Fluorescence Labeling And Fluorescence Confocal Imaging Of Hydrogen-bonding Assembly Film

In this paper, the widely used water-soluble polymer of polyvinyl pyrrolidone (PVPON) and polyacrylic acid (PAA) were used to fabricate the hydrogen-bonded complex film. Amino fluorescein dyes and rhodamine dyes were used to stain the film chemicaly and physicaly, respectively. And then, the confocal laser scanning microscopy was used to study the morphology and structural changes of the films after annealing in different vapour environment. The main findings are as follows:1. The widely used water-soluble polymer polyvinylpyrrolidone (PVPON) and polyacrylic acid (PAA) were used as building unit, the quartz or silicon substrate as the carrier to prepare the layer-by-layer hydrogen-bonded (PVPON/PAA)n multilayer film.2. Firstly, the amino fluorescein reacted with acrylic acid to prepare of the fluorescent monomer AA-FAM, and then AA-FAM was copolymerized with acrylic acid at a ratio of1:100to prepare the fluorescence-labeled PAA-FAM. Finally, PAA-FAM and PVPON were used to fabricate the chemically stained hydrogen bonding LBL assembly (PVPON/PAA-FAM)n films.3. The physical staining process of rhodamine dye on hydrogen-bonded composite film was studied detailly. The effects of a range of different environmental conditions on the film, such as the different dyes, pH, temperature, concentration, different solvents and so on. The results show that:a) For the vrious rhodamine dyes series, the dyeing capacity of Rhodamine B on the hydrogen-bonded PVPON/PAA film is better than Rhodamine6G and R110.b) When RB was adsorbed into the film, its maximum absorption peaks and fluorescence excitation and emission peaks occurred red shift, and its fluorescence intensity in the film was significantly higher than that in the bulk solution. These results shows when RB is adsorbed into the film, it occurs interaction with the inside polymer chain of the film and the adsorption concentration of small RB dye molecules in the film is much higher than its bulk solution.c) The thin film has a certain pH dependence adsorption of RB dye molecules. At higher pH values, the ability of films of adsorbing the dye will be stronger and the adsorption rate will be faster,too. And at a pH between2.0to3.0, the interaction between the RB dye molecule and the hydrogen PVPON/PAA film enhanced, there is a sharp increase process of the adsorption concentration. Similarly, the higher pH, the less RB dyes were desorbed from the film, the desorption rate is slower, the fixing ability of the dye in the film molecules is stronger.d) The thin film has a certain temperature-dependent adsorption of RB dye molecules. As the temperature increases, the adsorption concentration of the film on the dye decreased gradually. Further more, at certain temperature, the adsorption ability of the film on the RB dye molecules is certain.e) Although the concentration of the dye adsorbed into the film is much higher than the bulk absorption solution. However, the existed form of RB dye molecules in the film are not dispersed in the form of aggregates, but mainly in the form of a single molecule.f) The presence form of Rhodamine B in different solvents is different. Five kinds of different solvents (such as concentrated sulfuric acid, phosphoric acid, acetone, methanol, and DMF) were used to dissolve RB dyes. According to observe the different destruction status on the film, we found that RB dye can be not only dissolved in acetone solution, it can also stain the film successfully in case that it does not destroy the hydrogen-bonded film.4. The hydrogen-bonded PVPON/PAA films were chemically and physically stained by FAM and RB respectively. When FAM was covalently linked to PAA chain, its fluorescent quantum efficiency was greatly enhanced. When RB was adsorbed into the thin films, its absorbance peak and fluorescent emitting peak exhibited red shift, indicating the interaction with polymer chain in the film.The FAM or RB stained films can meet the fluorescence imaging demand of CLSM. The results from CLSM and AFM indicated that after incubation in neutral or basic vapor, the hydrogen-boned PVPON/PAA films would form porous structures and the majority pores should be through the whole film.