Measurements of FRET in a glucose-sensitive affinity system with frequency-domain lifetime spectroscopy
Measurements of FRET in a glucose-sensitive affinity system with frequency-domain lifetime spectroscopy
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DOI：
10.1562/2005-02-14-ra-440
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发表时间：
2005-11-01
影响因子：
3.3
通讯作者：
Sevick-Muraca, EM
中科院分区：
文献类型：
--
作者：
Liang, F;Pan, TS;Sevick-Muraca, EM
我们报告的测量荧光共振能量转移（FRET）葡萄糖传感在一个既定的伴刀豆球蛋白A-葡聚糖亲和系统使用频域寿命光谱。用小荧光供体分子Alexa Fluor 568标记的右旋糖酐（MW 2000000）在葡萄糖存在下与用受体分子Alexa Fluor 647标记的糖结合蛋白伴刀豆球蛋白A竞争性结合。从频域测量作为葡萄糖和受体蛋白质浓度的函数，使用福斯特型衰减动力学模型的供体的FRET淬灭动力学进行了分析。结果表明，频域测量和供体衰减动力学可以定量地指示在葡萄糖的存在下，在0至224 mg/dL的浓度范围内，在10.67 μ M的溶液浓度下，0.09 μ M葡聚糖与标记的伴刀豆球蛋白A的竞争性结合的变化。
We report measurements of fluorescence resonance energy transfer (FRET) for glucose sensing in an established concanavalin A-dextran affinity system using frequency-domain lifetime spectroscopy. A dextran (MW 2000000) labeled with a small fluorescent donor molecule, Alexa Fluor 568, was used to competitively bind to a sugar-binding protein, concanavalin A, labeled with acceptor molecule, Alexa Fluor 647, in the presence of glucose. The FRET-quenching kinetics of the donor were analyzed from frequency-domain measurements as a function of both glucose and acceptor-protein concentrations using a Forster-type decay kinetics model. The results show that the frequency-domain measurements and donor decay kinetics can quantitatively indicate changes in the competitive binding of 0.09 mu M dextran to labeled concanavalin A at a solution concentration of 10.67 mu M in the presence of glucose at concentrations ranging from 0 to 224 mg/dL.