Perfusion and chemical monitoring of living cells on a microfluidic chip
Perfusion and chemical monitoring of living cells on a microfluidic chip
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DOI：
10.1039/b404974h
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发表时间：
2005-01-01
期刊：
影响因子：
6.1
通讯作者：
Kennedy, RT
中科院分区：
文献类型：
--
作者：
Shackman, JG;Dahlgren, GM;Kennedy, RT
一个微流控装置，采用连续灌注和在线电泳免疫分析的开发，其特征在于，并应用于监测胰岛素分泌从单个胰岛。在该装置中，用细胞培养基或平衡盐溶液以0.6至1.5穆尔min（-1）灌注细胞室。流动由芯片外施加的气体压力驱动。通过电渗透通过芯片上的单独通道以2 nL min（-1）连续采样灌注液。将灌注液与异硫氰酸荧光素标记的胰岛素（FITC-胰岛素）和单克隆抗胰岛素抗体在线混合，并使混合物沿4 cm长的反应通道向下移动时反应60 s。细胞室和反应通道保持在37 ℃。以每6秒一次的速度将反应混合物注射到1.5 cm分离通道上，并在500至600 V cm（-1）的电场下分离游离FITC-胰岛素和FITC-胰岛素-抗体复合物。该免疫测定法具有0.8 nM的检测限和6%的相对标准偏差，在2小时的连续操作与标准溶液。当用不同浓度的葡萄糖灌注时，监测单个胰岛长达1小时。免疫测定允许定量监测经典的双相和振荡的胰岛素分泌与6秒的采样频率以下的葡萄糖从3到11 mM的阶跃变化。2.5 cm x 7.6 cm的微流体系统允许监测胰岛在一个高度自动化的方式。这项技术应该适用于涉及释放化学物质的其他组织或细胞的研究。
A microfluidic device that incorporates continuous perfusion and an on-line electrophoresis immunoassay was developed, characterized, and applied to monitoring insulin secretion from single islets of Langerhans. In the device, a cell chamber was perfused with cell culture media or a balanced salt solution at 0.6 to 1.5 muL min(-1). The flow was driven by gas pressure applied off-chip. Perfusate was continuously sampled at 2 nL min(-1) by electroosmosis through a separate channel on the chip. The perfusate was mixed on-line with fluorescein isothiocyanate-labeled insulin (FITC-insulin) and monoclonal anti-insulin antibody and allowed to react for 60 s as the mixture traveled down a 4 cm long reaction channel. The cell chamber and reaction channel were maintained at 37 degreesC. The reaction mixture was injected onto a 1.5 cm separation channel as rapidly as every 6 s, and the free FITC-insulin and the FITC-insulin-antibody complex were separated under an electric field of 500 to 600 V cm(-1). The immunoassay had a detection limit of 0.8 nM and a relative standard deviation of 6% during 2 h of continuous operation with standard solutions. Individual islets were monitored for up to 1 h while perfusing with different concentrations of glucose. The immunoassay allowed quantitative monitoring of classical biphasic and oscillatory insulin secretion with 6 s sampling frequency following step changes in glucose from 3 to 11 mM. The 2.5 cm x 7.6 cm microfluidic system allowed for monitoring islets in a highly automated fashion. The technique should be amenable to studies involving other tissues or cells that release chemicals.