The Dynamical Light Scattering Was Used To Study The Conformation Of Nanogold Surface Oligonucleotides And The Screening Technique Of Small Molecule Target Nucleic Acid Aptamers

This article focuses on the two key technical problems facing in the nano-biosensing technologies:controllable preparation of solid-phase sensing interface and the selection of aptamers against small molecule targets. The content of the thesis includes the following two parts:1. DNA/gold nanoparticle (AuNP) complex is one of the most common used sensing interfaces in solid-phase biosensors. DNA/AuNP holds the great potential in various applications including nanostructure assembly, biosensors, diagnosis, and therapies. As the conformation of DNA on the surfaces of AuNPs affects the hybridization efficiency and the ability of molecular recognition, the precise control of it is very important. Here, for the first time, we use Dynamic Light Scattering (DLS) as a sensitive tool to study the dynamic immobilization process of DNA onto gold nanoparticles. The results support the dynamic inner-layer and outer-tail (DILOT) conformation of the linear DNAs on AuNPs. According to the DILOT model, the length of bases that can't efficiently hybridize with the complementary strands can be calculated, which is benificial for precisely controlling the conformation, improving hybridization efficiency and molecular recognitions of DNA on AuNPs. In addition, we found that when the NaCl was lower than the certain concentration (critical concentration,[Na+]c), the aptamers and the linear DNA with the same lengths showed no difference in the assembly kinetics, suggesting that the conformation of aptamers also follows the DILOT model. However, when the salt concentration is above [Na+]c, the aptamers on AuNP start to fold into the secondary structures, resulting in their higher surface capacities and the smaller sizes of aptamer/AuNP complex. The conformation of DNA/AuNP no longer meets the DILOT model any more.2. Most of the traditional aptamer selection techonologies (Systematic Evolution of Ligands by Exponential Enrichment (SELEX)) for small molecule targets need to attached the targets on the solid surface. Thus the chemical modification of the targets is required, which would strongly change the structures of the target. Furthermore, the introduction of the solid surface can also increase the nonspecific adsorption of oligonucleotides and therefore cause the increased background and the low enrichment efficiency. In this thesis, we invented a novel aptamer selection method without the need to attach the target on the solid phase, referred as enzymatic-SELEX. The method is based on the significant reduction of digestion efficiency of aptamers by Exnulease I (Exo I) at the presence of targets to achieve the selection of aptamers. Our research was focused on the two key steps of enzymatic-SELEX:the optimization of the nuclease digestion conditions and finding the efficient ligation methods to ligate the random sequence with the primer. We also conducted one cycle of enzymatic-SELEX using hemin as the target.