Online fluorescence anisotropy immunoassay for monitoring insulin secretion from islets of Langerhans.
Online fluorescence anisotropy immunoassay for monitoring insulin secretion from islets of Langerhans.
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DOI：
10.1039/c6ay02899c
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发表时间：
2017-01-07
期刊：
影响因子：
--
通讯作者：
Roper MG
中科院分区：
文献类型：
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作者：
Schrell AM;Mukhitov N;Yi L;Adablah JE;Menezes J;Roper MG
郎格汉斯胰岛的胰岛素分泌是一个动态的过程，对维持血糖稳态是必不可少的。从单个胰岛测量葡萄糖刺激下胰岛素水平的动态变化的能力将有助于测试治疗方法，并研究在代谢性疾病中观察到的分泌缺陷的机制。到目前为止，大多数测量胰岛素水平快速变化的方法都依赖于分离，这使得这种分析很难转移到非专业实验室。为了能够以更适合转移到非专业实验室的方式快速测量单个胰岛的分泌动力学，开发了一种微流控在线荧光各向异性免疫分析方法。单个胰岛被安置在微流体室内，并由基于重力的灌流系统用不同的葡萄糖水平进行刺激。将含有胰岛分泌物的胰岛腔总流出物与重力驱动的胰岛素抗体和Cy5标记的胰岛素溶液混合。混合完成后，用线偏振的635 nm激光激发免疫分析混合物，将发射分成平行和垂直分量，以确定各向异性。优化了包括温度均匀性和流速稳定性在内的可重复性各向异性测量的关键因素，使得胰岛素的检测下限为4 nm，各向异性值的RSD为-lt；1%。通过刺激不同葡萄糖水平的胰岛，显示了该系统测量单个胰岛胰岛素分泌的能力。由于整个分析都是光学进行的，这个系统应该可以很容易地转移到其他实验室。
Insulin secretion from islets of Langerhans is a dynamic process that is essential for maintaining glucose homeostasis. The ability to measure dynamic changes in insulin levels upon glucose stimulation from single islets will allow testing of therapeutics and investigating mechanisms of defective secretion observed in metabolic diseases. Most approaches to date for measurement of rapid changes in insulin levels rely on separations, making the assays difficult to translate to non-specialist laboratories. To enable rapid measurements of secretion dynamics from a single islet in a manner that will be more suitable for transfer to non-specialized laboratories, a microfluidic online fluorescence anisotropy immunoassay was developed. A single islet was housed inside a microfluidic chamber and stimulated with varying glucose levels from a gravity-based perfusion system. The total effluent of the islet chamber containing the islet secretions was mixed with gravity-driven solutions of insulin antibody and Cy5-labeled insulin. After mixing was complete, a linearly polarized 635 nm laser was used to excite the immunoassay mixture and the emission was split into parallel and perpendicular components for determination of anisotropy. Key factors for reproducible anisotropy measurements, including temperature homogeneity and flow rate stability were optimized, which resulted in a 4 nM limit of detection for insulin with <1% RSD of anisotropy values. The capability of this system for measuring insulin secretion from single islets was shown by stimulating an islet with varying glucose levels. As the entire analysis is performed optically, this system should be readily transferable to other laboratories.