Study Of The Conformational Change Of Nucleic Acids On Interface And Biosensing Application

The conformation of biomolecule plays a significant role in its biological function modulation. For instance, the biomolecular recognition capability of surface-bound DNAs highly relies on their conformations. Different interactions, such as the adsorption between DNA bases and the surface, can stimulate the conformational change of DNAs and thus influence their sensing efficiency. Therefore,developing methods to explore the orientation and conformation of nucleic acids on different interfaces and the related recognition kinetics will help us understand the interfacial behaviors of biomolecules and provide fundamentals for construction of high-performance biosensors.The specific characteristics of gold nanoparticles including quantum size effect,surface effect, tunneling effect and so on endow nanoparticles with unique optical,magnetic and electrical properties. Localized surface plasmon resonance of single nanoparticles, also known as nanoparticle plasmon resonance (NPPR), can amplify the weak chemical signals within the nano-scale environment around the nanoparticle surface through the near-field electromagnetic enhancement effect of transition metals.On the other hand, the assembled biomolecules will influence the NPPR. Thus,exploring and understanding of the interactions between NPPR and assembled biomolecules will provide solid fundamentals for establishing novel concept based bioanalytical methods to achieve amplified weak chemical information of biomolecules.In this thesis, considering the outstanding bio-compatibility of gold nanoparticles,we construct a surface plasmonic near-field electromagnetic enhanced bio-nano interface. With the help of various analytical techniques such as NPPR, dark field microscopy (DFM), UV-vis and electrochemical methods, we studied the effect of conformational change, molecular recognition event and biocatalysis processes on the NPPR characters of single nanoparticle on one hand, and on the other hand, we studied the magnification and modulation mechanism of the localized electromagnetic field on transition metal surfaces toward bio-recognition and bio-catalysis processes.This dissertation mainly includes the following three parts:1.Fabrication and application of a reversible plasmonic pH probe based on i-motif-modulated morpholino-AuNPs assemblyA novel colorimetric pH probe has been developed based on the i-motif-modulated morpholino-AuNPs assembly. With the help of UV-vis and resonance scattering spectroscopy, we studied the impact of DNA conformations on the surface plasmonic properties of metal nanoparticles. The substitution of morpholino to normally used DNA during the fabrication process greatly increases the hybridization efficiency of i-motif DNA on the morpholino-modified AuNPs under weak buffer molarity and avoids the use of high salt concentration which might lead to the aggregation of AuNPs. Meanwhile, morpholino can protect the complementary DNAs against nuclease enzymolysis. The unique plasmonic resonance property of AuNP endows the pH probe with high sensitivity to bring out stable response toward pH change in bulk solution. By recording the intense Rayleigh resonance scattering spectra of AuNPs, we achieved the pH detection in micro/nano-environments. The fabricated i-motif-MO-AuNP assembly bears a superior stability and reversibility to the corresponding i-motif-DNA-AuNP assembly, which may have a promising potential in micro/nano scaled pH detection in real biological systems.2. Study of the conformational change of biomolecules and biocatalysis process on gold nanoparticlesBy combining the nanoparticle plasmon resonance scattering spectra (NPPR)with dark-field microscopy imaging technology, a label-free analytical method with high spatial and temporal resolution was developed to study the effect of conformational change and biocatalysis processes on the NPPR char.acters of single nanoparticle. Judging from the shift of NPPR spectra, we achieved the direct observation of conformational change of G-rich DNA from a random coil structure to a parallel G-quadruplex one on single gold nanoparticle as well as the molecule recognition event of hemin to G-quadruplex. In addition, the established technique enables to monitor the specific binding process of thrombin to TBA aptamer. The formed GNP-DNAzyme or GNP-HRP complex exhibits localized catalytic response toward H2O2-DAB and the relatively weak chemical signal can be visibly amplified by the surface localized electromagnetic field of gold nanoparticle. Therefore, through the combination of NPPR and dark field imaging technology, we achieve bio-sensing on single nanoparticle, which may pave a new and efficient way for bio-analysis on single molecule level.3. Study of the special properties of DNA/Morpholino monolayerFast scan rate cyclic voltammetry was used to study the characteristics of end-grafted DNA/Morpholino monolayer on a gold electrode. The impact of passivation layer properties (e.g., charge or alkanethiol chain length) on morpholino-DNA hybridization efficiency and the elastic bending of DNA chains were carefully studied. Meanwhile, the kinetics of electron transfer of ferrocene-terminated DNA monolayer on different passivation layers were also studied. Since DNA and morpholino chains are confined within the nano spaces around the surface of gold electrode, the nano characteristics of DNA chains are more prominent than that of the DNA chains in bulk solution. Neutrally-charged passivation layer has the least limitation on the flexibility and free motion of morpholino chains and the resultant MO-DNA duplex. The surface-immobilized morpholino monolayer with neutrally-charged passivation layer bears the highest hybridization efficiency with the target ferrocene-terminated DNA. At the same time, ferrocene exhibits a most reversible electron transfer behavior on neutrally-charged passivation layer. It is also found that the elastic bending of DNA monolayer will be affected by the alkanethiol chain length. With the increase of alkanethiol chains,the growing steric hindrance will interfere the free bending of DNA chains and weaken their Brown motion. Moreover, the bending diffusion speed of FA-DNA chains will have a direct impact on the voltammetric behavior of ferrocene.