Single-Molecule Fluorescence Imaging At Liquid-solid Interfaces

Single molecule detection (SMD) has been applied to study the change of the molecular conformation, dynamics, interactions between molecules and the control of single molecules. The analysis of single molecules can provide different kinds of information at molecular level. Traditional analytical methods just can offer average value of complicated systems and some individual information is enshrouded. However, SMD can obtain individual information under non-equilibrium conditions and the fluctuation of the system under equilibrium conditions. It also can be use to track the process of microcosmic dynamics which is enshrouded. Besides, the molecules do not have to be synchronized during SMD, and some rare intermediates or transitional states can be catched easily.It is very important to study biomolecule behaviors at the liquid-solid interface. It always has to investigate the non-specific adsorption of biomolecules in developing biochips and biosensors. So, the detailed dynamics of adsorption and desorption at the liquid-solid interface is vital to designing new biosensors and substrates for chromatography. This thesis investigates single molecule behaviors at the liquid-solid interface using fluorescence imaging technology, which contains the following three sections:(1) The powerful imaging processing software Image J was applied to single molecule fluorescence images analysis. The correct use of Image J is very important, because it affects the reliability of data interpretation. For DNA molecules, when the number of DNA molecules is small, it is not a complicated thing to analyze these DNA molecules manually. However, if there are lots of DNA molecules, it is hard to analyze all of them accurately and quickly. So, it is very important to study how to gain credible and high throughput data analysis results using special software such as Image J. According to our study, the average pixel size of DNA, the radius of rolling ball and the threshold value are the most important parameters for single particle analysis using Image J. Based on the characteristics of fluorescence images of single DNA molecule obtained experimentally, optimum parameters were found for background deduction, adsorbed and free single DNA molecules counting, single DNA molecule dynamics analysis, single molecule fluorescence intensity measurement and so on.(2) Glass surfaces modified with (3-mercaptopropyl)trimethoxysilane (3-MPTS), n-Octyltriethoxysilane (OTES) and 1H, 1H, 2H, 2H-Perfluorooctyltriethoxysilane (PFOTES) functional groups through silanization were studied using objective-type total internal refelction microscopy (TIRFM) with YOYO-1 labeled singleλ-DNA molecule as a probe. The three silanization-modified glass surfaces were also characterized using contact angle (CA) measurement. For glass surfaces modifed with 3-MPTS and OTES, SMD results show that allλ-DNA molecules were randomly diffusing and freely moving at pH 8.20. As pH decreased, the DNA molecules were gradually dragged and adsorbed onto the glass surfaces. For glass surface modified with PFOTES, however, several DNA molecules adsorbed at the surface at pH8.20. As pH decreased, the fractions of DNA molecules adsorbed at glass surfaces increased. According to the results of CA measurements, the glass surfaces modified with 3-MPTS and OTES were hydrophilic, but the surfaces modified with PFOTES were hydrophilic. At the same pH, the fractions of DNA molecules adsorbed at three glass surfaces follow the order: 3-MPTS﹤OTES﹤PFOTES. This is consistent with the hydrophobicity difference of the three silanization-modified glass surfaces. These results show clearly that the difference of DNA molecule behaviors near chemically modified surfaces mainly result from hydrophobic interactions.(3) Silica nanoparticle films were characterized using objective-type total internal refelction microscopy (TIRFM) with YOYO-1 labeled singleλ-DNA molecule as a probe, and the results were compared with CA and atomic force microscope (AFM) measurements. SMD results show that allλ-DNA molecules were randomly diffusing and freely moving on the silica nanoparticle film surface at pH 8.20. As pH decreased, the DNA molecules were gradually dragged and adsorbed onto the silica nanoparticle film surface. The kinetics of adsorption/desorption of individual DNA molecules was examined to elucidate the contributions of hydrophobic interaction and electrostatic repulsion, and the driving forces were found to be mainly hydrophobic interaction. However, at the same pH, the fraction of DNA molecules adsorbed on the silica nanoparticle film surface is much bigger than that on the glass surface. It is because that the total surface area of the silica nanoparticle film is larger than the bare glass surface and the silica nanoparticle has more active sites for DNA molecule adsorption/desorption.