Ultrasensitive Electrochemical Biosensor For Specific Detection Of DNA Based On Molecular Beacon Mediated Circular Strand Displacement Polymerization And Hyperbranched Rolling Circle Amplification

The detection of biomolecules in clinical diagnosis,gene therapy and mutation analysis has become increasingly important.Therefore,it is significant for establishing a simple,rapid and sensitive detection method.Electrochemical biosensor has gained increasing interest because of its advantages such as simplicity,portable,easy to operate and low cost in the field of biomolecules detection.With the combination of biochemistry and molecular biology,isothermal nucleic acid amplification techniques was researched and developed to improve the sensitivity of detection method.This study has been performed by integrating two isothermal nucleic acid amplification techniques for ultrasensitive DNA detection based on electrochemical biosensor.Using a cascade signal amplification strategy,an ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon(MB)mediated circular strand displacement polymerization(CSDP)and hyperbranched rolling circle amplification(HRCA)was proposed.The hybridization of MB probe to target DNA resulted in a conformational change of the MB and triggered the CSDP in the presence of bio-primer and Klenow fragment(KF exo-),leading to multiple biotin-tagged DNA duplex.Furthermore,the HRCA was implemented to product amounts of double-stranded DNA(ds-DNA)fragments using phi29 DNA polymerase via biotin-streptavidin interaction.After the product of HRCA binded numerous biotinylated detection probes,an ultrasensitive electrochemical readout by further employing the streptavidin-alkaline phosphatase.The proposed biosensor exhibited excellent detection sensitivity and specificity with a log-linear response to target DNA from 0.01 f M to 10 p M as low as 8.9 a M.The proposed method allowed DNA detection with simplicity,rapidness,low cost and high specificity,which might have the potential for application in clinical molecular diagnostics and environmental monitoring.