Single Molecule Spectral Analysis Of Gold Nanoparticles And Gold Nanorods Using Dark Field Microscopy

Localized Surface Plasmon Resonance can occur at the surface of metal nanoparticles, which exhibits some special light absorbance and scattering properties. For example, the resonance light-scattering spectrum of a metal nanoparticle depends on its size and shape and the refractive index of the surrounding medium, which can be used in developing new sensing techniques. Gold nanoparticles were widely used in bio-molecular recognition and detection because it is simple to synthesize, easy to modify, and have better stability and biocompatibility compared to other metal nanoparticles. Single metal particle LSPR study usually needs to use a dark field microscopy plus a spectrograph/CCD combination. While single nanoparticle spectra with high spectral resolution can be obtained with this arrangement, only the spectrum of immobilized nanoparticles can be acquired, and the spectral change of nanoparticles in motion can not be tracked. To address this problem, we developed a new setup by taking advantage of the optical property of the transmission grating, which allowed us to aquire both the realtime darkfield image of nanoparticles in motion and their dynamic LSPR spectra with high throughput. This thesis includes the following three sections:(1) Experimental evidence of single nanoparticles sensitivity of the dark field microscopyIn order to investigate the sensitivity of the dark field microscopy and to find out whether we can observe single 18 nm gold nanoparticle or not, we perfomed a concentration gradient analysis. Similar experiments were also perfomed using fluorescent polystyrene nanospheres. After having identified and optimized factors that affect the signal-to-noise ratio and the distribution of gold nanoparticles, good linear relationship was obtained between the count of nanoparticles and the concentration of its solution. We concluded that single 18 nm nanoparticle on amino-silane modified glass substrates can only be observed with very long exposure time of the CCD camera using our dark field microscopy under current experimental conditions.(2) Real-time spectral analysis of gold nanoparticles using dark field microscopyBy simply insert a transmission grating the certain distance before the CCD camera, the image of an individual gold nanoparticle shows a zero-order spot and a first-order stripe, and its LSPR scattering spectrum can be obtained from the position and intensity of the zero-order spot and the first-order stripe. When individual gold nanoparticles in free solution got absorbed onto APTMS modified glass substrate, their spectral maximum shifted due to the change of local environment. Our results demonstrate that our instrument is capable to track the spectral change of multiple nanoparticles in motion.(3) Single particle spectral analysis of gold nanorods using dark field microscopyGold nanospheres always present in gold nanorod solution. However, traditional darkfield microscopy can not differentiate because only intensity information is obtained. Using the darkfield spectral imaging system developed, we successfully separated individual gold nanospheres from individual gold nanorods based on their LSPR spectral difference. This proved that our technique can be potentially applied as a new detector for chromatography.