The high blood glucose levels associated with diabetes affect various cells and proteins in the body. In response to high blood glucose collagen and keratin proteins experience glycation. This work aims to establish if the intrinsic fluorescence of collagen and keratin could be used to monitor the glycation of said compounds, and thus offer an alternative method to monitoring long term glycaemic control. We have studied the evolution of the intrinsic fluorescence of both compounds in response to glucose in vitro, using steady state and time-resolved fluorescence spectroscopy techniques. Changes in the intrinsic fluorescence of both collagen and keratin were observed. For collagen, contrary to the traditional fluorescence intensity decay measurement at arbitrarily selected excitation and detection wavelengths, we conducted systematic wavelength- and time-resolved measurements to achieve time-resolved emission spectra (TRES). These showed changes in the intrinsic fluorescence kinetics, caused by both collagen aggregation and glycation. In keratin, the addition of glucose caused an increase in the fluorescence intensity at the characteristic wavelength of 460 nm, due to faster formation of new cross-links. The results also suggest that glucose may cause the formation of two new fluorescent complexes with peak fluorescence at ~525 nm and ~575 nm. In conclusion, monitoring the intrinsic fluorescence of collagen or keratin could be used as a method to monitor long term glycaemic control in patients with diabetes.
KEYWORDS: Luminescence, Absorption, Fluorescence resonance energy transfer, Proteins, Molecules, Time resolved spectroscopy, Fluorescence spectroscopy, Spectroscopy, Physics, Energy transfer
Human serum albumin (HSA) complexation with quercetin, a flavonoid commonly present in human diet, was monitored by means of fluorescence decays of the single HSA tryptophan - Trp214. Data analysis based on fitting to multiexponential functions and determining the lifetime distributions revealed a high sensitivity of tryptophan fluorescence to binding quercetin. Results are discussed in terms of the rotamer model for tryptophan, HSA-quercetin complexation and potential HSA to quercetin energy transfer. Evidence for quercetin stabilising tryptophan rotamers in HSA is presented.
KEYWORDS: Fluorescence resonance energy transfer, Luminescence, Systems modeling, Molecules, Cobalt, Ions, Time resolved spectroscopy, Microelectromechanical systems, Molecular energy transfer, Spectroscopy
We demonstrate a new fluorescence resonance energy transfer (FRET) based approach to determine the donor-acceptor
distributions and apply it to two model molecular systems: double stranded DNA labeled with Hoechst 33258 and FAM, and
perylene randomly surrounded by cobalt ions in a bulk solution. The approach makes some generic assumptions regarding
the FRET kinetics, but no a priori assumptions regarding the distribution function.
Fluorescence nanotomography (FN) is a newly developed method for determining molecular distributions on a nanometre scale in soft solids, biological macromolecules and medically important systems. FN uses fluorescence resonance energy transfer (FRET) for the recognition of the separations between molecules. By using a fluorescence lifetime measurement of sub-nanosecond time resolution, the spatial resolution of the resulting distribution function can be better than 1 Å. In this paper the theoretical background of the method is outlined and the results of simulations on model molecular distributions presented. This is followed by demonstration of several applications of FN to real molecular systems, including bulk solutions of molecules of different sizes, complexes, porous polymers, phospholipids and sugar-protein competitive binding sensors glucose. The experimental requirements of FN as a structural tool for wide class of biomedical systems are discussed.
Fluorescence resonance energy transfer (FRET) from donor to acceptor molecules is one of the most powerful techniques for monitoring structure and dynamics. This is because FRET has a strong spatial dependence with angstroms resolution. This dependence includes the simplest case of a random distribution of acceptors for which an analytical solution exists for the fluorescence impulse response I(t). However, in general the acceptor distribution function p(r) is not random and a unique solution cannot be found for I(t). In many important applications of FRET eg in proteins, the simple random treatment is quite inappropriate and yet the information concerning conformation changes is preserved in p(r). One approach, which as been applied to the problem of determining p(r), is to make some assumptions as to its form eg Gaussian and then try to use this to describe I(t).
KEYWORDS: Glucose, Luminescence, Molecules, Fluorescence resonance energy transfer, Magnesium, Sensors, Absorption, Energy transfer, Molecular energy transfer, Sensing systems
A new glucose sensing system based on near infra-red fluorescence resonance energy transfer (FRET) from CocanavalinA-allophycocyanin to dextran labelled malachite green is demonstrated. Single-photon timing fluorescence lifetime measurements have enabled us to investigate and understand the quenching kinetics in terms of the dimensionality of energy transfer.
We have developed a fluorescence lifetime based energy transfer sensor for detecting copper(II) ions. Rhodamine 800 in Nafion selectively transfers its energy to copper ions when excited at 670 nm. By fitting to the fluorescence decay we can resolve copper concentrations in water down to the level of 10 ppb. Time-correlated single-photon counting is used for detection. Good discrimination against interference by cobalt, nickel and chromium ions is obtained by virtue of the reduced spectral overlap of these ions as compared to copper. Possibilities for an energy transfer sensor for both copper(I) and copper(II) ions are discussed. Application of the theory of resonance energy transfer is investigated for use in sensor matrices using a range of donors and acceptors and the appropriateness of current models discussed.
Fluorescence sensors for transition metal ions are demonstrated using the principle of Forster energy transfer from a fluorophore embedded in a Nafion polymer to a metal ion in aqueous solution. The resonance requirement for energy transfer and hence specificity will be demonstrated for cobalt, copper and uranyl ions. Diffusion of these ions in the polymer matrix was also determined with, a diffusion coefficient for Co2+ in Nafion of 4.91 X 10-7 cm2s-1 and for Cu2+ 7.72 X 10-7 cm2s-1.
The fluorescence quenching of molecules by analytes of interest, is a widely employed phenomenon in fluorescence sensing technology. Forster type dipole-dipole energy transfer from dye molecules to transition metal ions, provides a method of monitoring the concentration of these ions with some degree of selectivity. Each metal ion has a different absorption spectrum, hence, in principle it is possible to choose different fluorophores for each metal ion. In the present work, quenching studies of the carbocyanine dye DTDCI by transition metal ions in a viscous solvent and a Nafion polymer matrices are reported. The potential for fabricating near-infrared energy transfer sensors is assessed, particularly with regard to detecting copper ions in solution.
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