The Preparation And Analytical Applications Of Novel Electrochemical DNA Biosensors Based On Signal Increases

As the main components of chromosome in the nucleus, DNA is vital to human genetics and health. The detection of human body, viruses and specific sequences of bases in the nucleic acids bacteria in the field of environmental monitoring, disease diagnosis, food contamination and forensic identification will play an increasingly significant role, has attracted a lot of attention. Due to the electrochemical DNA sensors can not only achieve more simple, faster, cheaper determination, but also can significantly improve the sensitivity and specificity of detection, providing a kind of more effective detection methodology for DNA hybridization detection. In this study, we developed several novel "signal-on" DNA biosensors for the highly sensitive and selective detection of heavy metals (mercury ions and lead ions) and DNA fragments.This thesis is mainly divided into four chapters.In chapter one, the classification of DNA biosensor, the significance of research and progress is introduced and summarized. Additionally, this section focuses on the electrochemical DNA biosensors and its application in metal ions, hybridization detection techniques and application in the study of gene diagnosis methodology. Finally, the research purposes and main contents of this dissertation is presented.In chapter two, a novel simple, signal-enhanced electrochemical sensor was designed for sensitive and selective determination of mercury ions by using target-triggered conformational change of DNA pseudoknots with the assistance of auxiliary DNA strands. Upon addition of Hg2+ ions, the auxiliary DNA strands are selectively complementary to DNA pseudoknots owing to the strong coordination of T-Hg2+-T between the mismatched bases. The binding reaction disrupts the DNA pseudoknots, and liberates a flexible, single-stranded element to strike the electrode, leading to a strong electrochemical signal due to the approaching ferrocene tags. By using square wave voltammetry (SWV) to detect change of the peak current between the presence and absence of target ions (Hg2+), the target can be sensitively detected.In chapter three, a new signal-on amplification strategy for sensitive electronic detection of nucleic acid based on the isothermal circular strand-displacement polymerization(ICSDP) reaction is reported.The assay mainly involves a hybridization of ferrocene-labeled hairpin DNA with blocker DNA, a strand-displacement process with target DNA, and an ICSDP-based polymerization reaction. The signalis amplified by the labeled ferrocene on the hairpin probe with target recycling. Upon addition of target analyte, the blocker DNA is initially displaced by target DNA from the hairpin/blocker DNA duplex owing to the difference of the folding free energy, then the newly formed target/blocker DNA duplex causes the ICSDP reaction with the aid of the primer and polymerase, and then the released target DNA retriggers the strand-displacement for target recycling. Numerous ferrocene molecules are close to the electrode surface due to the reformation of hairpin DNA, each of which produces an electronic signal within the applied potentials, thereby resulting in the amplification of electrochemical signal. Under the optimal conditions, the ICSDP-based amplification method displays good electrochemical responses for detection of target DNA at a concentration as low as 0.03 pM.In chapter four, a portable and quantitative monitoring protocol for sensitive detection of lead ions is designed, based on target-responsive cargo release from Pb2+-specific DNAzyme-capped mesoporous silica nanoparticles(MSNs), by coupling with a widely accessible personal glucose meter (PGM). Initially, glucose molecules are loaded into the pores of the MSNs, the pores are then capped with Pb2+-specific DNAzymes. Upon target introduction, the molecular gates open, resulting in release of the cargo from the pores. The released glucose can be quantitatively monitored using a portable PGM. Under optimal conditions, the as-prepared sensing platform presents good analytical properties for the determination of the target Pb2+ ions, and allows detection of Pb2+ at concentrations as low as 1.0 pM. Importantly, the portable sensing platform has the advantages of simple, on-site, user-friendly and low-cost assessment and has tremendous potential for quantitative detection of non-glucose targets by the public.