Development of single molecule methods and synthesis of fluorescent probes for bioanalytical applications



Journal Title

Journal ISSN

Volume Title



Single molecule detection methods had enabled biochemical analyses to be conducted with high sensitivity and temporal resolution. One of the applications is the study of apoptosis. In this work, the detection of apoptosis was studied by fluorescence correlation spectroscopy (FCS) in living cells. Caspase activity was assayed using a new red fluorogenic probe, 2SBPO-Casp, that can avoid the spectral overlap of green fluorescent probes and cell autofluorescence. This new probe was synthesized by coupling a water-soluble Nile Blue derivative sulfonated benzo[a]-phenoxazines (2SBPO) to an aspartic acid residue. Upon apoptosis induction and caspase activation, free 2SBPO dye was shown to accumulate inside the cell after probe cleavage. The use of 2SBPO-Casp resulted in a higher signal to noise ratio as the dye emission did not overlap with cell autofluorescence. In another work, two single molecule fluorescence techniques, Fluorescence Correlation Spectroscopy (FCS) and fluorescence Intensity Histograms (IH), were evaluated under high and low background conditions. Working near the single molecule limit, the autofluorescence of cells can obscure changes in analyte concentrations and introduce false positive signals. Under high background conditions, using one parameter alone (whether IH or FCS) was insufficient to distinguish apoptotic cells from controls. To solve this limitation, FCS and IH techniques were used in tandem to reduce false positives after a careful selection of gating parameters. In low background conditions, no false positives were identified, and cells were deemed apoptotic using only one parameter (FCS or IH). Apoptotic cells were identified using two different fluorescent probes, resulting in high or low background conditions. The combined use of FCS and IH was also studied. Combining both methods to identify dye accumulation in cells expanded the dynamic range of probe detection. By improving sensitivity and determining which technique is best suited for different cellular environment conditions, better assays can be developed that lead to new enhanced detection and treatments for diseases. Finally, innovations in fluorescent probes were extended to nanoscale composite materials. A versatile approach for functionalizing core-shell Ag@SiO2 nanoparticles for live-cell imaging was developed. The approach uses physical adsorption during the Stöber process and does not need covalent linkage to synthesize antibody-based labels. Different fluorophores were incorporated into the silica architecture, resulting in fluorescence signal enhancement. Different methods of attaching antibodies to the glass surface were used based on the fluorophore characteristics, including a non-covalent attachment process and covalent linkage. Metal-enhanced nanoparticles doped with fluorophores were used as the luminescent reporter in immunofluorescence labeling. Antibody-conjugated nanoparticles showed fluorescence up to 30-fold enhancement fluorescence compared to control nanobubbles. This simple synthesis protocol combining physical conjugation and covalent linking methods can be applied to a variety of fluorophore types, and has broad potential in bioanalytical and biosensing applications.



Fluorescent probes, Single molecule methods