Screening Of Aptamers And Establishment Of A Detection Method For Aflatoxin B₁

Aflatoxin B₁?Aflatoxin B₁,AFB₁?is a highly toxic mycotoxin with the basic structure of difuran ring and oxaphthalenedione.It has been classified as a carcinogen by WHO cancer research institute.AFB₁ can cause many diseases,such as liver cancer,and easily contaminate peanut,corn,wheat and other agricultural products and foods.The development and application of a fast and sensitive AFB₁ detection method is the basis of preventing food safety incidents.Nucleic acid aptamer is a single-stranded oligonucleotide sequence that can bind to target molecules with high affinity and specificity.It is known as"chemical antibody"since it has stable secondary structure and special three-dimensional conformation.Aptamers can be screened and obtained from in vitro using the systematic evolution of ligands by exponential enrichment?SELEX?.A colorimetric assay method for detection of AFB₁,which will provide technical support for food safety,has been established in this experiment,using the obtained AFB1 aptamer as the specific recognition probe and gold nanoparticles as the indicator.Screening of the specific aptamers for AFB₁ with graphene oxide-SELEX.Based on the fact that graphene oxide can adsorb single-stranded oligonucleotide ssDNA by?-?conjugation and has weak adsorptive ability to ssDNA binding the target,a screening scheme for AFB₁ aptamer was designed.Firstly,a random ssDNA library with a full length of 80bp containig each of fixed sequence at both ends and a 40bp random sequence in the middle was designed and sythesized.The pretreated random libraries were incubated with AFB₁ and graphene oxide solution nextly to adsorb the ssDNA without binding the target.After the AFB₁-ssDNA complex formed,it was separated and retained in the supernatant by centrifugation and severed as as template for PCR.After PCR amplification,the products were recovered and purified.The candidate aptamers were obtained for each round.After 10 rounds of selection,inlucding the sixth and eighth rounds for counter selection with other mycotoxins,45 ssDNA sequences were obtained,cloned and sequenced from the eighth and final rounds of screening products.Six representative sequences were selected for affinity analysis.The dissociation constants?Kd?were 16.29,27.88,18.54,23.11,47.94 and 22.31 nmol/L,respectively.Affinity of AFB-5 and AFB-8was selected for specificity analysis.The results showed that the selected aptamers could specifically adsorb AFB1 with high specificity.A visual detection method of AFB₁ was established based on the discoloration effect of gold nanoparticles interacting with the nucleic acid aptamer.Monodispersed gold nanoparticles were wine red in solution.The gold nanoparticles and the aptamers were combined with each other under the electrostatic forces to protect the gold nanoparticles against salt-induced agglomeration and discoloration.When AFB₁ is added to the solution,AFB₁ specifically bound to the aptamers and replaced the gold nanoparticles which bound to the original aptamers under the competitive action.Therefore,the gold nanopaticles were separated from the aptamers and agglomerated under the action of NaCl.The detection limit of this method was 0.025 ng/mL and the AFB₁ concentration had a good linear relationship with the OD value at 520 nm over range of 0.025¹0 ng/mL.The regression equation was y=-0.00709x+0.46142?R²=0.9979?.The specificity test and the standard addition recovery of rice samples,which the recovery rate of standard addition was 83%¹05%,proved the feasibility of this method.