Study On Novel DNA Electrochemiluminescence Biosensors

In this dissertation we focused on the preparation of a new type ECL activated substance. Based on proximity-dependent surface hybridization assay and hairpin structure DNA, we have developed two new ECL methods for the determination of special DNA sequence. Compared with other methods the new methods were simple, rapid as well as sensitive and selective.The main research work is described as follows:In chapter one of this thesis, A new electrogenelated chemiluninescence method based on the proximity-dependent surface hybridization assay was proposed for detecting DNA by using Ru(bpy)32+-doped silica nanoparticles (Ru-DSNPs) as labels. The whole process involves two steps:the 3'thiolated capture probe was self-assembled on the gold electrode first, when the target DNA was introduced into the system, it was complementary to the 5'Ru-DSNPs labeled probe at the one-half-segment and complementary to the 3'short thiolated capture probe at the other half-segment, resulting in forming a stable duplex complex. The proximity-dependent surface hybridization assay depended on simultaneous recognition of a target DNA by capture probe and Ru-DSNPs labeled probe and formation of a duplex complex, which results in the luminophor adjoined the electrode surface, and increased the charge transport efficiency as well as improved the sensitivity remarkably. The ECL intensity of Ru-DSNPs increased about two orders of magnitude compared to the simple Ru(bpy)32+ molecules. Under the optimum condition, the intensity of ECL was linearly related to the concentration of the target sequence in the range of 2.0×10-15 to 2.0×10-11 mol/L. The detection limit was1.0×10-15mol/L.In chapter two, A novel DNA detection platform based on a probe DNA switch, and Ru(bpy)32+-doped silica nanoparticles(Ru-DSNPs) signal amplification for ultrasensitive detection of DNA hybridization has been developed in this work. Thiolated DNA probe was self-assembled on the electrode through Au-S bonding, labeled with Ru-DSNPs, and formed a stem-loop structure. After hybridization, the probe undergoes a significant conformational change, which forced the luminophore away from the surface of gold electrode. As a result, the ECL intensity significantly decreased. Single-base mismatch oligonucleotide and random oligonucleotide can be easily discriminated from complementary target DNA. By using this new strategy, we demonstrate that the DNA sensor has been able to detect as low as 5×10-14 mol/L.