Development Of Novel DNA Biosensor Based On Covalent Immobilization

There are three parts in this thesis. Two new and simple strategies were established for fabricating of deoxyribonucleic acid(DNA) electrochemical biosensor based on covalent immobilization of probe NH2–ssDNA by sol–gel coupling (the first chapter) and diazotization-coupling (the second chapter). A reagentless H2O2 biosensors was also fabricated based on covalent immobilization of Thionine in the third part.1. A new procedure for fabricating DNA electrochemical biosensor was developed based on covalent immobilization of target ssDNA on Au electrode that had been functionalized by direct coupling of sol–gel and self-assembled technologies. Two siloxanes, 3-mercaptopropyltrim ethoxysiloxane(MPTMS) and 3-glycidoxypropyltrimethoxysiloxane(GPTMS) were used as precursors to prepare functionally self-assembly sol–gel film on Au electrode. The thiol group of MPTMS allowed assembly of MPTMS sol–gel on gold electrode surface. Through co-condensation between silanols, GPTMS sol–gel with epoxide groups interconnected into MPTMS sol–gel and enabled covalent immobilization of target NH2–ssDNA through epoxide/amine coupling reaction. The concentration of MPTMS and GPTMS influenced the performance of the resulting biosensor due to competitive sol–gel process. The linear range of the developed biosensor for determination of complementary ssDNA was from 2.51×10?9 to 5.02×10?7M with a detection limit of 8.57×10?10 M. The fabricated biosensor possessed good selectivity and could be regenerated.2. A novel and simple strategy for fabricating of DNA electrochemical biosensor was developed based on covalent coupling of probe NH2–ssDNA(S1) on Au electrode that had been functionalized by diazotization of assembled 4-aminothiophenol(4-ATP) monolayer. The thiol group of 4-ATP allowed the stable assembly of 4-ATP monolayer. The following diazotization reaction was directly performed to prepare functional diazo–ATP film for covalent coupling of probe S1. Remarkably, it was noting that the diazo–ATP provided a surface with high conductibility for electron transfer. The complementary ssDNA was determined by using differential pulse voltammetry. The linear range of the developed biosensor was from 1.57×10 to 4.52×10 M-9-7 with a detection limit of 3.26×10 -10 M. The fabricated biosensor possessed good selectivity and could be regenerated. The covalent immobilization of probe S1 by simple diazotization-coupling on self-assembled 4-ATP monolayer could serve as a versatile platform for DNA immobilization and biosensors fabricating.3. A simple strategy for fabricating of a non-enzyme and reagentless H2O2 electrochemical biosensor was developed based on covalent coupling of Thionine(Th) on Au electrode that had been functionalized by diazotization of assembled 4-aminothiophenol(4-ATP) monolayer. The thiol group of 4-ATP allowed the stable assembly of 4-ATP monolayer. The following diazotization reaction was directly performed to prepare functional diazo–ATP film for covalent coupling of Th. Th covalently immobilized on the diazo–ATP film possessed high electrocatalytic activity to H2O2 and provided a fast amperometric response. The linear response of the as-prepared biosensor for the determination of H2O2 ranged from 1.0×10?6 to 6.4×10?3 M with a detection limit of 6.71×10?7 M. The fabricated biosensor possessed high stability.