Screening, Identification And Application Of Arsenic Aptamers In The Food

Arsenic(As) is a toxic heavy metal element of savings, which is common in nature. simple Substance itself is non-toxic, while arsenic compound are toxic, wherein the presence of four valences are toxic to As3+, is about 60 times that of other valence of As. Recent years have seen a risingly high frequency of events of food contamination, having posed a serious threat on the health and safety of Chinese people. Also, diseases such as cancer, cardiovascular and nervous systems, has become an important issue of food security. Aptamers(evolution of ligands by exponential enrichment system) screened by SELEX technology can recognize single-stranded oligonucleotide sequence specific target molecule, the secondary structure of a stable, high affinity, high specificity, spatial structure diverse, extensive and easy target molecule chemical modification, etc., are called "chimeric antibodies" and "fourth-generation antibody technology". In this study, to be achieved precise detection of arsenic by aptamer technology. Based on graphene oxide(Graphene Oxide, GO) is strong adsorption of single-stranded oligonucleotides(ss DNA), but it is weak adsorption to bind the target ss DNA of principle, formulated GO-SELEX screening program for arsenic. Graphene oxide as a separating medium, from the full length of 100 bp, containing 40 random sequence of initial ss DNA library was screened with high affinity, highly specific of ss DNA binding to arsenic. After the pretreatment of initial library was incubated with arsenic standard solution, then add the graphene oxide, arsenic can not bind ss DNA is adsorbed on graphene oxide, then centrifuged, the pellet is discarded and the supernatant is collected, and the supernatant retained of binding the ss DNA of arsenic, and then recovered by ethanol precipitation, PCR amplified, product was purified, and preparation and separation of single-stranded secondary library by the method of streptavidin-biotin agarose beads in the end. After each round of screening calculates ss DNA recovery, after five rounds of selection calculate recovery stabilized and then carry on a round of negative selection, remove other similar heavy metals,which is capable of binding to ss DNA, this process were carried out in 10 selections. After the tenth selection, the ss DNA is recovered by ethanol precipitation, and then PCR amplification, the products are purified, cloned and sequenced, the final obtains a total of 30 different aptamer sequences.For 30 different candidate aptamers were analyzed and compared, select 6 sequence Ars-3, Ars-12, Ars-16, Ars-17, Ars-22 and Ars-25. Graphene oxide has the properties of quencher fluorophore, based on graphene oxide and aptamers constructes a fluorescence detection of As3+, then identified the affinity and specificity of aptamers. After the FAM-labeled aptamers are denaturated at 95 ℃ 10 min, and refolded at room temperature(25 ℃) 5 min, then GO and aptamer incubate and bind(combined mass ratio of 1: 300). Adding different concentrations of As3+,and then incubate, centrifuge and the supernatant is measured fluorescence intensity, calculate the dissociation constant(Kd) of each sequence by Graph Pad Prism 5.0. The values are 105.7±6.13 nmol/L、126.0±19.05 nmol/L、153.5±53.27 nmol/L、207.7±12.22 nmol/L、52.68±32.17 nmol/L、194.9±21.03 nmol/L, conclusion that different concentrations of As3+ and different structures of aptamer have different binding activity, wherein the aptamers of Ars-22 has the most sensitive binding activity and high specificity with As3+. Finally fluorescence detection of Ars-22 actual tests of sample recovery, successfully detectes As3+ of bean paste with the recovery of 81.7% to 92.2%.