Study Of Adsorption Behavior On Surfaces At The Single Molecules Level

Before the development of single molecule detection (SMD) methods, most of the studies of molecules are based on an ensemble average, that is, to detect the average responses and average values in homogeneous or inhomogeneous sample. The average effects cover up the special individual information, however, SMD can study single molecules to reveal heterogeneities of the system by a way of analysising physical parameters as functions of time to get the distribution of molecular specific characteristics or the process of changes. SMD offers an effective means for scientific research fields such as molecular biology, chemistry, medicine and nano-materials and so on. Researches on single-molecule behaviors on the surfaces have a great significance on analysis of the nature of surfaces and molecules, molecular structure and conformation, kinetics of adsorption/desorption and lateral diffusion.In this paper, we used a standard fluorescence microscopy to study the single-molecule behavior in solid-liquid interface, including: the data statistical methods to quantitative and qualitative analysis at the single-molecule level were built up; the influence of different conditions on protein molecular adsorption/desorption behavior was compared; the interaction between quantum dots encapsulated into liposomes withλ-DNA was observed. The details are as follows:1. The existence of noise causes signal fluctuates in a certain range, leading to errors in statistical process, and it is significant in quantitative analysis. Both fluorescence signal intensity and background change with the incident light intensity, but the differences in the trends of their variations give rise to different signal-noise ratio, signal-background ratio. Herein, we used a fixed incident light intensity, chose the most appropriate exposure time to obtain the maximum signal-noise ratio and signal-background ratio; we ensured that the parameter settings are optimized in process of data acquisition and reduce the interference of error. We determined the degree of labeling, it implies that single-step fluorescence bleaching curve means the fluorescence dot was single molecule, and it provides the precondition for the next step to identify a single molecule; we conducted linear relationship between counts and concentration, the single-step photobleaching and the specificity of fluorescence to verify the dots we observed was single-molecule. In order to ensure the stability and reproducibility of data, we developed a data -processing procedure of average values from repeated measurements as more as we can and the frequency of statistical distributions to reduce the error.2. Bovine serum albumin (BSA) and lysozyme were labled with Alexa Fluor. 594 dye before observation, after that we counted the fluorescence points observed by fluorescence microscopy. A comparison of adsorption counts between "soft proteins" such as BSA on glass and PDMS (polydimethylsiloxane) at pH 3.91, 4.89, 6.78 and "hard protein" such as lysozyme on glass and PDMS at pH 4.03, 6.82, 11.05 were conducted respectively. As we all known, properties of glass and PDMS are hydrophilic and hydrophobic individually. Protein structure, distribution of protein net charges and electronic properties of surface show regularities in a certain degree, and the variations of their adsorption counts are closely related to these factors.3. Preliminary studies about liposomes encapsulated with quantum dots 525, 585, 655 influenced on adsorption behavior of YOYO-1-λ-DNA and EthD-2-λ-DNA on glass surface. When pH above 5.5,λ-DNA is in motion, but after adding liposome encapsulated with quantum dots would fixedλ-DNA to the glass surface, and the proportion of DNA interacts with liposomes exists a saturation value theoretically. We also studided the effects of reaction time, adding in organic surfactants or ethanol on their interaction.