Schrell Adrian M, Mukhitov Nikita, Yi Lian, Adablah Joel E, Menezes Joshua, Roper Michael G
Department of Chemistry and Biochemistry, Florida State University, 95 Chieftain Way, Tallahassee, FL 32306.
Anal Methods. 2017 Jan 7;9(1):38-45. doi: 10.1039/C6AY02899C. Epub 2016 Oct 28.
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.
胰岛β细胞分泌胰岛素是一个动态过程，对维持血糖稳态至关重要。能够测量单个胰岛在葡萄糖刺激下胰岛素水平的动态变化，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机制。迄今为止，大多数测量胰岛素水平快速变化的方法都依赖于分离技术，这使得这些检测方法难以应用于非专业实验室。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态，开发了一种微流控在线荧光偏振免疫分析法。将单个胰岛置于微流控腔室内，通过基于重力的灌注系统用不同葡萄糖水平进行刺激。含有胰岛分泌物的胰岛腔室的总流出物与重力驱动的胰岛素抗体溶液和Cy5标记的胰岛素混合。混合完成后，使用线偏振635nm激光激发免疫分析混合物，并将发射光分为平行和垂直分量以测定偏振度。对包括温度均匀性和流速稳定性在内的可重复偏振度测量的关键因素进行了优化，这使得胰岛素的检测限为4nM，偏振度值的相对标准偏差<1%。通过用不同葡萄糖水平刺激胰岛，展示了该系统测量单个胰岛胰岛素分泌的能力。由于整个分析是通过光学方式进行的，该系统应易于转移到其他实验室。
