Microfluidic chip for continuous monitoring of hormone secretion from live cells using an electrophoresis-based immunoassay
Microfluidic chip for continuous monitoring of hormone secretion from live cells using an electrophoresis-based immunoassay
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DOI：
10.1021/ac0346813
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发表时间：
2003-09-15
影响因子：
7.4
通讯作者：
Kennedy, RT
中科院分区：
文献类型：
--
作者：
Roper, MG;Shackman, JG;Kennedy, RT
建立了毛细管电泳法测定朗格汉斯胰岛分泌胰岛素的微流控装置。在线分析是通过对来自不同储存库的抗胰岛素抗体(Ab)、异硫氰酸荧光素标记的胰岛素(FITC-Insufin)和胰岛素进行电泳法采样，并允许它们在通过加热到38℃的4厘米反应通道时混合。从反应通道中，样品被注入一个1.5 cm长的电泳道，在500V/cm的电场下，FITC-Ins和FITC-Ins-Ab复合体在5 S中被分离。在此操作模式下，对胰岛素的检测下限为3 NM。分析可以以15-S间隔收集，连续采样和在线混合长达30分钟，无需干预。根据胰岛素浓度的不同，相对标准偏差为2-6%。对胰岛素浓度阶跃变化的反应时间为30 S。为了进行活细胞监测，将单个胰岛放入芯片上的储液器中，并连续采集附近的液体，检测胰岛的胰岛素分泌。用15-S间隔采集的电泳图监测胰岛素分泌，确定了第一和第二时相胰岛素分泌的特征。该方法应适用于其他类型的细胞或组织，用于高时间分辨率的释放测量。
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-insufin), and insulin from separate reservoirs and allowing them to mix as they traveled through a 4-cm reaction channel heated to 38 degreesC. 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.