A New Method And Pperformance Study Of Immobilization-free Electrochemical Nucleic Acid Sensing Analysis

In this paper,three different immobilization-free electrochemical DNA biosensors were constructed for the problems and challenges of electrochemical DNA biosensors.It demonstrates the considerable advantages of assay simplicity,rapidness and high sensitivity.It has opened up new ideas and methods for the research of immobilization-free electrochemical DNA biosensors,which is beneficial to the detection of early clinical diseases.1.We present the proof-of-principle of a unique and versatile immobilization-free electrochemical nucleic acid biosensor architecture based on the exonuclease ?(Exo ?)-catalyzed releasing of MB-tagged mononucleotide,and successive enriching onto dodecanethiol monolayer by hydrophobic force between the alkyl chain of dedecanethiol and the hydrophobic part of MB-tagged monucleotide.It demonstrates the considerable advantages of assay simplicity,rapidness and high sensitivity owing to the immobilization-free and homogenous operation for the biorecognition and amplification process.A low detection limit of about 1 pM toward target DNA could be achieved with an excellent selectivity.Furthermore,it should be easily extended for the detection of a wide spectrum of targets only if the MB-tagged mononucleotide could be released after the target-triggered operation.It thus provides a promising avenue for the development of immobilization-free and sensitive electrochemical biosensors.2.We present the proof-of-principle of a unique and versatile immobilization-free electrochemical nucleic acid biosensor architecture based on the T7exonuclease-catalyzed releasing of MB-tagged mononucleotide,and successive enriching onto dodecanethiol monolayer by hydrophobic force between the alkyl chain of dedecanethiol and the hydrophobic part of MB-tagged monucleotide.It demonstrates the considerable advantages of assay simplicity,rapidness and high sensitivity owing to the immobilization-free and homogenous operation for the biorecognition and amplification process.It is easy to distinguish between Wild-type target DNA and Mutant-type target DNA by detection of single base mismatches,which provided a novel,efficient and sensitive detection strategy for single base mutation detection.3.A label-free and ultrasensitive electrochemical detection of target DNA was developed by using a KF polymerization and Nt.BbVCI cascade target recycling and DNAzyme releasing amplification strategy.The DNA polymerase guided the target recycling and simultaneously triggered the KF polymerization and Nt.BbVCI accompanied by the cascade recycling of the released new complementary strand and the amplified liberation of the G-rich sequence of the HRP-mimicking DNAzyme,which play a electrocatalytic role toward H2O2 reduction for the electrochemical signal readout.Such strategy endows the developed biosensor with a high sensitivity and also a high confidence,it also exhibits the distinct advantages of simplicity in probe design and biosensor fabrication,and label-free electrochemical detection,thus may offer a promising avenue for the applications in disease diagnosis and clinical biomedicine.In conclusion,the electrochemical DNA biosensor constructed in this paper can be extended to the detection of other biomolecules,which is of great significance to the future research and diagnosis...