| Fluorescence correlation spectroscopy (FCS) has been widely applied in biological and biomedical fields, especially in DNA hybridization detection, gene expression and interaction of bio-molecules. So far, FCS becomes an indispensable tool to study some biological events in cells at single molecular level because of its high sensitivity and very small detection volume. However, most of bio-molecules have no native fluorescence (or very weak), and thus, they need to be labeled with organic dye or inorganic nanocrystal quantum dots when fluorescence methods such as FCS method are used. However, some applications are limited by their bleaching or blinking properties of organic fluorescent dyes. Additionally, quantum dots are not suitable for some applications in vivo because most of quantum dots are composed of toxic elements such as Cd, Pb, Hg, Te and Se.The gold nanoparticles (GNPs) have been regarded as an ideal nano-probe with potential applications in diagnosis and bio-assay due to their unique optical and chemical properties. The labeling procedure of GNPs method is fair simple. The GNPs can conjugate to most of biomolecules such as nucleotides and proteins by adsorption. Meanwhile, the bioactivity of biomolecules was no significant change during labeling process, and GNPs conjugates with biomolecule are fairly stable and have no toxicity. To date, GNPs have been widely applied in the DNA detection and immunoassay detection. In this dissertation, we first build a resonance light scattering correlation spectroscopy (RLSCS) system based on the principle of fluorescence correlation spectroscopy and laser confocal configuration. All kinds of parameters in the RLSCS system were systematically optimized. The confocal detection volume in RLSCS was about 1 fl. And then, RLSCS method was successfully applied for characterizing translational and rotational diffusion, particles size and concentration of GNPs. Finally, RLSCS method also was applied in DNA hybridization detection and interaction of proteins using GNPs as probes. The main work includes as following:(1) RLSCS is a new single particle method, which is based on the resonance light scattering properties of gold nanoparticles. Following the principle of FCS, we consider resonance scattering light in RLSCS as fluorescence in FCS and build a theoretical model of RLSCS. The setup of RLSCS is built on the laser confocal configuration. All kinds of parameters are studied and optimized systematically. The detection volume obtained was about 1 fL. The RLSCS of gold nanoparticles for particles size larger than 15 nm can be obtained by this method. Our theoretical model of RLSCS is very well in agreement with experimental data.(2) We developed a new method to characterize concentration, translational and rotational diffusion of GNPs with RLSCS technology. We systematically studied the effects of some parameters on translational and rotational diffusion. We discovered that the intensity of scattering light and the polarization anisotropy of GNPs were significantly dependent on the illumination wavelength of laser and shape of GNPs. In the resonance scattering band, the polarization anisotropy and the scattering light intensity are very strong to successfully obtain the rotational information of GNPs by RLSCS. Far from the resonance scattering band, the rotational information of GNPs can not be tracked. The RLSCS method was successfully used to characterize the rotational and translational behaviors of GNPs and gold nanorods in solution using 632.8 nm He-Ne laser as light source. Meanwhile, the concentration of GNPs was characterized by RLSCS according to the particle numbers in the known illumination volume. The numbers of GNPs in the confocal detection volume were obtained from the resonance light scattering autocorrelation function and the confocal detection volume was determined by the translational diffusion coefficient of a known fluorescent dye.(3) The RLSCS technology was successfully applied in DNA hybridization. In this experiment, the two different oligo-DNA was introduced onto the surface of GNPs with thiol functional group. The aggregation of GNPs was formed by adding complementary target DNA. The diffusion time and intensity of scattering-light from GNPs can be monitored by RLSCS. The degree of aggregation became larger with the concentration increase of target oligo DNA. RLSCS discriminated complementary target DNA and single-base mismatch DNA through the diffusion time and intensity of scattering light from GNPs. The limit of detection for target oligonucleotides reach 0.1 nM using 21 nm gold nanoparticles modified with oligonucleotides as probes. Meanwhile, it can offer quantitative analysis for DNA concentration range from 0-48 nM.(4) The interaction between HRP-biotin and streptavidin was characterized by RLSCS with GNPs as probes. This interaction was regard as an immune assay model. The optimal environment pH value 8.4 for HRP-biotin covalent conjugation to the surface of GNPs was obtained by RLSCS as well as the pH value in the reaction system. The degree of aggregation was increasing with increase of the concentrations of streptavidin. It is interesting that the average translational diffusion time reached the maximum value when streptavidin concentration is 65 nM. However, the average translation diffusion time will gradually decrease when the concentration of streptavidin will be increased continually from 65 nM to 780 nM. It found that it reached the optimum stoichimetric ratio when the streptavidin concentration was 65 nM. The surface of HRP-biotinylated GNPs was rapidly occupied by large quantity of streptavidin molecules when streptavidin concentration was over 65 nM, which inhibit the cross link between GNPs. The RLSCS was applied in studying the dynamic of reaction. We found the aggregation of GNPs induced by interaction of HRP-biotin and streptavidin coincided with the reaction-limited aggregation modeling.
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