Study On Non-immobilized Screening Of Aptamers Against Four Marine Biotoxins And The Application In Bioassay

In recent years,increasing occurrences of harmful algal blooms in the seas and lakes lead to increasing outbreaks of food poisoning due to the ingestion of aquatic products containing marine toxins,which have become serious threat to human health and aquatic products industry.Therefore,it is essential to detect the marine toxins in the aquatic products quickly and accurately.Antibody preparation in traditional immunoassay is not practical for low-molecular-weight marine toxins with weak immunogenicity.As a novel recognition molecule,aptamer is a single-strand oligonucleotide obtained via an in vitro process.Besides the comparable affinity and specificity with antibody,aptamer has distinct advantages,including wider variety of targets,higher stability,easier modification and more convenient screening and synthesis.Recently,an increasing number of aptamers against various food hazard factors have been screened and applied for detection.And the marine toxins determination with aptamer have just emerged.In this study,aptamers against four major wildspread marine toxins originally produced by algae or microorganisms were selected using improved non-immobilized SELEX.Then,combining with gold nanoparticles colorimetric technique,fluorescent technique,rolling circle amplification technique,nuclease-based signal amplification technique,magnetic separation technique,etc,a series of simple,sensitive,accurate and efficient aptamer based assays for marine toxins were established.These researches provide new ideas and means for enriching and improving the determination and monitoring technologies of marine toxins.Firstly,non-immobilized graphene oxide SELEX?GO-SELEX?was applied to select aptamer against Okdaic acid?OA?.Following the incubation between target and ssDNA library,GO was added to adsorb the unbound ssDNA,and let the target/ssDNA complex free.Centrifugation was carried out to separate the two classes of ssDNA.After thirteen rounds of alternating selection by three modes of GO-SELEX and subsequent cloning,sequencing,sequence analysis,affinity and specificity assay,an optimized sequence OA-27 was selected as OA aptamer with K_d value of 42±4 nM.Further comparing the simulated secondary structure of the complete sequence with that of the random region,it is found that they have the similar stem-loop structure.After analyzing the affinity and specificity of the random region,a truncated aptamer OA27-1 was obtained with K_d value of 40±13 nM.Secondly,based on GO-SELEX,multiple magnetic reduced graphene oxide SELEX?MRGO-SELEX?was established to screen aptamers against Saxitoxin?STX?,Domoic acid?DA?and Tetrodotoxin?TTX?simultaneously.MRGO was first prepared by one-pot synthesis,and then characterized.Due to its property of magnetic separation and adsorbing single strand oligonucleotides via?-?stacking,MRGO can be used to effectively separate target-bound ssDNA from unbound ssDNA and simplify the separation step of aptamer screening process.After initial twelve rounds of selection against mixed targets and subsequent respective four rounds of selection against single target,three obtained ssDNA pools were cloned and sequenced.The binding affinity and specificity of these aptamer candidates were evaluated by a fluorescence assay.Finally,DA-06?K_d=62.07±19.97 nM?,TTX-07?K_d=44.12±15.38 nM?and STX-41?K_d=61.44±23.18 nM?showed high affinity and good specificity to DA,TTX and STX,respectively.The feasibility of these aptamer for marine toxins detection was verified by a fluorescence resonance energy transfer?FRET?assay.In addition,we also found two multi-target aptamers,DA-01 and TTX-27,which exhibited affinity for both DA and TTX.Thirdly,a novel Au nanoparticles?AuNPs?based direct competitive enzyme-linked aptamer assay?dc-ELAA?was fabricated to detect OA.The aptamer OA27-1 obtained by GO-SELEX was immobilized on microplate.The catalase labelled complementary sequence was designed to compete with OA present in sample to bind with the immobilized aptamer.Different concentrations of H₂O₂ resulted from catalase degradation could reduce gold trichloric acid to AuNPs with different aggregation states,leading to different colours for the colorimetric detection of OA.Under the optimized conditions,this ELAA method showed low detection limit?LOD of 0.01ng/m L?,wide linear range?from 0.025 to 10ng/m L?,good recovery rate?92.86-103.34%?and repeatability?RSD of 2.28-4.53%?.The proposed method can be used to detect OA in seafood products with high sensitivity.Fourthly,rolling circle amplification?RCA?combined with fluorescent detection technology were used to design a more sensitive competitive aptamer assay for the determination of OA.The aptamer against OA as a capture probe was first immobilized on microplate.The fluorescent detection probe which consisted of RCA product and FAM labelled signal probe was hybridized with the fixed aptamer.Then,a competitive assay was performed to detect OA based on the competition between the fluorescent detection probe and OA for the immobilized capture probe.Owing to the signal amplification effect of RCA,the fluorescence intensity was positively proportional to the logarithm of OA concentration ranging from 1pg/m L to100 ng/m L.And the detection limitation?LOD?was estimated at 1 pg/mL.Detecting clam samples by the proposed method showed good recovery rates?97.12-108.93%?and good agreement with the commercial ELISA kit.Fifthly,a high sensitive FRET aptasensor assisted by nuclease-based target recycling signal amplification for STX detection was developed.The aptamer STX-41 conjugated with graphene quantum dots?GQDs?was adsorbed on MRGO to establish a fluorescence quenching system.The binding between STX and aptamer induced desorption of GQDs-Aptamer from MRGO and the restoring of fluorescence for the fluorescent determination of STX.The digestion of the target bound aptamer by DNase I could release target for recycling to achieve signal amplification.Under the optimized conditions,there was a strong linear correlation between fluorescence intensity and the logarithm of STX concentration in the range of 0.1ng/m L to 100 ng/mL,with the detection limitation of 0.1 ng/mL,indicating that the FRET aptasensor can be applied to detect STX with high sensitivityIn conclusion,the non-immobilized GO-SELEX methods suitable for selecting aptamer against small molecule targets have been improved.By these developed SELEX methods,the aptamers targeted to OA,STX,DA or TTX with high affinity and specificity have been screened.Combined with optical detection technology,signal amplification technology and magnetic sepatation technology,these aptamers have been used to detect marine toxins successfully,which provide technical supports to develop new technologies and relative products for the determination of marine toxins.