Abstract:A series of near-JR (NIR) fluorescent dyes have been prepared which contain an intramolecular heavy atom for altering the photophysics to produce a set of probes appropriate for single lane DNA sequencing applications. The identification of the terminal nucleotide base will be affected by temporal discrimination using fluorescence lifetime determination. The heavy-atom modification consists of an intramolecular halogen (mono-or disubstituted) situated on a remote section of the chromophore in order to minimize… Show more
“…Long wavelength excitation and emission are valuable because the background autofluorescence from biological samples and optical components typically decreases at longer incident wavelengths. Red and NIR probes are now routinely used in protein labeling (2,3), chromatography (4,5), measurements in blood (6,7), noninvasive medical testing (8 -10), and DNA analysis (11)(12)(13)). An advantage of the presently available red-NIR fluorophores is their high extinction coefficient, which in turn allows high sensitivity detection.…”
We describe an approach to creating a new class of luminophores which display both long wavelength emissions exceeding 600 nm and long lifetimes. These luminophores are based on resonance energy transfer (RET) from a long lifetime donor to a short lifetime but long wavelength acceptor. We demonstrated the possibility of obtaining these desirable spectral properties using donors and acceptors noncovalently bound to DNA. The donor was a ruthenium (Ru) metal-ligand complex in which one of the diimine ligands intercalated into double-helix DNA. The acceptors were either nile blue, TOTO-3, or TO-PRO-3. Upon binding of the acceptor to donor-labeled DNA, we found that the acceptor quantum yield was remarkably enhanced so that the wavelength-integrated intensities of the donor and acceptor bound to DNA were many-fold greater than the intensity of the donor and acceptor alone when separately bound to DNA. The origin of this effect is efficient energy transfer from the donor. Under these conditions the effective overall quantum yield approaches that of the acceptor. Importantly, the increased quantum yield can be obtained while maintaining usefully long apparent acceptor lifetimes of 30 to 80 ns. The effect of an increased quantum yield from a low quantum yield donor may find use in assays to detect macromolecular binding interactions. These results suggest the synthesis of covalently linked donor-acceptor pairs with the desirable spectral properties of long wavelength emission, high quantum yield, and moderately long lifetimes for gated detection.
“…Long wavelength excitation and emission are valuable because the background autofluorescence from biological samples and optical components typically decreases at longer incident wavelengths. Red and NIR probes are now routinely used in protein labeling (2,3), chromatography (4,5), measurements in blood (6,7), noninvasive medical testing (8 -10), and DNA analysis (11)(12)(13)). An advantage of the presently available red-NIR fluorophores is their high extinction coefficient, which in turn allows high sensitivity detection.…”
We describe an approach to creating a new class of luminophores which display both long wavelength emissions exceeding 600 nm and long lifetimes. These luminophores are based on resonance energy transfer (RET) from a long lifetime donor to a short lifetime but long wavelength acceptor. We demonstrated the possibility of obtaining these desirable spectral properties using donors and acceptors noncovalently bound to DNA. The donor was a ruthenium (Ru) metal-ligand complex in which one of the diimine ligands intercalated into double-helix DNA. The acceptors were either nile blue, TOTO-3, or TO-PRO-3. Upon binding of the acceptor to donor-labeled DNA, we found that the acceptor quantum yield was remarkably enhanced so that the wavelength-integrated intensities of the donor and acceptor bound to DNA were many-fold greater than the intensity of the donor and acceptor alone when separately bound to DNA. The origin of this effect is efficient energy transfer from the donor. Under these conditions the effective overall quantum yield approaches that of the acceptor. Importantly, the increased quantum yield can be obtained while maintaining usefully long apparent acceptor lifetimes of 30 to 80 ns. The effect of an increased quantum yield from a low quantum yield donor may find use in assays to detect macromolecular binding interactions. These results suggest the synthesis of covalently linked donor-acceptor pairs with the desirable spectral properties of long wavelength emission, high quantum yield, and moderately long lifetimes for gated detection.
“…Dyes of this class have been extensively characterized for use as long-wavelength probes and in DNA sequencing. 52 The dye Thiazole Orange can be excited at 735 nm and binds strongly to DNA.53 Dyes of this type are also used for staining DNA restriction fragments during capillary electrophoresis.…”
Section: Red and Near-infrared (Nir) Dyesmentioning
FluorophoresFluorescence probes represent the most important area of fluorescence spectroscopy. One can spend a great deal of time describing the instrumentation for fluorescence spectroscopy, including light sources, monochromators, lasers, and detectors. However, in the final analysis, the wavelength and time resolution required of the instruments are determined by the spectral properties of the fluorophores. Furthermore, the information available from the experiments is determined by the properties of the probes. Only probes with nonzero anisotropies can be used to measure rotational diffusion, and the lifetime of the fluorophore must be comparable to the correlation time of interest. Only probes which are sensitive to pH can be used to measure pH. And only probes with reasonably long excitation and emission wavelengths can be used with tissues which display autofluorescence at short excitation wavelengths.Thousands of fluorescent probes are known, and it is not practical to describe them all. This chapter contains an overview of the various types of fluorophores, their spectral properties, and their applications. Fluorophores can be broadly divided into two main classes, intrinsic and extrinsic. Intrinsic fluorophores are those which occur naturally. These include the aromatic amino acids, NADH, flavins, and derivatives of pyridoxal and chlorophyll. Extrinsic fluorophores are added to the sample to provide fluorescence when none exists or to change the spectral properties of the sample. Extrinsic fluorophores include dansyl chloride, fluorescein, rhodamine, and numerous other substances.
3.1.A. Fluorescent Enzyme CofactorsEnzyme cofactors frequently are fluorescent (Figure 3.1). NADH is highly fluorescent, with absorption and emission maxima at 340 and 460 nm, respectively (Figure 3.3). The oxidized form of NADH, NAD+, is nonfluorescent. The lifetime of NADH in aqueous buffer is near 0.4 ns. The 63 J. R. Lakowicz, Principles of Fluorescence Spectroscopy
“…Also, it should not readily undergo radiationless energy transfer with the absorbing indicator because the concentration of the latter used in this study was well below that required for energy transfer in unlinked donor−acceptor pairs. , Furthermore, the roughly 5-ns fluorescence lifetime of rhodamine 640 is shorter than the pulse width of the laser source, so it does not degrade spatial resolution appreciably . If required, an alternative fluorophore with a subnanosecond fluorescence lifetime could be used with an emission almost anywhere in the range from the near-ultraviolet to the near-infrared. , 4 Absorption and fluorescence spectra of indicators used for experimental verification of the absorption-modulated fluorescence effect and for enhancement of the back-propagated signal in OTOF distributed sensors: (A) Absorption band of ammonia-sensitive phenol red (acid form, in absence of analyte) immobilized into silicone cladding of PCS optical fiber; (B) analyte-induced hyperchromic effect and bathochromic shift of the absorption band of immobilized phenol red; (C) absorption spectrum of rhodamine 640 (overlaps with the analyte-induced absorption band of phenol red); and (D) fluorescence spectrum of rhodamine 640. The small peak at 532 nm is scattered laser light.…”
A continuous chemically sensitive optical fiber is used with optical time-of-flight chemical detection (OTOF-CD) for spatially resolved analyte mapping. To enhance signal levels and to improve their reproducibility, two novel principles for signal generation and processing are introduced. In the first, the fluorescene of an analyte-insensitive fluorophore is monitored as a function of the evanescent wave absorption of an analyte-sensitive indicator. The resulting signal levels are well above those encountered in optical time domain reflectometry methods that rely upon backscattering for spatially resolved detection. As a result, the method could significantly expand the range of species that can be detected with absorption reagents used in OTOF sensors. The second method raises signal-to-noise ratios by 3-4.5-fold for measurements made at the far ends of the sensing fiber. It functions by sending probe laser pulses into and monitoring their return sequentially from both ends of the sensing fiber. Because the two pulses provide complementary information, only the first half of each of the collected waveforms is used for analyte quantitation. The introduced concepts were experimentally verified with a distributed sensor constructed from a 40-m-long continuous chemically sensitive optical fiber. This sensing element was produced by immobilization of an ammonia-sensitive absorbing reagent (phenol red) and an analyte-insensitive fluorophore (rhodamine 640) into the original silicone cladding of the plastic-clad silica fiber.
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