Specific binding of biotinilated bovine serum albumin (bBSA) and tetramethylrhodamine-labeled streptavidin (SAv−TMR) was observed by conjugating bBSA to CdSe−ZnS core−shell quantum dots (QDs) and observing enhanced TMR fluorescence caused by fluorescence resonance energy transfer (FRET) from the QD donors to the TMR acceptors. Because of the broad absorption spectrum of the QDs, efficient donor excitation could occur at a wavelength that was well resolved from the absorption spectrum of the acceptor, thereby minimizing direct acceptor excitation. Appreciable overlap of the donor emission and acceptor absorption spectra was achieved by size-tuning the QD emission spectrum into resonance with the acceptor absorption spectrum, and cross-talk between the donor and acceptor emission was minimized because of the narrow, symmetrically shaped QD emission spectrum. Evidence for an additional, nonspecific QD−TMR energy transfer mechanism that caused quenching of the QD emission without a corresponding TMR fluorescence enhancement was also observed.Fluorescence resonance energy transfer (FRET) is a process whereby the electronic excitation energy of a donor chromophore is nonradiatively transferred to a nearby acceptor molecule via a through-space dipole-dipole interaction between the donor-acceptor pair. 1-5 FRET occurs when there is appreciable overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor. The strong distance dependence of the FRET efficiency has been widely exploited in studying the structure and dynamics of proteins and nucleic acids, in the detection and visualization of intermolecular association, and in the development of intermolecular binding assays. 6 FRET-based studies involving pairs of organic dye molecules as the donoracceptor complexes are often limited by cross-talk caused by spectral overlap of the donor and acceptor emission. The need for significant overlap between the emission and absorption spectra of the donor and acceptor, coupled with the narrow absorption spectrum of conventional organic dye molecules, makes it difficult to avoid direct excitation of the acceptor molecules at the excitation wavelength needed to efficiently excite the donor. In addition, the broad emission spectrum of the donor, with its long red tail, can often overlap significantly with the emission spectrum of the acceptor. Several recent reports have confirmed that luminescent semiconductor quantum dots (QDs), such as CdSe and CdTe, are able to participate in resonance energy transfer processes analogous to FRET, 7-10 which makes these materials good candidates to overcome some of the limitations associated with conventional organic dye molecules in FRET-based studies of biomolecular structure, ligand-receptor binding, etc.Semiconductor QDs are currently being investigated for their use as luminescent biological probes because of their high photostability relative to organic dye molecules and their unique, size tunable spectral properties. 11-14 QDs possessing high luminescence qu...
Individual fluorescent polystyrene nanospheres (<10-100-nm diameter) and individual fluorescently labeled DNA molecules were dispersed on mica and analyzed using time-resolved fluorescence spectroscopy and atomic force microscopy (AFM). Spatial correlation of the fluorescence and AFM measurements was accomplished by (1) positioning a single fluorescent particle into the near diffraction-limited confocal excitation region of the optical microscope, (2) recording the time-resolved fluorescence emission, and (3) measuring the intensity of the excitation laser light scattered from the apex of an AFM probe tip and the AFM topography as a function of the lateral position of the tip relative to the sample substrate. The latter measurements resulted in concurrent high-resolution (approximately 10-20 nm laterally) images of the laser excitation profile of the confocal microscope and the topography of the sample. Superposition of these optical and topographical images enabled unambiguous identification of the sample topography residing within the excitation region of the optical microscope, facilitating the identification and structural characterization of the nanoparticle(s) or biomolecule(s) responsible for the fluorescence signal observed in step 2. These measurements also provided the lateral position of the particles relative to the laser excitation profile and the surrounding topography with nanometer-scale precision and the relationship between the spectroscopic and structural properties of the particles. Extension of these methods to the study of other types of nanostructured materials is discussed.
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