Study On The Electrochemical Detection Of DNA With High Speciifcity

The sequence-specific DNA detection on the basis of hybridization with thecomplementary DNA probe has attracted wide interests and shown significant applications inthe field of microbial identification, food quality control, disease diagnosis, environmentalmonitoring and drug analyses. Though the mainly methods of ssDNA detection are based onfluorescent analysis nowadays, electrochemical DNA biosensor showed advantages such assimple, rapid, environment-insensitive and low-cost.In the first, an electrochemical sensor based on the molecule probe and E. coli DNAligase was constructed to detect dual-DNAs and LDH simultaneously. In this experiment, themolecule probe was modified with a thiol at its5’end and a methylene blue (MB) at its3’endand covalently immobilized to the surface of a gold electrode to form a stem-loop structurethrough facile gold-thiol affinity.6-Mercaptohexanol (MCH) was used to cover the remnantbare region. In the original state, the probe formed a “close” hairpin structure, which forcedMB group to approach the electrode surface and exchanged electrons freely with the electrode.In the presence of both targets (target DNA1and target DNA2), the hairpin structure shiftedfrom “close” state to “open” state with the help of Escherichia coli (E. coli) DNA ligase,which could be characterized by alternating current voltammetry (ACV). For target DNA1(T1) and target DNA2(T2), the linear response observed were ranging from1.0×10-7to1.0×10-6mol·L-1and5.0×10-9to1.0×10-6mol·L-1, respectively. Furthermore, the assay wascoupled with the activity of lactate dehydrogenase (LDH), which catalyzes the conversion ofNADH to NAD+. The latter acted as the co-substrate of E. coli DNA ligase and producedcurrent change in the presence of both target DNAs. Hence the sensor could analyze theco-existence of three target components.Secondly, the other DNA biosensor was introduced using electrochemical technique withG-quadruplex-hemin complexes as signal transduction probes and could be used to detect T1and T2with the help of E. coli DNA ligase. In this assay, Sig-DNA including theG-quadruplex oligonucleotides and Tem-DNAwere added to the solution of T1and T2, whichenabled the reaction of hybridization between Tem-DNAwith T1, T2and Sig-DNA. After thisstep, it formed two molecular nicking on the hybridized double-helix structure. In thepresence of E. coli DNA ligase, the two nicking were linked and a new long ssDNA of37ntwas generated. Then the solution was heated at90oC for5min, the long sequence wasreleased from hybridized structure and immediately be captured by the Cap-DNA, which hasbeen immobilized on the surface of gold electrode. The G-quadruplex oligonucleotide wasalso close to gold electrode, which could form complexes in the joint of hemin. TheG-quadruplex-hemin complexes behaving electrochemical activity could lead to peak currentsignal which would be detected by differential pulse voltammetry (DPV). The detection limitsof this method for detecting T1and T2were both2.0×10-12mol·L-1. Compare to the first DNAbiosensor, this approach was more sensitive for two orders of magnitude.