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ABSTRACT: We review progress in our laboratories toward developing in vivo glucose sensors for diabetes that are based on fluorescence labeling of glucose/galactose-binding protein. Measurement strategies have included both monitoring glucose-induced changes in fluorescence resonance energy transfer and labeling with the environmentally sensitive fluorophore, badan. Measuring fluorescence lifetime rather than intensity has particular potential advantages for in vivo sensing. A prototype fiber-optic-based glucose sensor using this technology is being tested.
Journal of diabetes science and technology 01/2013; 7(1):62-71.
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ABSTRACT: We describe the auto-oxidation of 3, 4-dihydroxy-L-phenylalanine (L-DOPA) in the synthesis of eumelanin to spontaneously produce fibrils upon drying. The self-assembled fibrils are of characteristic diameter ∼ 1 to 2 μm, composed of filaments, and are unidirectional, apart from branches that are formed at typically an angle of 20 to 22 deg. The fibrils are characterized using fluorescence spectroscopy, fluorescence decay times, scanning electron microscopy, atomic force microscopy, and fluorescence lifetime imaging microscopy. The fibrils mimic natural melanin in consisting of core eumelanin with efficient nonradiative properties, but they also display pockets of electronically isolated species with higher radiative rates on the nanosecond timescale. Eumelanin fibrils formed occasionally in solution are tentatively attributed to a scaffold of bacteria or fungus. Fabricating and characterizing novel synthetic eumelanin structures such as fibrils are of interest in helping to reveal a functional structure for eumelanin, in understanding its photophysics, in learning more about L-DOPA as it is used in the treatment of Parkinson’s disease, and in producing novel materials which might embody some of the diverse properties of eumelanin.
Journal of Biomedical Optics 07/2012; 17(7):075001. · 3.16 Impact Factor
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ABSTRACT: We report the use of CdSe/ZnS core/shell quantum dots QDot800 (Invitrogen) as luminescence lifetime sensors for copper ions Cu2+(H2O)8 in solution with a sensitivity of <1 ppb that is relevant to intracellular copper concentrations. Excitation of QDot800 at 485 nm was found to be optimum in that it did not cause any change in the level of luminescence intensity or luminescence lifetime in the absence of copper ions. When excited at 485 nm a bi-exponential luminescence decay of QDot800 was observed suggesting the presence of two distinct emitting states, both capable of undergoing metal ion quenching that facilitates Cu2+ detection. Selectivity for copper, as against other transition metal ions, as well as other evidence, suggests the primary origin of the quenching is luminescence resonance energy transfer to both free and bound copper ions. The luminescence kinetics of quantum dots and their optimization and applicability for resonance energy transfer-based lifetime sensing in general is discussed.
Measurement Science and Technology 04/2012; 23(5):055103. · 1.49 Impact Factor
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ABSTRACT: We have demonstrated energy transfer between 4’-6-Diamidino-2-phenylindole (DAPI), a commonly used DNA label, and gold nanoparticles under two-photon excitation in solution using fluorescence lifetime imaging microscopy (FLIM). With comparable size and concentration, gold nanorods (GNRs) are shown to provide more efficient energy transfer than gold nanospheres (GNSs). We attribute this transfer enhancement effect to the longitudinal surface plasmon mode of GNRs overlapping with the excitation wavelength. Energy transfer under two-photon excitation between GNRs and DAPI has also been observed in cell culture and found to be in accord with the solution phase results.
Applied Physics Letters 09/2011; 99(10):103701-103701-3. · 3.84 Impact Factor
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ABSTRACT: Aggregation of the peptide beta-amyloid is known to be associated with Alzheimer's disease. According to recent findings the most neurotoxic aggregates are the oligomers formed in the initial stages of the aggregation process. Here we use beta-amyloid's (Aβ's) intrinsic fluorophore tyrosine to probe the earliest peptide-to-peptide stages of aggregation, a region often merely labelled as a time lag, because negligible changes are observed by the commonly used probe ThT. Using spectrally resolved fluorescence decay time techniques and analysis we demonstrate how the distribution of 3 rotamer conformations of the single tyrosine in Aβ tracks the aggregation across the time lag and beyond according to the initial peptide concentration. At low Aβ concentrations (≤5 μM), negligible aggregation is observed and this is mirrored by little change in the fluorescence decay parameters, providing a useful baseline for comparison. At higher concentrations (≈50 μM), and contrary to what is generally accepted from ThT studies the rate of aggregation can be described by an exponential growth to a plateau in terms of the relative contributions of two of the three rotamers, with a characteristic aggregation time of ≈33 h.
Physical Chemistry Chemical Physics 03/2011; 13(14):6434-41. · 3.57 Impact Factor
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ABSTRACT: We report changes in the photophysical properties of core-shell type CdSe/ZnS quantum dots (QDs) under optical irradiation. QDs either in aqueous solution or immobilized in a silica sol gel matrix have been excited at different wavelengths and fluxes. Illumination of the sample with 140 fs 700 nm Ti:sapphire laser pulses of the peak power of the order of 4 GW/cm2 caused gradual increase in the luminescence lifetime from an initial value of 3.5 increasing to 4.5 ns and an increase in luminescence intensity by ∼ 8%. Using about 16 GW/cm2 peak power resulted in a shortening of the luminescence lifetime to 3 ns and a decrease in intensity by ∼ 75%. Both photobrightening and photodarkening were fully reversible. We discuss the kinetics of photobrightening and photodarkening and investigate the suitability of QDs as luminescence lifetime sensors with tunable parameters.
Applied Physics Letters 01/2011; 98(2):021108. · 3.84 Impact Factor
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BIODEVICES 2011 - Proceedings of the International Conference on Biomedical Electronics and Devices, Rome, Italy, 26-29 January, 2011; 01/2011
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ABSTRACT: Beta-amyloid (Abeta) aggregation, believed to be responsible for Alzheimer's disease, is monitored using its intrinsic fluorescence decay. Alterations in the fluorescence decay of tyrosine correlate with the Abeta aggregation at a much earlier stage than the traditionally used fluorescence intensity of Thioflavin T (ThT). Potentially the finding may underpin progress towards an earlier diagnosis of the onset of Alzheimer's disease and an improved approach to developing intervention therapies.
Biosensors & bioelectronics 03/2010; 25(10):2249-52. · 5.43 Impact Factor
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ABSTRACT: We present a description of fluorescence decay kinetics in complex environments based on gamma functions rather than the conventional approach using exponentials. The gamma function description is tested in measurements on the temperature dependence of the protein human serum albumin (HSA), N-acetyl tryptophanamide (NATA), and 2, 5-dipenyl oxazole (PPO). The monitoring of macromolecular structure and dynamics is demonstrated by means of distinct tryptophan (Trp) rotamer populations and their interconversion in HSA.
Physical Review E 06/2009; 79(5 Pt 1):050901. · 2.26 Impact Factor
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ABSTRACT: We report the development of biophysical techniques based on circular dichroism (CD), diffuse reflectance infrared Fourier transform (DRIFT) and tryptophan (Trp) fluorescence to investigate in situ the structure of enzymes immobilised on solid particles. Their applicability is demonstrated using subtilisin Carlsberg (SC) immobilised on silica gel and Candida antartica lipase B immobilised on Lewatit VP.OC 1600 (Novozyme 435). SC shows nearly identical secondary structure in solution and in the immobilised state as evident from far UV CD spectra and amide I vibration bands. Increased near UV CD intensity and reduced Trp fluorescence suggest a more rigid tertiary structure on the silica surface. After immobilised SC is inactivated, these techniques reveal: a) almost complete loss of near UV CD signal, suggesting loss of tertiary structure; b) a shift in the amide I vibrational band from 1658 cm(-1) to 1632 cm(-1), indicating a shift from alpha-helical structure to beta-sheet; c) a substantial blue shift and reduced dichroism in the far UV CD, supporting a shift to beta-sheet structure; d) strong increase in Trp fluorescence intensity, which reflects reduced intramolecular quenching with loss of tertiary structure; and e) major change in fluorescence lifetime distribution, confirming a substantial change in Trp environment. DRIFT measurements suggest that pressing KBr discs may perturb protein structure. With the enzyme on organic polymer it was possible to obtain near UV CD spectra free of interference by the carrier material. However, far UV CD, DRIFT and fluorescence measurements showed strong signals from the organic support. In conclusion, the spectroscopic methods described here provide structural information hitherto inaccessible, with their applicability limited by interference from, rather than the particulate nature of, the support material.
ChemPhysChem 05/2009; 10(9-10):1492-9. · 3.41 Impact Factor
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ABSTRACT: By using the fluorescent dye 6-propionyl-2-(N,N-dimethylamino) naphthalene (PRODAN) to monitor methanol generated during tetramethyl orthosilicate polymerization we have
optimised the encapsulation of protein in silica sol–gel monoliths with respect to completion of hydrolysis and distillation
in order to remove methanol such that protein can be added without denaturation. A minimum of 24h at +4°C was found to be
required before hydrolysis is complete and 3–5min of vacuum distillation at 50°C and 300mbar needed to remove methanol
before the gel is formed. The biocompatibility of a tetramethyl orthosilicate sol–gel monolith was demonstrated by preserving
the trimer protein allophycocyanin (APC) in its native form for up to 500h. This obviates the previously essential requirement
of covalently binding the trimer together in order to prevent dissociation into monomers and has enabled observation of native
APC trimer in a sol–gel pore for the first time down to the single molecule level using combined fluorescence spectroscopy
and confocal microscopy. The higher stability afforded by the protocol we describe could impact on the application of sol–gel
materials to single-molecule studies of wider bearing such as protein folding and aggregation.
Journal of Sol-Gel Science and Technology 01/2009; 49(3):380-384. · 1.63 Impact Factor
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ABSTRACT: We demonstrate nanoparticle size measurement using time-resolved fluorescence anisotropy decay in relation to establishing a nanometrology standard. The rotational correlation time equivalent to the isotropic Brownian rotation of a fluorescent 6-methoxyquinolinium dye attached to amorphous silica nanoparticles was determined in three different LUDOX 2 colloids from the complex fluorescence anisotropy decay observed. Once competing depolarization and nanoparticle aggregation had been taken into account, good agreement was found of 4.0 ± 0.4 nm, 6.4 ± 0.5 nm and 11.0 ± 1.6 nm corresponding to the manufacturer's reported particle radii of 3.5 nm, 6 nm and 11 nm, for LUDOX SM30, AM30 and AS40 respectively. We describe the measurement science required for acquisition and interpretation of fluorescence anisotropy decay data in order to determine nanoparticle size while highlighting the limitations and useful range of measurement.
Meas. Sci. Technol. 01/2009; 20:25310-11.
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ABSTRACT: Some fluorescence dyes in complex media, such as those found in biology, demonstrate nonextensive kinetics, which implies representing their fluorescence decays in terms of lifetime distributions rather than simple exponentials. Complex kinetics usually discourage application to lifetime sensors, as it is believed, that additional molecular mechanisms employed for detection of an analyte will make the resulting kinetics ambiguous and the sensor response inconclusive. In this paper we investigate theoretically the applicability of complex dye kinetics as a fluorescence resonance energy transfer based lifetime sensor and demonstrate that the nonextensive nature of its kinetics does not decrease the sensing performance, and indeed even provides richer structural information than a simple exponential behavior.
The Journal of chemical physics 11/2008; 129(14):144507. · 3.09 Impact Factor
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ABSTRACT: Nanomedicine involves measurement and therapy at the level of 1-100 nm. Although the science is still in its infancy, it has major potential applications in diabetes. These include solving needs such as non-invasive glucose monitoring using implanted nanosensors, with key techniques being fluorescence resonance energy transfer (FRET) and fluorescence lifetime sensing, as well as new nano-encapsulation technologies for sensors such as layer-by-layer (LBL) films. The latter might also achieve better insulin delivery in diabetes by both improved islet encapsulation and oral insulin formulations. An 'artificial nanopancreas' could be an alternative closed-loop insulin delivery system. Other applications of nanomedicine include targeted molecular imaging in vivo (e.g. tissue complications) using quantum dots (QDs) or gold nanoparticles, and single-molecule detection for the study of molecular diversity in diabetes pathology.
Diabetes/Metabolism Research and Reviews 10/2008; 24(8):604-10. · 3.37 Impact Factor
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ABSTRACT: Recently, we described the characteristics and application of a 265-nm AlGaN light-emitting diode (LED) operated at 1-MHz repetition rate, 1.2-ns pulse duration, 1.32-microW average power, 2.3-mW peak power, and approximately 12-nm bandwidth. The LED enables the fluorescence decay of weakly emitting phenylalanine to be measured routinely in the condensed phase, even in dilute solution. For a pH range of 1-11, we find evidence for a biexponential rather than a monoexponential decay, whereas at pH 13, only a monoexponential decay is present. These results provide direct evidence for the dominance of two phenylalanine rotamers in solution with a photophysics closer to the other two fluorescent amino acids, tyrosine and tryptophan, than has previously been reported. Although phenylalanine fluorescence is difficult to detect in most proteins because of its low quantum yield and resonance energy transfer from phenylalanine to tyrosine and tryptophan, the convenience of the 265-nm LED may well take protein photophysics in new directions, for example, by making use of this resonance energy transfer or by observing phenylalanine fluorescence directly in specific proteins where resonance energy transfer is inefficient.
Annals of the New York Academy of Sciences 02/2008; 1130:300-4. · 3.15 Impact Factor
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ABSTRACT: A nonextensive model of decay kinetics has been used to describe fluorescence behavior of tryptophan in human serum albumin on binding two flavonoids, quercetin and morin. We demonstrate that this approach, alternative to multiexponential representation of usually complex decays of tryptophan, is more adequate and can be beneficial in noninvasive lifetime sensing based on intrinsic fluorescence.
Annals of the New York Academy of Sciences 02/2008; 1130:314-9. · 3.15 Impact Factor
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ABSTRACT: Here we describe progress toward our objective of detecting single nonfluorescent hydrated metal ions. Single-ion detection represents detection and spectroscopy at the ultimate sensitivity level of approximately 1.6 x 10(-24) M. Achieving this goal would provide a breakthrough in analytical science and allow much more detailed insight into sensor-ion interaction than that available with conventional bulk detection methods. We combine recent advances in confocal microscopy with the sensitivity and the noninvasive nature of fluorescence by analyzing Förster resonance energy transfer between sensor fluorophores and transition metal ions.
Annals of the New York Academy of Sciences 02/2008; 1130:62-7. · 3.15 Impact Factor
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ABSTRACT: Single molecule level detection of the near-infrared fluorescent protein allophycocyanin (APC) has been achieved using surface enhanced resonance Raman scattering (SERRS). The detection limit using the peak height of the 440 cm(-1) band was 1 x 10(-13) mol l(-1), compared to 2 x 10(-12) mol l(-1) for the fluorescence peak at 660 nm.
The Analyst 08/2007; 132(7):633-4. · 4.23 Impact Factor
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ABSTRACT: The authors show that p H increase, due to removal by condensation of silicic acid, correlates with nanoparticle growth during the initial stages of silica hydrogel formation and becomes constant at a time t<sub>p H </sub> , the point when other particle growth mechanisms dominate. Absorption of common phthalein indicators is shown to allow effectively instantaneous tracking of the p H and nanoparticle size in alkaline and acidic hydrogels. Particle sizes are calibrated using the hydrodynamic radius determined from the fluorescence anisotropy decay. Tracking p H complements fluorescence anisotropy nanometrology by offering a lower cost, speedier, and simpler method of studying particle growth during silica hydrogel fabrication.
Applied Physics Letters 10/2006; · 3.84 Impact Factor
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ABSTRACT: The authors describe the characteristics and application of a 265 nm AlGaN light-emitting diode (LED) operated at 1 MHz repetition rate, 1.2 ns pulse duration, 1.32 μ W average power, 2.3 mW peak power, and ∼12 nm bandwidth. The LED enables the fluorescence decay of weakly emitting phenylalanine to be measured routinely, even in dilute solution. For p H of 6–9.2, the authors find evidence for a biexponential rather than monoexponential decay, providing direct evidence for the presence of phenylalanine rotamers with a photophysics closer to the other two fluorescent amino acids tryrosine and tryptophan than has previously been reported.
Applied Physics Letters 09/2006; · 3.84 Impact Factor
