Diabetes Research Group, King's College London School of Medicine, Guy's Hospital, London SE11UL, UK.
Biochem Biophys Res Commun. 2013 Aug 30;438(3):488-92. doi: 10.1016/j.bbrc.2013.07.111. Epub 2013 Aug 6.
Near-infrared (NIR) fluorescent dyes that are environmentally sensitive or solvatochromic are useful tools for protein labelling in in vivo biosensor applications such as glucose monitoring in diabetes since their spectral properties are mostly independent of tissue autofluorescence and light scattering, and they offer potential for non-invasive analyte sensing. We showed that the fluorophore 651-Blue Oxazine is polarity-sensitive, with a marked reduction in NIR fluorescence on increasing solvent polarity. Mutants of glucose/galactose-binding protein (GBP) used as the glucose receptor were site-specifically and covalently labelled with Blue Oxazine using click chemistry. Mutants H152C/A213R and H152C/A213R/L238S showed fluorescence increases of 15% and 21% on addition of saturating glucose concentrations and binding constants of 6 and 25mM respectively. Fluorescence responses to glucose were preserved when GBP-Blue Oxazine was immobilised to agarose beads, and the beads were excited by NIR light through a mouse skin preparation studied in vitro. We conclude GBP-Blue Oxazine shows proof-of-concept as a non-invasive continuous glucose sensing system.
近红外（NIR）荧光染料对环境敏感或溶剂致变色，是用于体内生物传感器应用中蛋白质标记的有用工具，例如糖尿病中的葡萄糖监测，因为它们的光谱性质在很大程度上独立于组织自发荧光和光散射，并且它们为非侵入性分析物感应提供了潜力。我们表明，荧光团 651-蓝色恶嗪是极性敏感的，随着溶剂极性的增加，NIR 荧光显著降低。使用点击化学将葡萄糖/半乳糖结合蛋白（GBP）的突变体作为葡萄糖受体进行了位点特异性和共价标记与 Blue Oxazine。突变体 H152C/A213R 和 H152C/A213R/L238S 在添加饱和葡萄糖浓度时荧光增加了 15%和 21%，结合常数分别为 6 和 25mM。当 GBP-Blue Oxazine 固定在琼脂糖珠上时，对葡萄糖的荧光响应得以保留，并且通过在体外研究的小鼠皮肤制剂用近红外光激发珠。我们得出结论，GBP-Blue Oxazine 作为非侵入性连续葡萄糖传感系统具有概念验证。
