|dc.description.abstract||Electrochemical proximity assay (ECPA), was recently developed for direct protein quantitation as low as 10 fM. In this assay, the loop-like structure holds methylene blue (MB) in close proximity with the gold surface when the target is present, ensuring efficient electron transfer. However, for studying DNA binding dynamics on gold surfaces, investigations were carried out by immobilizing thiolated DNA onto the gold surface of the SPR sensor chip to form self-assembled monolayer. DNA loop oligonucleotides and MB-DNA sequences were alternately hybridized to the complementary strands to form a loop-like structure. Investigations on the formation of DNA loop like structure based on ECPA were carried out using surface plasmon resonance (SPR). SPR, is a well-established optical technique for studying mechanisms of DNA immobilization and interactions of DNA molecules with gold surfaces in real time. The result shows obvious refractive angle change (∆RA) corresponding to the DNA hybridization of the whole system while there is no discernible signal observed upon elimination sequences (thiolated DNA or DNA loop) of DNA Loop based ECPA system. Furthermore, investigation on optimization of concentration and condition of SH-DNA in flow mode was carried out. Before thiolated DNA was self-assembled on the gold surface, it was treated with a reductant TCEP for cleaving the disulfide precursor. This leads to the release of free thiols and other sulphur derivatives. In order to study the effect of interference of free thiols, the SH-DNA after treatment was purified and compared with non-purified DNA. Further study was conducted to observe the effect of purified DNA on the sensitivity and enhancement of signals on DNA Loop based ECPA system. Mixture of different ratios of free thiols (MCH) and purified SH-DNA was investigated for immobilization on gold surface. Through studying surface dynamics of DNA Loop based on ECPA system, we expect a significant improvement on sensitivity with ECPA- based biosensor.
Furthermore, effect of the presence of free thiols, produced by the presence of reductant often used to cleave disulfide precursors on SH-DNA self-assembly process, was investigated. We found that the SH-ssDNA binding on the gold surface was affected by the presence of these free thiols. With purification it was observed that purified SH-DNA led to surface hybridization enhancement in target binding signal by 39%. To study the presence of contaminants in non-purified and purified SH-DNA, the surfaces were characterized by UV-vis spectroscopic techniques. Different ratios of purified SH-DNA to free thiols demonstrated that the effect of binding intensities corresponding by ∆RA and the surface coverage. With this proof-of-concept study on the dynamics of the DNA loop model system, we anticipate the feasibility of this study in improving the speed and sensitivity of ECPA-based protein recognition.||en_US