Design And Application Of Proportional Electrochemical Biosensors

Electrochemical sensors have attracted increasing attention because of high sensitivity,rapid detection,easy fabrication and low cost but some issues still remain unsolved such as poor reproducibility and signal fluctuation caused by complex assay conditions.The ratiometric electrochemical biosensor can effectively tackle the problems mentioned above due to the introduction of two electrochemical redox labels which may offer a built-in correction toward signal transduction for accurate detection of biomolecules or metal ions.First,a ratiometric electrochemical biosensor for sensitive detection of DNA based on DNA-four way junction?DNA-4WJ?formation and enzyme-assisted recycling amplification was developed.We designed a triple-helix molecular beacon?THMB?which consisted of a methylene blue?MB?-labeled hairpin DNA?HP?as a center,two unmodified strands of?-and?-specific DNA sequences flanked by two arm segments and a ferrocene?Fc?-labeled universal trigger?UT?oligonucleotide.The ratiometric probe was formed via the integration of Fc labeled UT and MB labeled HP in the THMB.The formation of the DNA-4WJ structure can be triggered by the target DNA may induce and the THMB structure was break simultaneously,leading to the release of MB from the electrode surface and the close proximity of Fc to the electrode surface.Moreover,the structure of DNA-4WJ may be cleaved by RNase HII to initiate recycling signal amplification.This biosensor exhibits high sensitivity with a detection limit as low as 0.063 pM and a long dynamic range from 0.1 pM to 100 nM.In addition,it can be easily fabricated by one-step assembly of ratiometric probes and simply performed by a one-step incubation procedure.Then,we fabricated a reusable ratiometric electrochemical biosensor on the basis of the binding of methylene blue to DNA with alternating AT base sequence for sensitive detection of adenosine.We designed a strand 1 with MB-modified thymine?T?base in the proximal 3?termini as the capture probe for its immobilization on the gold?Au?electrode and a 3?termini ferrocene?Fc?-modified aptamer for the recognition of adenosine.In the presence of adenosine,the aptamer can specifically bind with it,inducing the release of Fc from the electrode surface and the close of MB to the electrode surface,generating a large value of I_MB/I_Fc(I_MB and I_Fc are the peak currents of MB and Fc,respectively).The ratiometric signal(I_MB/I_Fc)can be used for accurate quantification of adenosine.This ratiometric biosensor exhibits high sensitivity with a limit of detection of as low as 90.8 pM and a large dynamic range from 0.1 nM to 100?M,and it also demonstrates excellent selectivity,regeneration capability,high reliability and good reproducibility.