Study Of The DNA Electrochemical Biosensor Based On Enzyme-assisted Current Change Amplification

In this thesis, the DNA electrochemical biosensor based on several tools of enzymatic-assisted current change amplification were designed, constructed and characterized. The proposed biosensors were applied to ultra-sensitive detection of DNA sequences of certain microorganisms and heavy metal ions.This thesis is divided into four chapters to elaborate:The first chapter introduces the features and the advantages of DNA electrochemical biosensor, DNA signal amplification method and the progress of the enzymatic DNA biosensors. Meanwhile, the research ideas were summarized.In second chapter, a simple, sensitive and specific electrochemical sensor was designed for determination of given DNA sequence from bacillus subtilis by nicking endonuclease assisted current change amplification and target-induced mode. First, the capture probe (CP) with a recognition sequence hybridized with the signal probe (SP) with ferrocene (FC) at the 5’-terminus to form the self-assembled DNA sensor. In the absence of target DNA (TD, the sequence from hypervariant region of 16s rDNA of bacillus subtilus), the double-strand formation of the DNA biosensor showed an unobvious faradic current from FC. When the TD was introduced, the N.BstNB I identified the double strand but just cut the single strand after the CP hybridized the SP. Then the TD could go through many cycles, resulting in a high peak current. The dynamic concentration range spanned from 0.1 fM to 20 fM and the detection limit 0.084 fM at the 3sblank level under optimum conditions. Moreover, the compare between our proposed biosensor and Real-time Quantitative PCR Detecting System (QPCR) with SYBR Green for detecting bacillus subtilis was investigated. The relative deviation is about-7.8%.In third chapter, a DNA biosensor was proposed to detect Hg2+ based on exonuclease III amplification mechanism and dual-signal model. Firstly, the sensor enhanced the signal through increasing the quantity of DNA (1) in the electrode surface by gold nanoparticles. Secondly, the Hg2+ can go through many cycles because of the interaction between the T-Hg2+-T formation of DNA (1) and EXO Ⅲ, resulting in a high peak current. Thirdly, the dual-signal was superimposed on the Hg2+ quantification. Therefore, the sensor could achieve the ultra-low detection limit (aboutl.8 aM) when the linear range is from 0.01 to 4.5 fM. In addition, the compare between our proposed biosensor and ICP-MS for detecting real samples was investigated. The relative deviation is about 2.6%.In fourth chapter, a signal-on DNAzyme biosensor based on hybridization chain reaction was designed to detect Pb2+. The Pb2+-DNAzyme can specificly recognize Pb2+, which can lead Dz/SS duplexes to dissolve. At this moment, TBA that from the 3’terminal of Dz binds Tb effectively induce DNA (3) and DNA (4) to take place hybridization chain reaction. Finally, with a large number of G-quadruplex was formed, a large number of hemin DNAzyme catalyze H2O2-TMB to achieve rapidly amplification catalytic currents. The linear range is from 0.08 to 6 fM, and the sensor detection limit is 2 aM. Moreover, the compare between our proposed biosensor and ICP-MS for detecting real samples was investigated. The relative deviation is about-7.8%.