Department of Bioengineering, University of California Berkeley , Berkeley, California 94720, United States.
Anal Chem. 2017 Sep 19;89(18):9643-9648. doi: 10.1021/acs.analchem.7b02406. Epub 2017 Sep 7.
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 O and the presence of O 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 O, we coat the PDMS channel with a 10% benzophenone solution, which quenches the inhibiting effect of O 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.
鉴于聚二甲基硅氧烷 (PDMS) 在快速制造微流控网络方面的广泛应用，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 分子筛分凝胶的技术。在开发制造协议时，我们权衡了这两种聚合物材料的材料性能限制：PDMS 对 O 是可渗透的，而 O 的存在会抑制聚丙烯酰胺的聚合。我们提出了一种与在复合 PDMS-玻璃微器件中进行 PAGE 蛋白质分离兼容的制造方法，该方法可从用于 PAGE 和印迹的“封闭”微通道切换到用于免疫探测和读出的“开放”PA 凝胶通道。为了克服 O 的抑制作用，我们用 10%的苯甲酮溶液涂覆 PDMS 通道，当暴露于 UV 时，苯甲酮溶液会猝灭 O 的抑制作用，从而形成 PDMS 中的 PAGE 装置。然后，我们对 PAGE 分离性能进行了表征。使用从小分子到大分子的一系列蛋白质（胰蛋白酶抑制剂 (TI)；卵清蛋白 (OVA)；牛血清白蛋白 (BSA)），我们观察到标志物在 <60 s 内得到分离，分离分辨率超过 1.0，BSA-OVA 的 CV 值为 8.4%，OVA-TI 的 CV 值为 2.4%，与玻璃微器件 PAGE 的可重复性相当。我们表明，通过丙烯酰胺掺入凝胶中的苯甲酮基团可以通过 UV 多次激活以光捕获蛋白质。PDMS 微通道网络可可逆地键合到玻璃载玻片上，从而可以直接访问分离的蛋白质，随后进行原位扩散驱动的免疫探测和总蛋白 Sypro red 染色。我们认为，这种 PDMS 中的 PAGE 制造技术扩展了微流控 PAGE 的应用和使用，而无需玻璃微制造基础设施。
