Rapid Detection Of Microcystins In Aquatic Products Using The Aptamer-Based Nanoprobes

Microcystins（MCs）are a class of monocyclic heptaeptide cyanobacteria toxins commonly find in eutrophic water environment,in which Microcystin-LR（MC-LR）is the most toxic and widespread variant of MCs.MC-LR has stable chemical properties,it is resistant to high temperature,strong acid and alkali,so it cannot be removed by conventional treatments.MC-LR has hepatotoxicity as it can cause acute liver damage and is closely related to the occurrence of hemorrhage and primary cancer in liver.MC-LR can accumulated through the food chains and ultimately has a detrimental effect on humans.Therefore,it is particularly important to develop a simple,efficient and sensitive detection method for MC-LR.In this study,a novel colorimetric aptasensor based on aptamer and nanomaterials was developed to realize the convenient,rapid and sensitive detection of MC-LR.Meanwhile,to obtain higher binding affinity between MC-LR and its aptamer,the sequences of aptamer were optimized by a series of truncation and site-directed mutation.And the binding sites between MC-LR and aptamer were predicted by molecular docking.Finally,a lateral flow strip assay via competition model based on aptamer and AuNPs was developed for naked-eyes detection of MC-LR.The mainly results were as follows:Based on the reported aptamer specific binding to MC-LR（Apt-1）,a new Apt-3was designed and synthesized by the site-directed mutation,which has a completely different secondary structure from Apt-1.The AuNPs colorimetric detection system was constructed using both Apt-1 and Apt-3,and the concentration of each component in the reaction mixture was optimized.Results indicated that the visual detection limit（LOD）of MC-LR was 70 ng/m L using the（Apt-1）-AuNPs system,whereas this value was 30ng/m L by the（Apt-3）-AuNPs system.When the concentration of Na Cl was 0.05 mol/L and Apt-3 was 0.06μmol/L,a good linear calibration relationship was obtained within the range of 1-120 ng/m L.The linear regression equation is y=0.0066x+0.0595（R²=0.995）,and the theoretical LOD was calculated to be 5.88 ng/m L.These data indicated that the aptamer-based gold nanoparticles colorimetry can realize the visual detection of MC-LR in a rapid and simple means.Compared with the Apt-1,Apt-3 has higher binding specificity and affinity to MC-LR,which is more suitable for the subsequent development of aptasensors.Based on mutation and reasonable truncation of the original aptamer（Apt-1）,a series of aptamers with segmented structure and sequence were obtained,and their binding affinity to MC-LR was compared with each other using the AuNPs colorimetric detection system.Through study the structure characteristics and quantization performance of the original Apt-1 and the truncated aptamers,we found that 20 mer Apt-2C region was the main binding domain for MC-LR.By molecular docking,the hydrogen bond interaction was observed between the aptamer sequence and the carbonyl group of Adda residue or the guanidyl group of Arg residue of MC-LR.In addition,D-me-Asp and D-Glu of MC-LR also contribute to its binding to aptamers.Among the aptamers,the 20-mer Apt-2C presented the highest binding affinity and specificity to MC-LR.Under the optimum conditions,the Apt-2C colorimetric aptasensor worked well with MC-LR concentrations ranged from 10 to 100ng/m L,and it had a good linear calibration relationship（R²=0.9908）.The theoretical LOD and visual LOD was 0.55 ng/m L and 10 ng/m L respectively.The colorimetric sensor based on aptamer Apt-2C was used to detect the MC-LR in the pool water after adding standard solution.The recovery was 9.42%-107.5%in the range of 10-100 ng/m L,and the standard deviation was 1.2%-5.8%.The results show that the method has high sensitivity and accuracy for the determination of MC-LR in real water samples.Thus,it provided a rapid,reliable and convenient approach for visual detection of MC-LR,which is of great significance for food safety control by assessing potential health risks related to seafood consumption.According to the principle of literal flow strip assay（LFSA）,a colloidal gold strip of test paper was established with competition mode,and it was firstly used for the detection of MC-LR.The thiolated aptamer Apt-2C was conjugated with colloidal gold by sodium chloride aging method to form a conjugated complex as the binding pad,and characterized by Mey’s stability test and UV spectroscopy.The complementary sequence of Apt-2C and DNA _{Ploy A}were used as detection line and quality control line,respectively.The reaction system of the strip was optimized.Under the optimum conditions,the test strip has high specificity,the LOD of LFSA constructed by Apt-2C for MC-LR is 50 ng/m L,and the detection time is less than 15 min.The detection concentration of MC-LR ranges from 50-1000 ng/m L.The method is simple,accurate,and low-cost,and can realize on-site visualization and rapid detection of MC-LR.