Olaf J. Rolinski

In vivo glucose sensing for diabetes management: progress towards non-invasive monitoring

A device for continuous in vivo monitoring of glucose concentration in people with diabetes has been a clinical and research priority for many years but now has an urgency which is probably unquestioned in diabetes care. The purpose of this article is to explain recent advances in technology that are bringing glucose sensors closer to routine use and to highlight some of the remaining problems. Important new technologies include artificial receptors for glucose, tissue fluid sampling techniques, and new approaches to non-invasive sensing, such as fluorescence lifetime measurements.

Fluorescence lifetime spectroscopy and imaging of nano-engineered glucose sensor microcapsules based on glucose/galactose-binding protein

We aimed to develop microsensors for eventual glucose monitoring in diabetes, based on fluorescence lifetime changes in glucose/galactose-binding protein (GBP) labelled with the environmentally sensitive fluorophore dye, badan. A mutant of GBP was labelled with badan near the binding site, the protein adsorbed to microparticles of CaCO(3) as templates and encapsulated in alternating nano-layers of poly-L-lysine and heparin. We used fluorescence lifetime imaging (FLIM) with two-photon excitation and time-correlated single-photon counting to visualize the lifetime changes in the capsules. Addition of glucose increased the mean lifetime of GBP-badan by a maximum of approximately 2 ns. Analysis of fluorescence decay curves was consistent with two GBP states, a short-lifetime component (approximately 0.8 ns), likely representing the open form of the protein with no bound glucose, and a long-lifetime component (approximately 3.1 ns) representing the closed form with bound glucose and where the lobes of GBP have closed round the dye creating a more hydrophobic environment. FLIM demonstrated that increasing glucose increased the fractional proportion of the long-lifetime component. We conclude that fluorescence lifetime-based glucose sensing using GBP encapsulated with nano-engineered layer-by-layer films is a glucose monitoring technology suitable for development in diabetes management.

Determination of acceptor distribution from fluorescence resonance energy transfer: Theory and simulation

A new method for determining the donor–acceptor distribution function in fluorescence resonance energy transfer (FRET) systems is presented. The approach is based on time-resolved fluorescence experiments with nanosecond resolution. Potential applications of this method include: determination of the morphology of porous materials (e.g., polymers, sol-gels, resins, etc.), monitoring processes occurring on the nanometer scale including biomolecules labeled with the donor/acceptor species, and FRET sensors based on competitive binding. In this paper a theoretical derivation of the method is presented and the method is tested in a series of numerical simulations. The experimental conditions regarding this approach are discussed and its applicability to real measurement systems is demonstrated.

Fluorescence lifetime sensor of copper ions in water

We demonstrate an optical method for the selective detection of Cu(II) ions in water using time‐resolved fluorescence resonance energy transfer from the dye rhodamine 800 encapsulated in a sensor. In comparison to copper, quenching by other metal ions such as cobalt, nickel, and chromium is shown to be negligible. The experimental arrangement incorporates picosecond diode laser excitation and time‐correlated single‐photon counting for detection. Down to 5 mM of copper deposited on the sensor can be measured and a linear response is obtained up to at least 50 mM. A lower limit of detection for the sensor in the region of 10 ppb is shown to be readily achievable with good resolution.

A time-resolved near-infrared fluorescence assay for glucose: opportunities for trans-dermal sensing.

We report a time-resolved near-infrared fluorescence assay for glucose detection that incorporates pulsed diode laser excitation. Reduction in fluorescence resonance energy transfer to a malachite green-Dextran complex from allophycocyanin bound to concanavalin A (ConA) due to displacement of the complex by glucose from ConA provides the basis of the assay. The fluorescence quenching kinetics are analysed and discussed in detail. The change in fluorescence decay kinetics in the presence of glucose is found from dimensionality studies to be brought about by a change in the distribution of malachite green-Dextran acceptors. Glucose concentrations are measured in solution to within +/- 10% over the range 0-30 mM.

Optical spectroscopic methods for probing the conformational stability of immobilised enzymes

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.

Non-Invasive Glucose Monitoring by NAD(P)H Autofluorescence Spectroscopy in Fibroblasts and Adipocytes: A Model for Skin Glucose Sensing

The aim of this study was to develop an in vitro cell-culture model of skin-component cells to test the hypothesis that glucose can be monitored non-invasively by measuring NAD(P)H-related fluorescence changes in tissues. 3T3-L1 fibroblasts and adipocytes were grown in culture, and the response to added glucose was assessed by changes in steady-state autofluorescence at 400-500 nm [excitation at 340 nm, an index of NAD(P)H]. We also studied glucose-related fluorescence changes in cells stained with the mitochondrial marker, rhodamine-123. Fibroblasts and adipocytes showed glucose-dependent increases in autofluorescence with both short- and long-term exposure. Spectral properties indicated that the fluorescence was due to NAD(P)H production. With 5-h exposure to glucose, the maximal response was at 10-15 mmol/L glucose. Cells stained with the fluorescent mitochondrial marker, rhodamine-123, showed an immediate and marked decrease in fluorescence when exposed to glucose. We conclude that glucose can be sensed non-invasively by cellular fluorescence changes in fibroblasts and adipocytes. This is a model for the further exploration of fluorescence-based non-invasive metabolic monitoring in human diabetes.

Human serum albumin and quercetin interactions monitored by time-resolved fluorescence: evidence for enhanced discrete rotamer conformations

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.

Early detection of amyloid aggregation using intrinsic fluorescence

Beta-amyloid (Abeta) aggregation, believed to be responsible for Alzheimers 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 Alzheimers disease and an improved approach to developing intervention therapies.

A fluorescence lifetime sensor for Cu(I) ions

We have developed a new approach to selectively detecting copper(I) ions in aqueous solutions with a detection limit down to 0.1 ppb. The concentration of Cu(I) ions is determined from the change in fluorescence decay of perylene caused by energy transfer to the complex of Cu(I) ions with a chelating agent, 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline. Complex formation and energy transfer in two different sensor matrixes, porous Nafion 117 film and Amberlite resin, are discussed.