Study On Denatured Vesicle-mediated Chain Exchange Amplification Combined With Electric Field-assisted DNA Sensor

Nucleic acids,as carriers of genetic information and the material basis for gene expression,are essential in organisms.Currently,nucleic acid detection has become a research hotspot for scientific researchers,and has been applied to various fields such as medicine,environment,food,and so on.Although nucleic acid amplification methods are sensitive and reliable,the existing nucleic acid detection methods still require large instruments,professional personnel,and operation space.Time-consuming detection has become the most shortcoming of electrochemical sensing technology which is sensitive and simple.Therefore,establishing a simple,rapid,sensitive,and miniaturizable scheme for detecting nucleic acid is the focus of this research.Our research group has independently developed a new approach for amplifying nucleic acid at a constant temperature-denaturing bubble-mediated strand exchange amplification(SEA).Only a pair of primers and a kind of enzyme,Bst DNA polymerase,are needed to achieve rapid detection of DNA or RNA.In order to improve the sensitivity of detection,this study combined SEA with electrochemical sensing technology,and the established method amplified the signal by SEA on the electrode.Then,Streptavidin modified horseradish peroxidase(SA-HRP)was connected with the amplification products on the electrode through the specific recognition of streptavidin and biotin.The reduction current signal would be generated in the presence of 3,3,5,5-Tetramethyl benzidine substrate solution(TMB).With the above operation process,the target could be detected quantitatively.In addition,the amplification mixture includes 1 n M forward primers and the modification of electrodes with gold nanoparticles(Au NPs)could improve the efficiency of amplification and the performance of the sensor.The established sensor method took beef genomic DNA as the target,and then the concentration of the probe and SA-HRP,coupling time,and modification conditions were optimized.The sensitivity was 10~4times higher than SEA and there was a good linear relationship within the concentration ranging from 1 fg/μL to 10 ng/μL,with a minimum detection concentration of 1 fg/μL.At the same time,the study had also established a standard curve between electrical signals and beef content in mixed meat products,which could detect beef content as low as 1%(w/w),demonstrating the potential of this sensor for detecting meat adulteration.In order to improve the detection rate,electric field assisted technology was introduced in the constructed sensor scheme.Under the optimized electric field,the coupling time of Streptavidin and biotin only takes 5 min,which greatly shortens the overall detection time and maintains its ultra-sensitive performance.Although the electric field has an impact on the activity of DNA polymerase,which leads to the failure of electric field assisted technology to promote SEA amplification reaction,it proves that the electric field can promote the hybridization and denaturation of nucleic acid chains.As a fast,sensitive and miniaturized detection method,this sensor method not only has the potential to be converted into a portable device for nucleic acid detection,but also has important research significance in the field of point-of-care testing(POCT)of nucleic acid.