Fluorescence Correlation Spectroscopy Of Gold Nanoparticles And Its Applications In Analytical Chemistry

Fluorescence correlation spectroscopy(FCS) is a single molecule detection method, and it has been used currently in chemical, biological and medical fields due to its high sensitivity, good selectivity, small sample requirements, simple procedure of sample preparation and short analysis time. However, in applications of FCS in life sciences and medicine, there are still two main challenges.The first challenge is that the photostability and biocompatibility of optical probes used currently are not good. The organic fluorescent dyes which are widely used perform serious photobleaching, and semiconductor quantum dots(QDs) have certain biological toxicity. The Second challenge is that commercial FCS instruments are too expensive, and cannot widely extend to general labs. To face the two challenges above, this dissertation is focused on the study of gold nanoparticles as optical probes, and the new detection method of combining FCS with three-dimensional scanning system. The main contributions are as follows:1. Fluorescent properties of gold nanoparticles(GNPs) were studied by ensemble fluorescence spectroscopy method and FCS method at the single particle level. We found that GNPs show the following optical properties:(1) GNPs with different sizes have the same emission wavelengths;(2) the fluorescence intensity of GNPs increases with an increase of excitation intensity, and GNPs have a high photo-saturation feature;(3) under illumination of stronger light, GNPs possess high photostability, no photobleaching and stronger brightness per particle over fluorescent dyes despite their low fluorescent quantum yields. Our results document that GNPs are potential fluorescent labeling probes in bioassay and bioimaging.2. Based on the unique fluorescent properties of GNPs and molecule recognition of aptamers, we developed a sensitive and homogenous method for determination of thrombin in human plasma by FCS technique. In assay of thrombin, we used a sandwich strategy and conjugated GNPs with two different aptamers(like antibodies) respectively. When two GNPs labeled with different aptamers are mixed in a sample containing thrombin, the binding reaction will cause GNPs to form dimers(or oligomers), which leads to a significant increase in the characteristic diffusion time of GNPs in the detection volume. FCS method can be used to sensitively detect the change in the characteristic diffusion time of GNPs before and after the binding reaction. The quantitative analysis is based on the relation between the change in the characteristic diffusion time of GNPs and the concentration of thrombin. In the optimal conditions, the linear range of this assay is from 0.5 nM to 110 nM and the detection limit is 0.16 nM for thrombin. This new method was successfully applied for direct determination of thrombin levels in human plasma.3. A new and sensitive method for determination of mercury ions(Hg2+) was developed on the basis of FCS and recognition of oligonucleotides. In this assay, 30 nm GNPs as fluorescent probes were modified with oligonucleotides containing thymine bases(T), and principle of this assay is based on the specific binding of Hg2+ by two DNA thymine bases. When two GNPs labeled with different oligonucleotides are mixed in a sample containing Hg2+, the T-Hg2+-T binding reaction will cause GNPs to form dimers(or oligomers), which leads to a significant increase in the characteristic diffusion time of GNPs in the detection volume. FCS method can sensitively detect the change in the characteristic diffusion time of GNPs before and after binding reactions. The quantitative analysis was preformed according to the relation between the change in the characteristic diffusion time of GNPs and the concentration of Hg2+. In the optimal condition, the linear range of this method is from 0.3 nM to 100 nM, and the detection limit is 0.14 nM for Hg2+. This new method was successfully applied for direct determination of Hg2+ levels in water and cosmetics samples.4. A single molecule detecting and imaging system was established by coupling of fluorescence correlation spectroscopy to confocal laser scanning microscopy(FCS-CLSM). The new system was based on the FCS system, and the objective lens was located on a piezoelectric ceramic three-dimensional scanning system. This FCS-CLSM system was optimized, and was successfully used for cell imaging and FCS measurements. The results demonstrated that FCS-CLSM system is characterized with high sensitivity, good stability and reproducibility. Furthermore, CdSeTeS QDs were synthesized and modified to Erbitux, then were incubated with SiHa cells and detected by the FCS-CLSM system for three-dimensional imaging and FCS analysis. The autocorrelation functions were well fitted, and the obtained diffusion coefficients of QDs modified to Erbitux were from 0.012 μm2/s to 0.097 μm2/s on the cell membrane.