Study On The Application Of DNA Electrochemical Biosensor For Analysis Of Mercury Ion And ATP Based On The Dual-hairpin And Y-shaped DNA Structure

The quick, low-cost, highly selective and sensitive detection of biologically active small molecules and proteins, pathogenically associated sequence-specific nucleic acids and highly toxic heavy metal ions is one of the most important topics in modern biochemical analysis. In recent years, with the rapid development of science and technology, DN A electrochemical biosensor is a new cross-disciplinary which relates to the field of biochemistry,medicine,electrochemistry and information technology and so on. DNA electrochemical biosensor has been widely used in genetic diagnosis, environmental monitoring, drug research and other fields. An electrochemical nucleic acid based biosensor is a biosensor that integrates a nucleic acid as the biological recognition element and an electrode as the physicochemical transducer. The nucleic acid molecule probe has the advantages of strong specificity, simple structure, target molecules widely and is easy to synthesis quantify, etc. Electrochemical nucleic acid based biosensor combines the high specificity of nucleic acid probes and the excellent sensitivity of electrochemical detection techniques, it has become the most important branch of biosensors. In addition, the discovery of the new performance of nucleic acid modified electrode、the design and preparation of new nucleic acid probe and the research for development of high efficient DNA electrochemical sensor are concerned increasingly. Four kinds of DNA electrochemical biosensor based on the dual- hairpin structure and Y-shaped structure DNA were developed through combining the ratiometric method and Exo-III based signal amplification in this paper, realizing the high sensitive selective detection of mercury ion and ATP. The main work is as follows: Part 1 A simple electrochemical method for the detection of ATP using target-induced conformational change of dual-hairpin DNA structureA novel method based on both insertion approach for the repeatability improvement and the dual- hairpin structure as a signal amplifier for the sensitivity improvement was investigated for adenosine triphosphate（ATP） detection. For the preparation of the aptasensor, the moiety and methylene blue（MB） labeled aptamer probe were inserted into a loosely packed cyclic-dithiothreitol（DTT） monolayer, and the MB labeled adjunct probe could hybridize with the aptamer probe to form the dual- hairpin structure on the electrode. In the absence of the target, the stable dual- hairpin conformation enabled the MB to stay close to the electrode surface, generating a strong current signal. However, when the target was introduced, the high affinity binding of aptamer/target complex kept the MB of aptamer probe distanced from the electrode surface and forced the adjunct probe to dissociate from the modified electrode, decreasing the current signal obviously. The current intensity was inversely proportional to the concentration of ATP in the range of 5 nmol L-1 to 1 μmol L-1 with a detection limit of 1.4 nmol L-1. Part 2 A regenerative electroche mical biosensor for mercury（II） by using the insertion approach anddual-hairpin-based amplificationA simple and effective biosensor for Hg²⁺ determination was investigated. The novel biosensor was prepared by the insertion approach that the moiety-labeled DNA inserted into a loosely packed cyclic-dithiothreitol（DTT） monolayer, improving the hybridization efficiency. Electrochemical impedance spectroscopy studies of two biosensors（single hairpin and dual- hairpin structure DN A modified electrodes） used for Hg²⁺ detection indicated that the dual-hairpin modified electrode had a larger electron transfer resistance change（ΔRct）. Consequently, the dual-hairpin structure was used as a signal amplifier for the preparation of a selective Hg²⁺ biosensor. A linear relation was observed between the ΔRct and Hg²⁺ concentrations in a range from 0.1 nmol L-1 to 5 μmol L-1 with a detection limit of 28 p mol L-1. Part 3 A regenerative ratiometric electrochemical biosensor for selective detecting Hg²⁺ based on Y-shaped/hairpin DNA transformationInspired by dual-signaling ratiometric mechanism which could reduce the influence of the environmental change, a novel, convenient, and reliable method for the detection of mercury ions(Hg²⁺) based on Y-shaped DN A（Y-DNA） was developed. Firstly, the Y-DNA was formed via the simple annealing way of using two different redox probes simultaneously, omitting the multiple operation steps on the electrode. The Y-DNA was immobilized on the gold electrode surface and then an obvious ferrocene（Fc） signal and a weak methylene blue（MB） signal were observed. Upon addition of Hg²⁺,the Y-DNA structure transformed into hairpin structure based on the formation of T-Hg²⁺-T complex. During the conformational transduction, the redox MB gets close to and the redox Fc gets fa r away from the electrode surface, respectively. This special design allows a reliable Hg²⁺ detection with a detection range from 1 nmol L-1 to 5 μmol L-1 and a low detection limit down to 0.094 nmol L-1. Part 4 A regenerative ratiometric electroche mical biosensor for sensitive and selective Hg²⁺ detection based on Exonuclease III assisted signal amplificationIn this work, a regenerative electrochemical biosensor based on exonuclease III（Exo III） catalyzed target recycling was developed for Hg²⁺ detection. The proposed biosensor adopted dual-signaling ratiometric strategy. As to the construction of the biosensor, methylene blue（MB） modified thiolated probe（MB-probe） could hybridize with the ferrocene（Fc） labeled probe（Fc-probe） to form Y-shaped DNA structure, hence emerged a weak MB signal and a strong Fc signal. In the presence of Hg²⁺, Fc-probe could form double-stranded structure with an assist probe（H-probe） by the formation of T-Hg²⁺-T, leading to the dissociation of Fc-probe from the electrode and the formation of hairpin structure by MB-probe. Exo III could selectively digest Fc-probe in double-stranded structure and released Hg²⁺ and H-probe to realize target recycling. As a consequence, the electrochemical signal of MB enhanced significantly and the peak current of Fc decreased distinctly, realizing the dual-signaling ratiometric strategy. The employment of Exo III and dual-signaling ratiometric method significantly improved the sensitivity of the biosensor. This special design allows a novel and reliable method with high sensitivity and good selectivity for Hg²⁺ detection in the range of 5 p mol L-1 to 20 nmol L-1, and a low detection limit down to 1.63 pmol L-1.