Leng Weijia, Evans Kimberly, Roper Michael G
Department of Chemistry and Biochemistry, Florida State University, 95 Chieftan Way, Dittmer Building, Tallahassee, FL 32306, USA.
Anal Methods. 2019 Dec 7;11(45):5768-5775. doi: 10.1039/C9AY01758E. Epub 2019 Oct 29.
Pancreatic islets of Langerhans release glucagon to maintain blood glucose levels, and release of this peptide is dysregulated in diabetes mellitus. Although the importance of proper secretion of this peptide has been shown, no measurement of its release at the single islet level has been reported. In previous work, a non-competitive assay for glucagon was developed with a 6 pM limit of detection, low enough to measure from a single islet. To incorporate this method in an online assay, a microfluidic system with several distinct features was developed. To maintain appropriate flow rates in the presence of the high concentration of salt that was required for the assay, a piezo-actuated pressure transducer with in-line flow sensors was used to drive sample flow through 80 × 50 μm (width × depth) channels, while electroosmotic flow was used to gate the sample away from 15 × 5 μm separation channel. Flow rates tested with this system were 50 - 200 nL min with relative standard deviations (RSDs) ranging from 1 - 4 %. Use of the pressure-driven flow was found to increase the amount of clogs in the system, so a method to incorporate in-line filters into the channels was developed. A total of 4 low resistance, in-line microfabricated filters were evaluated, with all designs prolonging the operation time of the microfluidic device to more than 4 hours without clogs observed. Use of this system enabled highly reproducible injections (3-6% RSD). During initial incorporation of the noncompetitive assay for glucagon, it was determined that Joule heating was problematic and temperature measurements revealed the separation channel increased to more than 50°C during operation. A 3D-printed manifold was used to hold a Peltier cooler in place on the microfluidic device which produced a 2.6-fold improvement in the amount of the noncovalent glucagon complex that was detected compared to without cooling. These features are expected to be useful for not only long-term monitoring of the glucagon release from islets of Langerhans, but has the potential to be applied to a number of other microfluidic separation-based assays as well.
胰岛释放胰高血糖素来维持血糖水平，而在糖尿病中这种肽的释放会失调。尽管已表明这种肽正常分泌的重要性，但尚未有在单个胰岛水平上对其释放进行测量的报道。在之前的工作中，开发了一种胰高血糖素的非竞争性检测方法，检测限为6 pM，低到足以从单个胰岛进行测量。为了将该方法纳入在线检测，开发了一种具有几个独特特征的微流控系统。为了在检测所需的高盐浓度下保持适当的流速，使用了带有在线流量传感器的压电驱动压力传感器来驱动样品流过80×50μm（宽×深）的通道，同时使用电渗流将样品从15×5μm的分离通道中分流。用该系统测试的流速为50 - 200 nL/min，相对标准偏差（RSD）范围为1 - 4%。发现使用压力驱动流会增加系统中的堵塞量，因此开发了一种将在线过滤器纳入通道的方法。总共评估了4个低阻力的在线微加工过滤器，所有设计都将微流控装置的运行时间延长到4小时以上且未观察到堵塞。使用该系统实现了高度可重复的进样（RSD为3 - 6%）。在最初将胰高血糖素的非竞争性检测方法纳入时，确定焦耳热是个问题，温度测量显示在运行期间分离通道温度升高到50°C以上。使用3D打印的歧管将珀耳帖冷却器固定在微流控装置上，与不冷却相比，检测到的非共价胰高血糖素复合物的量提高了2.6倍。预计这些特征不仅对长期监测胰岛中胰高血糖素的释放有用，而且有可能应用于许多其他基于微流控分离的检测方法。
