a base plate in sequence, and the sample pad, the binding pad, the nitrocellulose membrane and the water absorption pad are arranged in sequence with their ends partially overlapped. The sample pad and the binding pad are made of glass fiber. The binding pad is sprayed with fluorescently labeled microcystin aptamer probes. The nitrocellulose membrane is provided with a detection line and a quality control line arranged in parallel and at intervals.

2. The time-resolved fluorescence quantitative detection microcystin test strip according to claim 1, characterized in that, The fluorescently labeled microcystin aptamer probe is obtained by coupling rare earth element labeled time-resolved nanometer microspheres with microcystin aptamer, and the rare earth element is europium. 3.The time-resolved fluorescence quantitative detection microcystin test strip according to claim 2, characterized in that, The microcystin aptamer sequence is designed as 5'-GGGGCCATATGCCTACCATCTCTTTTTTTTTTTT-NH2-3'; the detection line aptamer sequence is a partial complementary sequence of the microcystin aptamer, specifically 5'-GAGATGTGCTGCATA-Biotin-3'; and the quality control line aptamer sequence is a poly A sequence, specifically 5'-Biotin-AAAAAAAAAAAAAAAAA-3'.

4. The preparation method of a time-resolved fluorescence quantitative detection microcystin test strip according to claim 2 or 3, characterized in that, The test strip comprises the following process steps: S1: glass fiber is selected as the material of the sample pad and the binding pad, the sample pad and the binding pad are soaked in PBS solution, dried in an oven, and then sealed and stored for later use; S2: the fluorescently labeled microcystin aptamer probe solution is sprayed on the binding pad by a membrane scribing and gold spraying instrument, dried in an oven, and then sealed and stored for later use; S3: the prepared detection line solution and the quality control line solution are sprayed on the nitrocellulose membrane by a membrane scribing and gold spraying instrument to obtain the detection line and the quality control line, which are dried in an oven and then sealed and stored for later use; S4: the test strip is assembled by sequentially arranging the treated sample pad, the binding pad, the nitrocellulose membrane and the water absorption pad on the base plate in the chromatographic direction.

5. The method for preparing a test strip for time-resolved fluorescence quantitative detection of microcystins according to claim 4, characterized in that, In step S2, the fluorescently labeled microcystin aptamer probe is prepared by the following process: a certain amount of carboxylated europium labeled time-resolved fluorescent microspheres and aminoated microcystin aptamer are coupled at 25-30℃ for 12-16h, and then purified by centrifugation, resuspended in 0.01mol / L PBS solution, and stored at 4-5℃.

6. The method for preparing a test strip for time-resolved fluorescence quantitative detection of microcystins according to claim 4, characterized in that, In step S3, the preparation process of the detection line solution is as follows: a certain concentration of streptavidin solution is prepared by using PBS solution, a certain volume of biotinylated detection line aptamer solution is added to the streptavidin solution, and the mixture is reacted at 4-5℃ for 2-3h; after the reaction, the mixture is centrifuged in an ultrafiltration centrifuge tube, washed with PBS solution, and finally the solution in the tube is collected and resuspended in PBS solution to the original volume for later use; the preparation process of the quality control line solution is as follows: a certain concentration of streptavidin solution is prepared by using PBS solution, a certain volume of biotinylated quality control line aptamer solution is added to the streptavidin solution, and the mixture is reacted at 4-5℃ for 2-3h; after the reaction, the mixture is centrifuged in an ultrafiltration centrifuge tube, washed with PBS solution, and finally the solution in the tube is collected and resuspended in PBS solution to the original volume for later use.

7. The method for preparing a test strip for time-resolved fluorescence quantitative detection of microcystins according to claim 4, characterized in that, In step S4, the overlap is arranged as follows: the sample pad is overlapped at one end of the bonding pad, the other end of the bonding pad is overlapped at one end of the nitrocellulose membrane, and the water absorption pad is overlapped at the other end of the nitrocellulose membrane, with an overlap length of 2-3 mm between each other, and the test paper strip is cut into a width of 3-4 mm by a slitting machine; the bonding pad is arranged on the nitrocellulose membrane on the side of the detection line, and the water absorption pad is arranged on the nitrocellulose membrane on the side of the quality control line.

8. The method of claim 2 or 3, wherein the microcystin detection method is a time-resolved fluoroimmunoassay. The method comprises the following steps: S1: preparing microcystin standard solution with different concentration gradients, and the solvent is PBS buffer solution; S2: preparing a fluorescence enhancer: the fluorescence enhancer is an ethanol-water solution containing β-diketone, tri-n-octylphosphine oxide and Triton X-100, wherein the concentration of β-diketone is 0.1-0.3 mol / L, and the concentration of tri-n-octylphosphine oxide is 0.05-0.15 mol / L; S3: adding the microcystin standard solution with different concentration gradients to the sample pad of the test paper strip respectively, and adding the fluorescence enhancer, reacting at room temperature for a period of time, and using a time-resolved fluorescence detector to measure the fluorescence intensity ratio of the corresponding detection line and quality control line; taking the standard solution with different concentrations as the abscissa and the fluorescence intensity ratio of the corresponding detection line and quality control line as the ordinate, a standard curve is drawn, and a regression equation is obtained; S4: obtaining the fluorescence intensity ratio of the detection line and the quality control line corresponding to the sample after pretreatment of the sample by the process of step S3, and then substituting the fluorescence intensity ratio into the regression equation to calculate the concentration of microcystin in the sample.

9. The method according to claim 8, wherein the microcystin detection method is a time-resolved fluoroquantitation method. The sample is a water sample or an algal sample, and the pretreatment process of the corresponding water sample is: taking the water sample into a centrifuge tube, centrifuging, and taking the supernatant; the pretreatment process of the corresponding algal sample is: taking the algal sample and adding PBS buffer solution, homogenizing and centrifuging, and taking the supernatant; the supernatant is filtered through a filter membrane to obtain the sample solution.

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