Direct detection of glucose by surface plasmon resonance with bacterial glucose/galactose-binding protein
Direct detection of glucose by surface plasmon resonance with bacterial glucose/galactose-binding protein
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DOI：
10.1016/s0956-5663(03)00271-9
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发表时间：
2004-02-15
影响因子：
12.6
通讯作者：
Pitner, JB
中科院分区：
文献类型：
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作者：
Hsieh, HV;Pfeiffer, ZA;Pitner, JB
血糖水平的监测和管理是维持糖尿病患者健康的关键组成部分。传统上，葡萄糖监测基于使用电化学和酶（例如葡萄糖氧化酶或葡萄糖脱氢酶）的间接检测。在这里，我们展示了直接检测葡萄糖使用表面等离子体共振（SPR）生物传感器。通过位点特异性和共价连接一个已知的葡萄糖受体，葡萄糖/半乳糖结合蛋白（GGBP），SPR表面，我们能够检测葡萄糖结合，并确定平衡结合常数。通过将GGBP上的单个氨基酸突变为半胱氨酸并随后进行巯基缀合来实现位点特异性偶联。所得SPR表面具有与GGBP的已知性质一致的葡萄糖特异性结合性质。引入进一步的修饰以减弱GGBP结合亲和力，以更紧密地匹配生理相关的葡萄糖浓度（1-30 mM）。一个蛋白质的响应接近这个葡萄糖范围被确定，GGBP三重突变体E149 C，A213 S，L238 S与0.5 mM的平衡解离常数。这些结果表明，生物传感器的SPR或类似的折射检测方法的基础上直接葡萄糖检测，如果小型化，有潜力发展为连续葡萄糖监测设备。(C)2003 Elsevier B. V.保留所有权利。
The monitoring and management of blood glucose levels are key components for maintaining the health of people with diabetes. Traditionally, glucose monitoring has been based on indirect detection using electrochemistry and enzymes such as glucose oxidase or glucose dehydrogenase. Here, we demonstrate direct detection of glucose using a surface plasmon resonance (SPR) biosensor. By site-specifically and covalently attaching a known receptor for glucose, the glucose/galactose-binding protein (GGBP), to the SPR surface, we were able to detect glucose binding and determine equilibrium binding constants. The site-specific coupling was accomplished by mutation of single amino acids on GGBP to cysteine and subsequent thiol conjugation. The resulting SPR surfaces had glucose-specific binding properties consistent with known properties of GGBP. Further modifications were introduced to weaken GGBP-binding affinity to more closely match physiologically relevant glucose concentrations (1-30 mM). One protein with a response close to this glucose range was identified, the GGBP triple mutant E149C, A213S, L238S with an equilibrium dissociation constant of 0.5 mM. These results suggest that biosensors for direct glucose detection based on SPR or similar refractive detection methods, if miniaturized, have the potential for development as continuous glucose monitoring devices. (C) 2003 Elsevier B.V. All rights reserved.