Simultaneous Monitoring of Insulin and Islet Amyloid Polypeptide Secretion from Islets of Langerhans on a Microfluidic Device
Simultaneous Monitoring of Insulin and Islet Amyloid Polypeptide Secretion from Islets of Langerhans on a Microfluidic Device
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DOI：
10.1021/ac401625g
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发表时间：
2013-08-20
影响因子：
7.4
通讯作者：
Roper, Michael G.
中科院分区：
文献类型：
--
作者：
Lomasney, Anna R.;Yi, Lian;Roper, Michael G.
开发了一种方法，允许同时监测急性分泌动态的胰岛素和胰岛淀粉样多肽（IAPP）从胰岛使用微流控系统与双色检测。流动切换功能使灌注介质在5秒内发生变化，允许快速交换输送到胰岛组的葡萄糖浓度。通过电渗流连续采样灌注液，并在8.15 cm混合通道中与Cy 5标记的胰岛素、异硫氰酸荧光素（FITC）标记的IAPP、抗胰岛素和抗IAPP抗体在线混合，混合通道维持在37 ℃。将免疫测定混合物以11.75秒的间隔注射到1.5 cm分离通道上0.3秒，并使用488和635 nm激光检测免疫测定试剂，其中两个独立的光电倍增管用于检测FITC和Cy 5信号。结合-游离免疫测定比值的RSD范围为2 - 7%，胰岛素的LOD为20 nM，IAPP为1 nM。在葡萄糖浓度的多步变化期间，从4-10个胰岛的组监测两种肽的同时分泌概况。胰岛素和IAPP以约10：1的比例分泌，并且对从3至11或20 mM葡萄糖的阶跃变化显示出类似的响应。以高度自动化的方式监测来自胰岛的多种肽的分泌动力学的能力预期是用于调查葡萄糖稳态的激素调节的有用工具。
A method was developed that allowed simultaneous monitoring of the acute secretory dynamics of insulin and islet amyloid polypeptide (IAPP) from islets of Langerhans using a microfluidic system with two-color detection. A flow-switching feature enabled changes in the perfusion media within 5 s, allowing rapid exchange of the glucose concentrations delivered to groups of islets. The perfusate was continuously sampled by electroosmotic flow and mixed online with Cy5-labeled insulin, fluorescein isothiocyanate (FITC)-labeled IAPP, anti-insulin, and anti-IAPP antibodies in an 8.15 cm mixing channel maintained at 37 degrees C. The immunoassay mixture was injected for 0.3 s onto a 1.5 cm separation channel at 11.75 s intervals and immunoassay reagents detected using 488 and 635 nm lasers with two independent photomultiplier tubes for detection of the FITC and Cy5 signal. RSD of the bound-to-free immunoassay ratios ranged from 2 to 7% with LODs of 20 nM for insulin and 1 nM for IAPP. Simultaneous secretion profiles of the two peptides were monitored from groups of 4-10 islets during multiple step changes in glucose concentration. Insulin and IAPP were secreted in an approximately 10:1 ratio and displayed similar responses to step changes from 3 to 11 or 20 mM glucose. The ability to monitor the secretory dynamics of multiple peptides from islets of Langerhans in a highly automated fashion is expected to be a useful tool for investigating hormonal regulation of glucose homeostasis.