Roper Michael G, Shackman Jonathan G, Dahlgren Gabriella M, Kennedy Robert T
Department of Chemistry and Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
Anal Chem. 2003 Sep 15;75(18):4711-7. doi: 10.1021/ac0346813.
A microfluidic device has been developed for the determination of insulin secreted from islets of Langerhans by a capillary electrophoresis competitive immunoassay. Online assays were performed by electrophoretically sampling anti-insulin antibody (Ab), fluorescein isothiocyanate-labeled insulin (FITC-insulin), and insulin from separate reservoirs and allowing them to mix as they traveled through a 4-cm reaction channel heated to 38 degrees C. From the reaction channel, samples were injected onto a 1.5-cm-long electrophoresis channel where the FITC-insulin and FITC-insulin-Ab complex were separated in 5 s using an electric field of 500 V/cm. Detection limits for insulin were 3 nM in this mode of operation. Assays could be collected at 15-s intervals with continuous sampling and online mixing for up to 30 min with no intervention. Relative standard deviation was 2-6% depending on the insulin concentration. Response time to a step change in insulin concentration was 30 s. For live cell monitoring, single islets were placed into a reservoir on the chip and fluid in the immediate vicinity was continuously sampled to detect insulin secretion from the islet. Monitoring of insulin secretion with electropherograms taken at 15-s intervals resolved secretory profiles characteristic of first- and second-phase insulin secretion. The method should be amenable to other cell or tissue types for measurements of release with high temporal resolution.
一种微流控装置已被开发出来，用于通过毛细管电泳竞争免疫测定法测定胰岛分泌的胰岛素。在线测定是通过电泳方式从单独的储液器中取样抗胰岛素抗体（Ab）、异硫氰酸荧光素标记的胰岛素（FITC -胰岛素）和胰岛素，让它们在流经加热至38摄氏度的4厘米长反应通道时混合。从反应通道中取出的样品被注入到1.5厘米长的电泳通道中，在500 V/cm的电场下，FITC -胰岛素和FITC -胰岛素 - Ab复合物在5秒内被分离。在这种操作模式下，胰岛素的检测限为3 nM。测定可以每隔15秒收集一次，进行连续采样和在线混合，长达30分钟无需干预。相对标准偏差根据胰岛素浓度在2% - 6%之间。对胰岛素浓度阶跃变化的响应时间为30秒。对于活细胞监测，将单个胰岛放入芯片上的一个储液器中，并对紧邻的液体进行连续采样，以检测胰岛分泌的胰岛素。以15秒间隔采集的电泳图监测胰岛素分泌，解析出了第一阶段和第二阶段胰岛素分泌的特征性分泌曲线。该方法应该适用于其他细胞或组织类型，以高时间分辨率测量释放情况。
