RESUMO
Alternative, non-electrochemistry-based technologies for continuous glucose monitoring are needed for eventual use in diabetes mellitus. As part of a programme investigating fluorescent glucose sensors, we have developed fibre-optic biosensors using glucose/galactose binding protein (GBP) labelled with the environmentally sensitive fluorophore, Badan. GBP-Badan was attached via an oligohistidine-tag to the surface of Ni-nitrilotriacetic acid (NTA)-functionalized agarose or polystyrene beads. Fluorescence lifetime increased in response to glucose, observed by fluorescence lifetime imaging microscopy of the GBP-Badan-beads. Either GBP-Badan agarose or polystyrene beads were loaded into a porous chamber at the end of a multimode optical fibre. Fluorescence lifetime responses were recorded using pulsed laser excitation, high speed photodiode detection and time-correlated single-photon counting. The maximal response was at 100 mM glucose with an apparent K(d) of 13 mM (agarose) and 20 mM (polystyrene), and good working-day stability was demonstrated. We conclude that fluorescence lifetime fibre-optic glucose sensors based on GBP-Badan are suitable for development as clinical glucose monitors.
Assuntos
Técnicas Biossensoriais/métodos , Proteínas de Ligação ao Cálcio/química , Fluorescência , Glucose/análise , Proteínas de Transporte de Monossacarídeos/química , Proteínas Periplásmicas de Ligação/química , Técnicas Biossensoriais/instrumentação , Proteínas de Ligação ao Cálcio/metabolismo , Microscopia de Fluorescência , Microesferas , Proteínas de Transporte de Monossacarídeos/metabolismo , Ácido Nitrilotriacético/química , Fibras Ópticas , Proteínas Periplásmicas de Ligação/metabolismo , Poliestirenos/química , Sefarose/química , Fatores de TempoRESUMO
We synthesized mutants of glucose/galactose-binding protein (GBP), labeled with the environmentally sensitive fluorophore Badan, with the aim of producing a fluorescence-based glucose sensing system with an operating range compatible with continuous glucose monitoring in patients with diabetes mellitus. From five mutants tested, the triple mutant H152C/A213R/L238S-Badan showed a large (200%) maximal increase in fluorescence intensity on the addition of glucose, with a binding constant (K(d)) of 11 mM, an operating range of approximately 1-100 mM, and similar responses in buffer and serum. The mean fluorescence lifetime of this mutant also increased by 70% on the addition of glucose. We conclude that the GBP mutant H152C/A213R/L238S, when labeled with Badan, is suitable for development as a robust sensor for in vivo glucose monitoring in diabetes.
Assuntos
2-Naftilamina/análogos & derivados , Glicemia/análise , Proteínas de Ligação ao Cálcio/metabolismo , Ensaios Enzimáticos/métodos , Corantes Fluorescentes/química , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Periplásmicas de Ligação/metabolismo , 2-Naftilamina/química , Substituição de Aminoácidos , Técnicas Biossensoriais , Proteínas de Ligação ao Cálcio/genética , Diabetes Mellitus/diagnóstico , Humanos , Proteínas de Transporte de Monossacarídeos/genética , Mutagênese Sítio-Dirigida , Proteínas Periplásmicas de Ligação/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
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.
Assuntos
Técnicas Biossensoriais/métodos , Galactose/análise , Galactose/química , Glucose/análise , Glucose/química , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Nanoestruturas/química , Engenharia Biomédica/métodos , Cápsulas , Ativação Enzimática , Estabilidade Enzimática , Cinética , Teste de Materiais , Microscopia de Fluorescência , Nanomedicina/métodos , Nanoestruturas/ultraestrutura , Tamanho da Partícula , Ligação ProteicaRESUMO
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.