Preparation of lateral flow chromatography test strip for colorimetric detection of protopolycoic acid-1 in shellfish based on litchi-shaped molybdenum disulfide and gold composite nanomaterial and split aptamer

Abstract

The invention discloses a preparation method of a lateral flow chromatography test strip for colorimetric detection of protodococeric acid-1 in shellfish based on a litchi-shaped molybdenum disulfide-gold composite nano material and a split aptamer, which comprises the following steps: (1) preparing the litchi-shaped molybdenum disulfide-gold composite nano material by a hydrothermal synthesis method; (2) analyzing the structure of the aptamer and splitting the aptamer; (3) preparing a signal probe; and (4) preparing and assembling the test strip. Molybdenum disulfide has a large specific surface area and can be connected with a large number of gold nanoparticles through S-S bonds, so that the sensitivity of the test strip is improved; the existence of irrelevant basic groups in the aptamer can reduce the affinity and the specificity of the aptamer, so that a false positive result is caused. The split aptamer can effectively avoid the situation, the binding stability and specificity can be improved through the split aptamer, and the reliability of a test result is improved. Compared with the traditional colloidal gold lateral flow chromatography test strip, the colloidal gold lateral flow chromatography test strip has the advantages of wide detection range, good stability, high sensitivity and the like. The method can realize rapid qualitative and quantitative detection of an actual sample, is simple and convenient in operation steps and strong in controllability, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the preparation of a lateral flow chromatography test strip for detecting protodinodinium-1 in molluskine based on litchi-shaped molybdenum disulfide@gold composite nanomaterials and splitting aptamers, and particularly to a method for preparing splitting aptamer probes. Background Technology

[0002] Azaspiracids (AZAs) are a class of lipophilic polyether marine toxins produced by *Prozaspira*, belonging to the AZA family. They were first discovered in 1995 when at least eight people in the Netherlands fell ill after consuming *Mytilus edulis* mussels from Killary Harbour, Ireland. They can accumulate in filter-feeding shellfish, and consuming contaminated shellfish can lead to severe acute gastrointestinal poisoning, characterized by dizziness, nausea, vomiting, diarrhea, and abdominal cramps. Furthermore, AZAs are relatively stable in toxicity, and their toxicity cannot be reduced under acidic, alkaline, or high-temperature conditions. Therefore, establishing effective methods for detecting AZAs is of great significance for improving the safety of aquatic products and protecting human health. In 2004, the Food and Agriculture Organization of the United Nations, the World Health Organization, and the Intergovernmental Oceanographic Commission jointly established the Working Group on Bivalve Mollusc Toxins serving the Codex Alimentarius Commission. AZAs toxins were classified as one of the eight categories of shellfish biotoxins. The European Union stipulates that the maximum permissible content of AZA1, AZA2, and AZA3 in bivalve seafood is 160 μg / kg to protect consumers.

[0003] Currently, the main methods for detecting AZAs (azoospiky toxins) in shellfish include high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS / MS), and gas chromatography (GC). These methods offer high sensitivity and accurate results; however, they require large-scale equipment, are expensive, have long testing cycles, and require specialized operators, making them unsuitable for rapid on-site detection and significantly limiting research on AZAs toxins. Lateral chromatography test strips have attracted widespread attention from researchers due to their simple operation, low cost, and ability to enable rapid on-site detection. Among them, aptamer-based lateral flow chromatography test strips offer advantages such as good stability and ease of synthesis and modification. However, not all bases in the aptamer participate in the target binding process; the presence of irrelevant bases in the aptamer may reduce its affinity and specificity, leading to false positive results. Splitting aptamers can effectively avoid this situation and increase the reliability of test results. Furthermore, lychee-shaped molybdenum disulfide with a large specific surface area can connect more gold nanoparticles, thereby improving the detection sensitivity of the test strip. Therefore, designing and synthesizing a splitting aptamer probe is the key to this side-flow chromatography test strip. Currently, there are no reports on the design and synthesis of a side-flow chromatography test strip for detecting protodinodinium-1 in mollusks using litchi-shaped molybdenum disulfide@gold composite nanomaterials and splitting aptamers. Summary of the Invention

[0004] This invention relates to the preparation of a lateral flow chromatography test strip for detecting protodinodinium-1 in mollusks based on litchi-shaped molybdenum disulfide@gold composite nanomaterials and splitting aptamers.

[0005] Preparation of the litchi-shaped molybdenum disulfide@gold composite nanomaterial: Litchi-shaped molybdenum disulfide was prepared by hydrothermal synthesis. First, 0.5-3 g of molybdenum source and 2-10 g of sulfur source were weighed and dissolved in 60-70 mL of ultrapure water. The mixture was then magnetically stirred at 800-1000 rpm for 30-40 min. The mixture was then poured into a high-pressure reactor lined with 100 mL of Teflon and reacted at 200-220 °C for 24-28 h. After natural cooling, the black precipitate was centrifuged at 12000-14000 rpm for 20-30 min in a high-speed refrigerated centrifuge and washed repeatedly with large amounts of anhydrous ethanol and ultrapure water 3-5 times. Finally, the sample was dried in a drying oven at 60-80 °C for 6-8 h to obtain litchi-shaped molybdenum disulfide solid powder. The synthesized solid powder was prepared into a 0.1-0.5 mol / L solution. Then, 500-800 μL of the litchi-like molybdenum disulfide solution was added to 90-100 mL of ultrapure water containing 300-500 μL of chloroauric acid (1%-1.5%) solution, and the mixture was heated for 25-50 min to obtain the litchi-like molybdenum disulfide@gold nanocomposite material.

[0006] Preparation of the splitting aptamers: Molecular docking was performed on the Autodock Vina cloud computing platform (https: / / vina.scripps.edu / ). First, the three-dimensional structures of AZAs toxins were retrieved from the PubChem database, and their energies were optimized. Second, the secondary structures of the AZAs toxin splitting aptamers were predicted using the Mfold online software (http: / / www.unafold.org / mfold / applications / dna-folding-form.php), identifying splitting sites without key binding sites. Then, the three-dimensional structures were obtained from the 3dRNA online prediction website (http: / / biophy.hust.edu.cn / 3dRNA), and energy was optimized using molecular dynamics. Stable structures were obtained by docking the two separated aptamer probes using the HDOCK server (http: / / hdock.phys.hust.edu.cn / ). The obtained structures were further processed to detect the binding activity of the recombinant aptamers to the target. Splitting aptamer 1 was modified with a thiol group, and splitting aptamer 2 was modified with biotin.

[0007] Preparation of the litchi-shaped molybdenum disulfide@gold-splitting aptamer 1 probe: First, 5-10 μL of activated 10-15 μM thiol-modified splitting aptamer 1 was added to 500-800 μL of the previously prepared litchi-shaped molybdenum disulfide@gold solution and mixed thoroughly. The mixture was allowed to stand in the dark at 0-4℃ for 24-28 h to allow Au-S bond linkage. Then, 80-120 μL of 10%-20% BSA solution was added and reacted for 1-2 h to block unbound sites on the colloidal gold surface. Next, the mixture was centrifuged at 10000-12000 rpm for 20-30 min in a high-speed refrigerated centrifuge at 0-4℃ to remove excess unreacted aptamer 1 supernatant. Finally, 50-60 μL of reconstitution solution was added and mixed thoroughly.

[0008] The lateral flow chromatography test strip is used for the detection of proto-alginate-1. First, the lateral flow chromatography test strip is prepared by pretreating the sample pad with buffer solution and drying it overnight at 60–80 °C. Then, the sample pad, nitrocellulose membrane, backing plate, and absorbent pad are cut into strips 30–40 cm long and 1–6 cm wide. 8–10 μM biotinylated apposition aptamer 2 is spread onto the nitrocellulose membrane at a rate of 0.6–1.2 μL / cm to form the detection zone. 10–15 μM complementary DNA 2 is spread onto the nitrocellulose membrane at a rate of 0.5–1.5 μL / cm to form the quality control zone, with a distance of 3–8 mm between the detection and quality control zones. Then, the sample to be tested is mixed with the litchi-shaped molybdenum disulfide@gold modified apposition aptamer 2 and incubated for 5–20 min before being dropped onto the sample pad. When both the T and C lines show color, the result is positive; when the T line does not show color but the C line does, the result is negative.

[0009] The molybdenum source is one or more of ammonium molybdate primary and secondary, ammonium molybdate tetrahydrate, and sodium molybdate dihydrate. The sulfur source is one or more of thiourea, thioacetamide, and sodium sulfide. The sequence of the thiol-modified cleavage aptamer 1 is: 5'-AATTGTGTGTTCGGTACCTATGCGTGCTACCGTGAA-SH-3' The biotinylated cleavage aptamer 2 sequence is: 5'-AGCAGCACAGAGGTCAGATGCAGCTCAAGTTCA-bio-3' The reconstitution solution is 0.01 mol / L PBS containing 5% sucrose, 5% PEG 20000, 5% Tween-20, 1% BSA, and 1% MgCl2.

[0010] The buffer solution is 0.01 mol / L PBS containing 2% sucrose, 0.5% Tween-20, and 1% BSA.

[0011] The complementary DNA2 sequence is: 5'-GGGGG-bio-3' The lateral flow chromatography test strip of this invention uses litchi-shaped molybdenum disulfide@gold and splitting aptamer 2 as probes. The prepared splitting aptamer effectively reduces false positives. Furthermore, compared with other test strips based on colloidal gold or similar materials as colorimetric probes, the prepared lateral flow chromatography test strip has advantages such as signal amplification, wide detection range, high sensitivity, fast response speed, good repeatability, and high accuracy. Attached Figure Description

[0012] Figure 1 The images show the transmission electron microscopy (TEM) characterization of the prepared colloidal gold and the electron microscopy (SEM) characterization of the litchi-shaped molybdenum disulfide composite nanomaterial. Figure 2 Eds mapping characterization diagram of the prepared litchi-shaped molybdenum disulfide composite nanomaterial; Figure 3 Zeta characterization diagrams before and after ligation of the litchi-shaped molybdenum disulfide@gold-splitting aptamer probe; Figure 4 A schematic diagram of the secondary structure of the polymethylalginate-1 toxin fission aptamer; Figure 5 A schematic diagram illustrating the principle of the test strip for detecting polymethylalginate-1 toxin. Detailed Implementation

[0013] The present invention will now be described in conjunction with specific embodiments: Example

[0014] The specific steps are as follows: (1) Hydrothermal synthesis of lychee-shaped molybdenum disulfide material: First, 0.53 g of ammonium molybdate primary and secondary molybdate and 2.1 g of thiourea were weighed and dissolved in 60 mL of ultrapure water. Then, the mixture was magnetically stirred at 1000 rpm for 30 min, and then poured into a high-pressure reactor lined with 100 mL of Teflon. The mixture was heated to 220 °C and reacted for 24 h. After natural cooling, the black precipitate was centrifuged at 12000 rpm for 20 min in a high-speed refrigerated centrifuge and washed three times with large amounts of water and ethanol, respectively. Then, it was dried in a vacuum drying oven at 60 °C for 6 h to obtain lychee-shaped MoS2 solid powder. The synthesized MoS2 powder was prepared into a 0.1 mol / L solution. 500 μL of the 0.1 mol / L MoS2 solution was added to 90 mL of ultrapure water containing 300 μL of chloroauric acid (1%) solution and heated for 30 min to obtain MoS2@Au composite nanomaterials.

[0015] (2) Preparation of litchi-shaped molybdenum disulfide@gold-splitting aptamer 1 probe: 5 μL of activated 10 μM thiol-modified splitting aptamer 2 was added to a centrifuge tube containing 500 μL of the previously prepared MoS2@Au composite material solution. After mixing, the tube was incubated at 4℃ in the dark for 24 h. The Au-S bond binds to the gold nanoparticles on the MoS2@Au composite material, thereby attaching the splitting aptamer 2 to the surface of the nanocomposite material. Then, 100 μL of 10% BSA solution was added and reacted for 60 min to seal the unbound sites on the colloidal gold surface. The mixture was then centrifuged at 10000 rpm for 20 min at 4℃ to remove excess unreacted aptamer 2 supernatant. Finally, 50 μL of a complex solution containing 5% sucrose, PEG20000, Tween-20, 1% BSA, and MgCl2 was added and mixed.

[0016] (3) Lateral flow chromatography test strips for the detection of proto-alginoic acid-1 in shellfish: First, the lateral flow chromatography test strips were prepared. The sample pad was pretreated with 0.01 mol / L PBS buffer containing 2% sucrose, 0.5% Tween-20, and 1% BSA, and dried overnight at 60 °C. Then, the sample pad, nitrocellulose membrane, backing plate, and absorbent pad were cut into strips 30 cm long and 2 cm wide. 10 μM biotinylated apposition aptamer 1 (sequence: 5'-AATTGTGTGTTCGGTACCTATGCGTGCTACCGTGAA-SH-3') was spread on the nitrocellulose membrane at a rate of 0.8 μL / cm to form the detection zone. 10 μM complementary DNA2 (sequence: 5'-GGGGG-bio-3') was spread on the nitrocellulose membrane at a rate of 0.8 μL / cm to form the quality control zone, wherein the distance between the detection zone and the quality control zone was 3 mm. Then, the sample to be tested was mixed with litchi-shaped molybdenum disulfide@gold modified splitting aptamer 2 (sequence: 5'-AGCAGCACAGAGGTCAGATGCAGCTCAAGTTCA-bio-3') and incubated for 20 min before being dropped onto the sample pad. When both T and C lines showed color, the result was positive; when T did not show color but C line showed color, the result was negative. Example

[0017] The specific steps are as follows: Hydrothermal synthesis of lychee-shaped molybdenum disulfide material: First, 3g of ammonium molybdate tetrahydrate and 5g of thioacetamide were dissolved in 70mL of ultrapure water. The mixture was then magnetically stirred at 1000 rpm for 40 min, and poured into a high-pressure reactor lined with 100 mL of Teflon. The reaction was carried out at 210℃ for 26 h. After natural cooling, the black precipitate was centrifuged at 12000 rpm for 30 min in a high-speed refrigerated centrifuge, and washed three times successively with large amounts of water and ethanol. The precipitate was then dried in a vacuum drying oven at 70℃ for 8 h to obtain lychee-shaped MoS2 solid powder. The synthesized MoS2 powder was prepared into a 0.2 mol / L solution. 600 μL of the 0.1 mol / L MoS2 solution was added to 100 mL of ultrapure water containing 350 μL of chloroauric acid (1%) solution, and the mixture was heated continuously for 40 min to obtain MoS2@Au composite nanomaterials.

[0018] Preparation of the litchi-shaped molybdenum disulfide@gold-splitting aptamer 1 probe: 10 μL of activated 12 μM thiol-modified splitting aptamer 2 was added to a centrifuge tube containing 800 μL of the previously prepared MoS2@Au composite material solution. After mixing, the mixture was incubated at 0℃ in the dark for 26 h. The Au-S bond binds to the gold nanoparticles on the MoS2@Au composite material, thus attaching the splitting aptamer 2 to the surface of the nanocomposite material. Then, 120 μL of 15% BSA solution was added and reacted for 1.5 h to seal the unbound sites on the colloidal gold surface. The mixture was then centrifuged at 12000 rpm for 30 min at 4℃ to remove excess unreacted aptamer 2 supernatant. Finally, 60 μL of a complex solution containing 5% sucrose, PEG20000, Tween-20, 1% BSA, and MgCl2 was added and mixed thoroughly.

[0019] (3) Side-flow chromatography test strips for the detection of proto-alginoic acid-1 in shellfish: First, the side-flow chromatography test strips were prepared. The sample pad was pretreated with 0.01 mol / L PBS buffer containing 2% sucrose, 0.5% Tween-20, and 1% BSA, and dried overnight at 80 °C. Then, the sample pad, nitrocellulose membrane, backing plate, and absorbent pad were cut into strips 32 cm long and 1.75 cm wide. 8 μM biotinylated apposition aptamer 1 (sequence: 5'-AATTGTGTGTTCGGTACCTATGCGTGCTACCGTGAA-SH-3') was spread on the nitrocellulose membrane at a rate of 0.8 μL / cm to form the detection zone. 12 μM complementary DNA2 (sequence: 5'-GGGGG-bio-3') was spread onto a nitrocellulose membrane at a rate of 0.8 μL / cm to form a quality control zone, with a distance of 5 mm between the test zone and the quality control zone. Then, the sample to be tested was mixed with litchi-shaped molybdenum disulfide@gold modified aptamer 2 (sequence: 5'-AGCAGCACAGAGGTCAGATGCAGCTCAAGTTCA-bio-3') and incubated for 15 min before being dropped onto the sample pad. A positive result was indicated when both the T and C lines showed color; a negative result was indicated when no color was observed on the T line but the C line showed color.

[0020] The prepared side-flow chromatography test strip exhibits a wide detection range, high sensitivity, fast response, good repeatability, and high accuracy in the detection of proto-alginate-1. Furthermore, the test results on actual samples demonstrate that the prepared side-flow chromatography test strip has significant practical application value.

[0021] The above embodiments are merely illustrative of the present invention and not intended to limit it. Many improvements and modifications can be made to the present invention based on the above description. Within the scope of the appended claims, the present invention can have other implementations different from those described above; the selection of other reagents and materials, adjustment of dispersion time, etc., are all within the scope of the claims of this invention.

Claims

1. A side-flow chromatography test strip for the colorimetric detection of proto-polydinocyanic acid-1 in mollusks based on litchi-shaped molybdenum disulfide@gold composite nanomaterials and splitting aptamers, characterized in that, Includes the following steps: (1) Preparation of lychee-like @gold composite nanomaterials: Lychee-like composite nanomaterials were prepared by hydrothermal synthesis. First, 0.5-3g of molybdenum source and 2-10g of sulfur source were weighed and dissolved in 60-70mL of ultrapure water. Then, the mixture was magnetically stirred at 800-1000 rpm for 30-40min. The mixture was then poured into a high-pressure reactor lined with 100mL of Teflon and reacted at 200-220℃ for 24-28h. After natural cooling, the black precipitate was centrifuged at 12000-14000rpm for 20-30min in a high-speed refrigerated centrifuge and washed repeatedly with a large amount of anhydrous ethanol and ultrapure water 3-5 times. Finally, the sample was dried in a drying oven at 60-80℃ for 6-8h to obtain a black solid powder, namely lychee-like molybdenum disulfide composite nanomaterials. The synthesized solid powder was prepared into a 0.1-0.5 mol / L solution. Then, 500-800 μL of the solution was added to 90-100 mL of ultrapure water containing 300-500 μL of chloroauric acid (1%-1.5%) solution, and the mixture was heated for 25-50 min to obtain litchi-shaped molybdenum disulfide@gold composite nanomaterials. (2) Preparation of splitting aptamers: Molecular docking was performed on the Autodock Vina cloud computing platform (https: / / vina.scripps.edu / ). First, the three-dimensional structures of AZAs toxins were retrieved from the PubChem database, and their energies were optimized. Second, the secondary structures of AZAs toxin splitting aptamers were predicted using the Mfold online software (http: / / www.unafold.org / mfold / applications / dna-folding-form.php), and splitting sites without key binding sites were identified. Then, the three-dimensional structures were obtained from the 3dRNA online prediction website (http: / / biophy.hust.edu.cn / 3dRNA), and the energies were optimized using molecular dynamics. The two separated aptamer probes were docked using the HDOCK server (http: / / hdock.phys.hust.edu.cn / ) to obtain stable structures. The obtained structures were further processed to detect the binding activity of the recombinant aptamers to the target. The obtained splitting aptamer 1 (sApt-1) was modified with thiol, and the splitting aptamer 2 (sApt-2) was modified with biotin. (3) Preparation of litchi-shaped molybdenum disulfide@gold-splitting aptamer 1 probe: First, take 5~10μL of activated 10~15μM thiol-modified splitting aptamer 1 (sApt-1) and add it to 500~800μL of the previously prepared litchi-shaped molybdenum disulfide@gold solution and mix well. Let it stand in the dark at 0~4℃ for 24~28h to connect Au-S bonds. Then add 80~120μL of 10%~20% BSA solution and react for 1~2h to block the unbound sites on the colloidal gold surface. Then place the mixture in a high-speed refrigerated centrifuge at 0~4℃ and centrifuge at 10000~12000rpm for 20~30min to remove the supernatant of excess unreacted aptamer 1. Finally, add 50~60μL of reconstitution solution and mix well. (4) Lateral flow chromatography test strips for the detection of proto-alginate-1: First, the lateral flow chromatography test strips were prepared. The sample pad was pretreated with buffer and dried overnight at 60-80 °C. Then, the sample pad, nitrocellulose membrane, backing plate, and absorbent pad were cut into strips 30-40 cm long and 1-6 cm wide. 8-10 μM biotinylated apposition aptamer 2 was spread onto the nitrocellulose membrane at a rate of 0.6-1.2 μL / cm to form the detection zone. 10-15 μM complementary DNA 2 was spread onto the nitrocellulose membrane at a rate of 0.5-1.5 μL / cm to form the quality control zone, with a distance of 3-8 mm between the detection zone and the quality control zone. Then, the sample to be tested was mixed with litchi-shaped @gold modified apposition aptamer 1 and incubated for 5-20 min before being dropped onto the sample pad. When both the T and C lines show color, the result is positive; when the T line does not show color but the C line does, the result is negative.

2. The preparation of a side-flow chromatography test strip for the colorimetric detection of proto-polydinocyanic acid-1 in mollusks based on litchi-like@gold composite nanomaterials and splitting aptamers according to claim 1, characterized in that, The thiol-modified splitting aptamer 1 (sApt-1) sequence in step (2) is: 5'-AATTGTGTGTTCGGTACCTATGCGTGCTACCGTGAA-SH-3', and the biotinylated splitting aptamer 2 (sApt-2) sequence is: 5'-AGCAGCACAGAGGTCAGATGCAGCTCAAGTTCA-bio-3'.

3. The preparation of a side-flow chromatography test strip for the colorimetric detection of proto-polydinocyanic acid-1 in mollusks based on litchi-like@gold composite nanomaterials and splitting aptamers, as described in claim 1, is characterized in that... The reconstitution solution mentioned in step (3) is 0.01 mol / L PBS containing 5% sucrose, 5% PEG 20000, 5% Tween-20, 1% BSA, and 1% MgCl2.

4. The preparation of a side-flow chromatography test strip for the colorimetric detection of proto-polydinocyanic acid-1 in mollusks based on litchi-like@gold composite nanomaterials and splitting aptamers according to claim 1, characterized in that, The buffer solution mentioned in step (4) is 0.01 mol / L PBS containing 2% sucrose, 0.5% Tween-20, and 1% BSA; the complementary DNA2 sequence is: 5'-GGGGG-bio-3'.

5. The test strip prepared by the method according to any one of claims 1-4.

6. The application of the test strip according to claim 5 in the detection of polyalginic acid-1.

Images