Label-and Modification-free-based In Situ Selection Of Bovine Serum Albumin Aptamer And The Establishment Of Biosensor

Aptamers are one kind of short single-stranded nucleic acid sequences that can bind to target molecules like antibody.Because of many advantages of long shelf life,low batch to batch variation,low immunity/non-immunity,chemical modification easily to enhance affinity and specificity,they have huge potential in many applications such as bioanalysis,food detection,drug delivery,biosensor and functional genomics.Aptamers were generated by SELEX technology,a systematic evolution of ligands by exponential enrichment in vitro.During the past years,scientists have proposed many novel SELEX strategies based on traditional SELEX technology.Despite these improvements,these strategies still have some intrinsic restrictions,such as long experimental period,complex operation of chemical modification,and the change of molecular space structure due to chemical modification.The chemical modification of ssDNA libraries or targets before selection may disturb their recognition and binding with their targets,affecting their binding process further.Therefore,in order to overcome the limitations,this study established a label-and modification-free-based in situ selection strategy through agarose gel electrophoresis to select potential specific ssDNA aptamers of bovine serum albumin(BSA),a typical pattern protein,from the random oligonucleotide library.Then,a fluorescent aptasensor was built to detect BSA in actual sample.(1)Establish a label-and modification-free-based in situ selection strategy.First,BSA was mixed with agarose gel,and then the BSA specific aptamers(No.1,No.2,No.3,No.4,No.5,No.6,No.7,No.8)were obtained after 8 rounds selection based on the principle of electrophoretic mobility change analysis.Subsequently,the traditional SELEX technology was built to obtain the aptamers S1,S2,S3 and S4 of BSA.These aptamers have high affinity to BSA and low homology with each other.Among them,aptamer No.4 showed the highest affinity,whose Kd value is 69.44±7.6nM.The affinity of aptamer S1 is next,whose Kd value is 87.51±17.89nM.By comparison,the label-and modification-free-based in situ selection strategy proposed in this paper can effectively screen specific aptamers of target,and its affinity is higher.(2)we constructed a fluorescent aptasensor to detect BSA using aptamer No.4.After optimization,the length of complementary probe was 13 bp,the optimal addition of aptamer was 3?M,the optimal combination time of dsDNA and magnetic beads was 80 min,and the optimal testing reaction time of BSA was 30 min.The linear detection range of the fluorescent aptasensor is 1-120 ng/mL with a limit of detection(LOD)of 0.24 ng/mL.In addition,the recovery rate of BSA in pear samples was between 96%and 99%.