Methods and Applications in Fluorescence, Dec 13, 2016
An oligopeptide: Lys-Gly-Pro-Arg-Ser-Leu-Ser-Gly-Lys-NH2, cleaved specifically by a matrix metall... more An oligopeptide: Lys-Gly-Pro-Arg-Ser-Leu-Ser-Gly-Lys-NH2, cleaved specifically by a matrix metalloproteinase 9 (MMP-9) at the Ser-Leu bond, was labeled on the ε-NH2 groups of lysine with donor (5, 6 TAMRA) and acceptor (HiLyte647) dye. The donor control was a peptide labeled with 5, 6 TAMRA only on the C-terminal lysine, and the acceptor control was free HiLyte647. Following three products were studied by dissolving in 10% (w/w) poly(vinyl alcohol) and dried on glass slides forming 200 micron films. Absorption spectra of the films show full additivity of donor and acceptor absorptions. A strong Fluorescence Resonance Energy Transfer (FRET) with an efficiency of about 85% was observed in the fluorescence emission and excitation spectra. The lifetime of the donor was shorter and heterogeneous compared with the donor control.
We present fluoroimmunoassays on plain metal coated surfaces (metal mirrors) enhanced by metal na... more We present fluoroimmunoassays on plain metal coated surfaces (metal mirrors) enhanced by metal nanoparticles (silver island films, SIFs). Continuous metal layers (gold, or silver protected with a thin silica layer) were coated with SIFs. The surfaces were characterized by atomic force microscopy (AFM). The immunoassay (model assay for rabbit IgG, or myoglobin immunoassay) was performed on these surfaces using fluorescently labeled antibodies. Our results showed that SIFs alone (on glass surface not coated with metal) enhance the immunoassay signal approximately 3 to 10-fold. Using a thin metal layer deposited on glass as support for SIFs leads to up to 50-fold signal enhancement. We also present a theoretical explanation of localized nanoparticle polaritons
Methods and Applications in Fluorescence, Jan 12, 2016
We measured the Forster resonance energy transfer (FRET) from Uranin (U) donor to Rhodamine 101 (... more We measured the Forster resonance energy transfer (FRET) from Uranin (U) donor to Rhodamine 101 (R101) acceptor in propylene glycol. Steady-state fluorescence measurements show a significant difference between mixed and unmixed fluorophore solutions. In the solution with mixed fluorophores, fluorescence intensity of the U donor decreases and intensity of R101 fluorescence increases. This is visualized as a color change from green to orange. Fluorescence anisotropy of the mixture solution increases in the donor emission wavelength region and decreases in the acceptor emission wavelengths; which is consistent with FRET occurrence. Time-resolved (lifetime) measurements show a decrease of the U lifetime in the presence of R101 acceptor. In the intensity decay of R101 acceptor appears a negative component indicating excited state process. All these measurements prove the presence of FRET in U/R101 mixture fluorescence.
Methods and Applications in Fluorescence, Nov 17, 2016
A fluorescence lifetime imaging probe with a long lifetime was used in combination with time-gati... more A fluorescence lifetime imaging probe with a long lifetime was used in combination with time-gating for the detection of hyaluronidase using hyaluronic acid as the probe template. This probe was developed by heavily labeling hyaluronic acid with long lifetime azadioxatriangulenium fluorophores (ADOTA). We used this probe to image hyaluronidase produced by DU-145 prostate cancer cells.
Fluorescence intensity changes were investigated theoretically and experimentally using self-asse... more Fluorescence intensity changes were investigated theoretically and experimentally using self-assembled colloidal structures on silver semitransparent mirrors. Using a simplified quasi-static model and finite element method, we demonstrate that near-field interactions of metallic nanostructures with a continuous metallic surface create conditions that produce enormously enhanced surface plasmon resonances. The results were used to explain the observed enhancements and determine the optimal conditions for the experiment. The theoretical parts of the studies are supported with reports on detailed emission intensity changes which provided multiple fluorescence hot spots with 2-3 orders of enhancements. We study two kinds of the fluorophores: dye molecules and fluorescent nanospheres characterized with similar spectral emission regions. Using a lifetime-resolved fluorescence/reflection confocal microscopy technique, we find that the largest rate for enhancement (~1000-fold) comes from lo...
Methods and Applications in Fluorescence, Dec 13, 2016
An oligopeptide: Lys-Gly-Pro-Arg-Ser-Leu-Ser-Gly-Lys-NH2, cleaved specifically by a matrix metall... more An oligopeptide: Lys-Gly-Pro-Arg-Ser-Leu-Ser-Gly-Lys-NH2, cleaved specifically by a matrix metalloproteinase 9 (MMP-9) at the Ser-Leu bond, was labeled on the ε-NH2 groups of lysine with donor (5, 6 TAMRA) and acceptor (HiLyte647) dye. The donor control was a peptide labeled with 5, 6 TAMRA only on the C-terminal lysine, and the acceptor control was free HiLyte647. Following three products were studied by dissolving in 10% (w/w) poly(vinyl alcohol) and dried on glass slides forming 200 micron films. Absorption spectra of the films show full additivity of donor and acceptor absorptions. A strong Fluorescence Resonance Energy Transfer (FRET) with an efficiency of about 85% was observed in the fluorescence emission and excitation spectra. The lifetime of the donor was shorter and heterogeneous compared with the donor control.
We present fluoroimmunoassays on plain metal coated surfaces (metal mirrors) enhanced by metal na... more We present fluoroimmunoassays on plain metal coated surfaces (metal mirrors) enhanced by metal nanoparticles (silver island films, SIFs). Continuous metal layers (gold, or silver protected with a thin silica layer) were coated with SIFs. The surfaces were characterized by atomic force microscopy (AFM). The immunoassay (model assay for rabbit IgG, or myoglobin immunoassay) was performed on these surfaces using fluorescently labeled antibodies. Our results showed that SIFs alone (on glass surface not coated with metal) enhance the immunoassay signal approximately 3 to 10-fold. Using a thin metal layer deposited on glass as support for SIFs leads to up to 50-fold signal enhancement. We also present a theoretical explanation of localized nanoparticle polaritons
Methods and Applications in Fluorescence, Jan 12, 2016
We measured the Forster resonance energy transfer (FRET) from Uranin (U) donor to Rhodamine 101 (... more We measured the Forster resonance energy transfer (FRET) from Uranin (U) donor to Rhodamine 101 (R101) acceptor in propylene glycol. Steady-state fluorescence measurements show a significant difference between mixed and unmixed fluorophore solutions. In the solution with mixed fluorophores, fluorescence intensity of the U donor decreases and intensity of R101 fluorescence increases. This is visualized as a color change from green to orange. Fluorescence anisotropy of the mixture solution increases in the donor emission wavelength region and decreases in the acceptor emission wavelengths; which is consistent with FRET occurrence. Time-resolved (lifetime) measurements show a decrease of the U lifetime in the presence of R101 acceptor. In the intensity decay of R101 acceptor appears a negative component indicating excited state process. All these measurements prove the presence of FRET in U/R101 mixture fluorescence.
Methods and Applications in Fluorescence, Nov 17, 2016
A fluorescence lifetime imaging probe with a long lifetime was used in combination with time-gati... more A fluorescence lifetime imaging probe with a long lifetime was used in combination with time-gating for the detection of hyaluronidase using hyaluronic acid as the probe template. This probe was developed by heavily labeling hyaluronic acid with long lifetime azadioxatriangulenium fluorophores (ADOTA). We used this probe to image hyaluronidase produced by DU-145 prostate cancer cells.
Fluorescence intensity changes were investigated theoretically and experimentally using self-asse... more Fluorescence intensity changes were investigated theoretically and experimentally using self-assembled colloidal structures on silver semitransparent mirrors. Using a simplified quasi-static model and finite element method, we demonstrate that near-field interactions of metallic nanostructures with a continuous metallic surface create conditions that produce enormously enhanced surface plasmon resonances. The results were used to explain the observed enhancements and determine the optimal conditions for the experiment. The theoretical parts of the studies are supported with reports on detailed emission intensity changes which provided multiple fluorescence hot spots with 2-3 orders of enhancements. We study two kinds of the fluorophores: dye molecules and fluorescent nanospheres characterized with similar spectral emission regions. Using a lifetime-resolved fluorescence/reflection confocal microscopy technique, we find that the largest rate for enhancement (~1000-fold) comes from lo...
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