Study On Highly Sensitive Electrochemical Dna Biosensors Based On Cascade Signal Amplification Strategy

In this thesis, we have constructed three highly electrochemical DNA biosensor based on cascade signal amplification strategy, accomplished detection of Small molecules, protein and DNA.The obtained main experimental results could be concluded as below:1. The isothermal amplification with nicking endonuclease/KF polymerase-based strategy was effectively married with the enzyme/gold nanoparticle-based post-amplifi-cation strategy to promote the detection performance towardtarget DNA. A hairpin DNA probe（HP） is designed that consists of an overhang at 3′-end as the recognition unit for target DNA, a recognition site for nicking endonuclease, and an alkane spacer to terminate polymerization reaction. The autonomous DNA replication–scission–displacement reaction operated by the nicking endonuclease/KF polymerase induced the autocatalytic opening of HP, which wasthen specifically bound by the enzyme/gold nanoparticles for further dual-signal amplification towardtarget-related sensing events.A low detection limit of 0.065 fM with an excellent selectivity towardtarget DNA could be achieved.2. A simple, highly sensitive and selective electrochemical DNAzyme sensor for Pb²⁺ was developed based on the basis of a 8-17 DNAzyme cleavage-induced template-independent polymerization amplification strategy. The hairpin-like substrate strand（HP DNA） of 8-17 DNAzyme was firstly immobilized onto the electrode. In the presence of Pb²⁺ and the catalytic strand of 8-17 DNAzyme, the HP DNA could be cleaved to expose the free 3′-OH terminal, which could be then utilized for the cascade operation by terminal deoxynucleotidyl transferase（TdTase） for the base extension to incorporate biotinylated dUTP（dUTP-biotin）. The further conjugated streptavidinlabeled alkaline phosphatase（SA-ALP） then catalyzes conversion of electrochemicallyinactive 1-naphthyl phosphate（1-NP） for the generation of electrochemical response signal. An impressive detection limit of 0.043 nM toward Pb²⁺ with an excellent selectivity could be ultimately obtained.3. An isothermal, enzyme-free, label-free and ultrasensitive electrochemical DNA biosensor was fabricated by coupling the DNA-fueled molecular machine and hybridization chain reaction strategy, which were deployed for target recycling and post-amplification process, respectively. The DNA duplex probes are self-assembled on the gold electrode surface to fabricate thesensor.The DNA target（TD） binds to the terminal toehold region of the probes,displaces the assistant DNA strands（AP） through toehold-mediated strand displacementreactions, and exposes the secondary toehold region for subsequent hybridization withthe DNA fuel strands（FS）, which further displace both the target DNA and the protection strands（PP）, and then the target recycling.FS toehold sequence as a rigger to the hybridization chain reaction of two hairpin DNA probes（HP-1 and HP-2）, and the dendritic DNA concatamers were autonomously formed. It was then used as the signal carrier by electrostatic adsorption of RuHex and played the signal amplification role toward target DNA detection.