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Fabrication of an Open Microfluidic Device for Immunoblotting
Analytical Chemistry ( IF 6.7 ) Pub Date : 2017-09-07 00:00:00 , DOI: 10.1021/acs.analchem.7b02406
Philippe Abdel-Sayed 1 , Kevin A. Yamauchi 1 , Rachel E. Gerver 1 , Amy E. Herr 1
Affiliation  

Given the wide adoption of polydimethylsiloxane (PDMS) for the rapid fabrication of microfluidic networks and the utility of polyacrylamide gel electrophoresis (PAGE), we develop a technique for fabrication of PAGE molecular sieving gels in PDMS microchannel networks. In developing the fabrication protocol, we trade-off constraints on materials properties of these two polymer materials: PDMS is permeable to O2 and the presence of O2 inhibits the polymerization of polyacrylamide. We present a fabrication method compatible with performing PAGE protein separations in a composite PDMS-glass microdevice, that toggles from an “enclosed” microchannel for PAGE and blotting to an “open” PA gel lane for immunoprobing and readout. To overcome the inhibitory effects of O2, we coat the PDMS channel with a 10% benzophenone solution, which quenches the inhibiting effect of O2 when exposed to UV, resulting in a PAGE-in-PDMS device. We then characterize the PAGE separation performance. Using a ladder of small-to-mid mass proteins (Trypsin Inhibitor (TI); Ovalbumin (OVA); Bovine Serum Albumin (BSA)), we observe resolution of the markers in <60 s, with separation resolution exceeding 1.0 and CVs of 8.4% for BSA-OVA and 2.4% for OVA-TI, with comparable reproducibility to glass microdevice PAGE. We show that benzophenone groups incorporated into the gel through methacrylamide can be UV-activated multiple times to photocapture protein. PDMS microchannel network is reversibly bonded to a glass slide allowing direct access to separated proteins and subsequent in situ diffusion-driven immunoprobing and total protein Sypro red staining. We see this PAGE-in-PDMS fabrication technique as expanding the application and use of microfluidic PAGE without the need for a glass microfabrication infrastructure.

中文翻译:

用于免疫印迹的开放式微流控设备的制造

鉴于聚二甲基硅氧烷(PDMS)被广泛用于微流体网络的快速制造以及聚丙烯酰胺凝胶电泳(PAGE)的实用性,我们开发了一种在PDMS微通道网络中制造PAGE分子筛凝胶的技术。在开发制造协议中,我们权衡这些两种聚合物材料的材料性质的制约:PDMS是可渗透至O 2和的O-存在2抑制聚丙烯酰胺的聚合反应。我们提出了一种与在复合PDMS玻璃微设备中执行PAGE蛋白质分离兼容的制造方法,该方法从“封闭”微通道进行PAGE转移并转移到“开放” PA凝胶泳道以进行免疫探测和读出。克服O 2的抑制作用,我们用10%的二苯甲酮溶液覆盖PDMS通道,从而消除了O 2的抑制作用当暴露在紫外线下时,会产生PAGE-in-PDMS装置。然后,我们表征PAGE分离性能。使用中小型蛋白质梯形蛋白(胰蛋白酶抑制剂(TI);卵清蛋白(OVA);牛血清白蛋白(BSA)),我们在小于60 s的时间内观察到了标记的分离,分离分辨率超过1.0,CV为BSA-OVA的含量为8.4%,OVA-TI的含量为2.4%,与玻璃微器件PAGE的可重复性相当。我们表明,通过甲基丙烯酰胺掺入凝胶中的二苯甲酮基团可以多次被UV激活以捕获光捕获蛋白。PDMS微通道网络可逆地结合到载玻片上,从而可以直接访问分离的蛋白质,并随后进行原位扩散驱动的免疫探测和总蛋白质Sypro红染色。
更新日期:2017-09-07
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