Fluorescence Correlation Spectroscopy System And Its New Applications On The Characterization Of Fluorescent Nanomaterials

To directly observe distributions and time trajectories of single molecules or single particles and then to analyze, sort, and describe them, are still one of analysts'dreams. In 1970s, in order to measure molecular diffusion and reaction kinetics of biomacromolecules in the highly diluted solution, fluorescence correlation spectroscopy (FCS) technique was introduced. And with the development of confocal microscope technique, optical detection technique and computer science in 1990s, FCS gradually become an advanced single molecule detection technique in the solution. Now it has recently experienced growing popularity in the life science, such as study on the interaction of DNA and protein, diffusion of particles in the inner of cell and on the cell membrane and high throughput screening of new drug (HTS). FCS is an ultrasensitive and noninvasive detection technique that uses statistical analysis of the fluctuations of fluorescence emitted from a small, optically well-defined open volume element. The autocorrelation function can provide us some important information, such as the average number of luminescent particles in the volume and the coefficient of diffusion etc. As a new single molecule detection technique, study on FCS instrument and application is scarce.In this work, based on the principles to construct one single molecule detection system: (1) a small excitation volume, to reduce the background;(2) high-efficiency collection optics;(3) the use of detectors with high quantum efficiency and low dark noise;(4) careful elimination of background fluorescence by various means, a sensitive and stable fluorescence correlation spectroscopy (FCS) setup was developed and used for study on some key problems of fluorescent nanomaterials. The paper contains the following parts:1. One fluorescence correlation spectroscopy system was developed with laser confocal optics as main optical structure, one high numerical aperture water-immersion objective as optical collecting device and a pinhole as space filter. The measurement on the diffusion of organic dyes shows that the laser highly illuminated volume is less than 1fL and ratio of signal to background (S/B) of single dye molecule is greater than 400. The high-sensitive and stable system can be used for C677T point mutation analysis of methylenetetrahydrofolate reductase gene.2. Combined FCS with ensemble molecular spectrometry, a new method was developed to characterize molecular weight, molar extinction coefficient and bright fraction of CdTe QDs synthesized in aqueous solution. The principle is mainly based on the measurements of hydrodynamic diameters of CdTe QDs and the number of bright QDs in a small illuminated volume element using FCS technique. Hydrodynamic diameters of a series of CdTe QDs were measured with FCS and the molecular weights were calculated assuming the measured hydrodynamic diameters as the diameters of QDs. The molar extinction coefficients of QDs at different excitonic absorption peak positions were calculated with the molecular weights. Furthermore, the fitted excitonic absorption peak dependence of extinction coefficients was used to study Ostwald ripening or defocusing of QDs in aqueous synthesis process. The bright fraction of QDs samples were characterized by measuring the concentration of the bright QDs and the total concentration of QDs, and it was observed that the bright fractions of CdTe QDs sample were proportional to quantum yields (QYs) and the ratio of the bright fraction to QY was close to 1. Our methods described herein are simple and universal, and are suitable for characterization of molecular weight, molar extinction coefficient and bright fraction of QDs synthesized in aqueous phase and organic phase. Meanwhile, the methods were used to characterize HRP protein tagged with QDs. It was observed that nearly one quantum dot was conjutated on the surface of each HRP protein.3. A new method was described for the measurement of surface charge of fluorescent particles, including QDs, by coupling FCS with microfluidic chip electrophoresis. This method has been successfully used to determine the surface charge of fluorescein and CdTe QDs. The measured charges of fluorescein in pH 7.4 and pH 8.4 buffers are 1.1±0.5 and 1.5±0.4, respectively. The surface charge of MPA-stabilized CdTe QDs in the TB buffer of pH 8.4, 9.3 and 10.0 was 6.2±0.4, 6.3±0.8, and 7.6±0.7. Meanwhile, it was found that the surface charge of QDs was remarkably associated with the type of stabilizers on QDs surface, buffer pH and other factors. After MPA-stabilized CdTe QDs dry powder was dissolved and displaced by reduced glutathione in the 10 mM borate buffer (pH 10.0) containing 10 mM reduced glutathione, surface charge of CdTe QDs changed into 11.8±0.5. Compared to the current methods, this technique is characterized by noninvasiveness, high sensitivity, effectiveness and versatility.4. Combination of FCS with some ensemble techniques was used to investigate the quenching process and mechanism of heavy metal ions (such as Ag+) on CdTe QDs. It is found that when silver ions (Ag+) quench QDs, the free Ag+ ions enter the trap sites, bind with bare Te atoms and form the AgTe structure in the surface, which results in the luminescent quenching of QDs. The FCS experimental results show that the quenching process is not the gradual reduction of fluorescence intensity of single QDs, but the decrease in the number of bright QDs with the addition of Ag+ ions. Furthermore, FCS was used to investigate the effect of dialysis process on CdTe QDs. It is observed that some dark QDs convert into the bright QDs in the dialysis experiments and the dialysis process can improve the brightness per QDs. And the results of FCS and fluorescence spectroscopy illustrate that the increase of the fluorescence quantum yield (QY) is mainly attributed to the removal of excess unreacted Cd-MPA complex and the possible chemical change of the QDs surface in the dialysis process. These new results can help us to further understand the complex surface structure of water-soluble QDs, improve their surface chemical features, and expand their applications in some field.5. The aggregation and the subsequent photo-activation process of MPA-capping CdTe QDs induced by laser were systematically studied with fluorescence correlation spectroscopy (FCS). It was observed that the photo-activation process was closely related with the aggregation of QDs in solution. The brightening of QDs samples was due to laser-induced aggregation and subsequent surface reconstruction. The aggregation process of QDs was dependent on sizes of QDs. The smaller QDs were apt to be aggregated together and be photo-activated by laser. And the higher concentration of salt could reduce and suppress the aggregation significantly, and then affected photo-activation process of QDs. Meanwhile, it was found that photo-activation process was also associated with the concentration of QDs. Only when the concentrations of QDs were greater than the certain concentration (called critical concentration), the photo-activation of QDs happened under laser irradiation.