Selection,Characterization And Application Of Aptamer Targeting Nodularin-R

Nodularin-R(NOD-R)is a kind of hepatotoxin isolated mainly from the filamentous,planktonic cyanobacterium,Nodularia spumigena.Recent researches indicate that NOD-R is a carcinogen,as well as a cancer promoter.NOD-R is water soluble,heat stable and can anti-pH changes.As a result,they cannot be completely eliminated via routine water treatment processes like precipitation,filtration and activated carbon adsorption.The major routes with regard to human exposure of NOD-R could be accidental swallowing of water during recreational activities and seafood consumption.The essential symptoms of NOD-R poisoning are gastroenteritis,fatigue,anorexia and so on.What's worse,it could lead to deaths.The toxicity of NOD-R is related to the inhibition of the catalytic subunits of serine/threonine-specific protein phosphatases(PPs)1 and 2A(PP1 and PP2A).Recently,the top three routin detection methods of NOD-R are bioanalysis,physical chemistry processes and immunochemistry methods.However,these techniques have more or less disadvantages,such as low specificity,high cost and time consuming.Therefore,there is still an urgent need to develop a novel mean to replace the traditional approaches.Aptamers are arising molecular recognition elements,and are renowned as chemical antibodies.Accordingly,they are combined with biosensors to develop alternative detection methods.There have been many reports of aptasensors applied in drug detection,food safety and diagnosis.Using magnetic beads selection(MB-SELEX),we acquired aptamer N13which can bind specifically to NOD-R with the highest affinity(K_D=138 nM).According to the secondary structure predicted by the mfold web server,we further truncated aptamer N13and obtained the core sequence N13-T-O2.After optimization,the capability of aptamer N13-T-O2 binding to NOD-R exhibited higher affinity(K_D=29.6 nM).Biolayer interferometry(BLI)technology is lable-free,and provides real-time as well as high-throughput biomolecular interaction information.As a consequence,we applied aptamer N13-T-O2 as a molecular recognition element and constructed a sensitive and specific aptasensor based on biolayer interferometry technology platform.The BLI aptasensor we propose displayed a linear range response from 40 to 200 nM NOD-R.Detection limit of the BLI aptasensor was as low as 167 pM NOD-R.In addition,the BLI aptasensor was shown to possess high selectivity.In other words,MC-LR(the analog of NOD-R),GTX,STX,BTX and PTX exhibited no interaction with the BLI aptasensor.Furthermore,the coefficient of variation(CV)was 8.23%,which indicates that the aptasensor possessed good reproducibility.To evaluate the usefulness of the newly developed BLI aptasensor with a real sample,tap water with different concentrations of NOD-R were studied.Results indicate that a good recovery percentage of 91.92 to 102.16%was obtained.Meanwhile,the CV value was acceptable(1.31~3.73%).In conclusion,we harvested aptamer N13-T-O2 which can bind specifically to NOD-R with the highest affinity.And we developed a BLI aptasensor based on aptamer N13-T-O2.The aptasensor exhibited merits like high specificity,good sensitivity and acceptable reproducibility.Consequently,we are confident that this novel BLI aptasensor provides a potential prototype for the accurate and rapid detection of NOD-R,and may serve for the updation of traditional NOD-R detection methods.