Wang Joseph, Ibáñez Alfredo, Chatrathi Madhu Prakash
Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico 88003.
J Am Chem Soc. 2003 Jul 16;125(28):8444-5. doi: 10.1021/ja036067e.
A microfluidic device coupling immunological and enzymatic assays within a single microchannel has been developed for simultaneous measurements of insulin and glucose. Such a dual-mode (enzyme/immuno) protocol involves precolumn reactions of insulin and glucose with the enzyme-labeled anti-human insulin and glucose-dehydrogenase/NAD+, respectively, followed by the electrophoretic separation of the free antibody, antibody-antigen complex, and the NADH product of the enzymatic reaction. The separation is followed by a postcolumn reaction of the alkaline-phosphatase tracer with the p-NPP substrate and a downstream amperometric detection of the p-nitrophenol and NADH products. Despite the huge concentration difference [millimolar (glucose) and nanomolar (insulin)] and the use of different assay principles, the new biochip responds independently to the corresponding target analytes, with linear dynamic ranges over their clinically relevant ranges. Complete assays, carried out within less than 4 min, lead to good precision (RSD 0.36%) for the insulin/glucose ratio. The resulting biochip allows simultaneous testing for insulin and glucose to be performed more rapidly, easily, and economically, and hence it holds great promise for improved management of diabetes.
一种在单个微通道内耦合免疫分析和酶分析的微流控装置已被开发出来，用于同时测量胰岛素和葡萄糖。这种双模式（酶/免疫）方案包括胰岛素和葡萄糖分别与酶标记的抗人胰岛素和葡萄糖脱氢酶/NAD+进行柱前反应，随后对游离抗体、抗体-抗原复合物以及酶促反应的NADH产物进行电泳分离。分离之后是碱性磷酸酶示踪剂与对硝基苯磷酸酯（p-NPP）底物的柱后反应，以及对硝基苯酚和NADH产物的下游安培检测。尽管存在巨大的浓度差异[毫摩尔级（葡萄糖）和纳摩尔级（胰岛素）]且使用了不同的分析原理，但这种新型生物芯片能独立响应相应的目标分析物，在其临床相关范围内具有线性动态范围。在不到4分钟内完成的完整分析，胰岛素/葡萄糖比值的精密度良好（相对标准偏差为0.36%）。由此产生的生物芯片能够更快速、轻松且经济地同时检测胰岛素和葡萄糖，因此在改善糖尿病管理方面具有巨大潜力。
