Department of Bioengineering, University of California, Berkeley, California 94720, USA.
Anal Chem. 2011 Sep 1;83(17):6573-9. doi: 10.1021/ac200982j. Epub 2011 Aug 11.
We introduce a fully integrated multistep protein assay that reports both protein identity and size. To report these two properties, a microfluidic design strategy integrates pore limit electrophoresis (PLE) with a heterogeneous immunoassay in a single microchannel (PLE-IA). PLE-IA was applied in a study of follistatin, a 31.5 kDa glycoprotein regulating mammalian cell proliferation and differentiation. In a single-channel multistage assay approach, an antibody to follistatin was first immobilized in a polyacrylamide PLE gradient gel, near the origin of the separation axis. Immobilization relies on pore-limit exclusion of the antibody and not on chemical functionalization of either the sieving matrix or the antibody, making assay customization by an end-user straightforward. Subsequently, target and ladder protein species were electrophoretically introduced into the antibody-patterned PLE channel. Species having an affinity for the immobilized antibody were detected via heterogeneous immunoassay. Noninteracting and, thus, unbound species electromigrated past the patterned antibodies, along the separation axis, and finally separated according to the pore-size limit of each, yielding a log-linear dependence of molecular weight on migration distance. Separations of 10 min yielded an average peak capacity of 18 ± 1.3 (separation resolution (SR) = 1) in a 10 mm separation distance. Comparison of the separated peaks in two parallel PLE channels in the presence or absence of capture antibody with a protein size ladder revealed good agreement of the target molecular weight with reported values. In addition, a more than 50-fold decrease in the detection limit (0.078 vs 5 nM) was achieved using an electrophoretic "continuous injection" technique in which sample material was continuously loaded for 40 min. On the basis of this proof-of-principle demonstration with follistatin, PLE-IA should find application in study of cell signaling, including questions related to aging and regeneration.
我们介绍了一种完全集成的多步蛋白质分析方法，可同时报告蛋白质的身份和大小。为了报告这两个属性，微流控设计策略将孔限电泳（PLE）与异质免疫测定法集成在单个微通道（PLE-IA）中。PLE-IA 应用于研究卵泡抑素，一种 31.5 kDa 的糖蛋白，调节哺乳动物细胞的增殖和分化。在单通道多步分析方法中，首先将抗卵泡抑素抗体固定在聚丙酰胺 PLE 梯度凝胶中，靠近分离轴的原点。固定依赖于抗体的孔限排除，而不是筛基质或抗体的化学功能化，使得最终用户可以轻松进行检测定制。随后，目标和梯级蛋白物种通过电泳引入到抗体图案化 PLE 通道中。与固定化抗体具有亲和力的物种通过异质免疫测定法检测。非相互作用的，因此，未结合的物种沿分离轴电泳迁移过图案化抗体，最后根据每个分子的孔径限制进行分离，得到分子量与迁移距离的对数线性依赖性。10 分钟的分离产生了 10mm 分离距离内 18 ± 1.3（分离分辨率（SR）= 1）的平均峰容量。在存在或不存在捕获抗体的情况下，将两个平行 PLE 通道中分离的峰与蛋白质大小梯级进行比较，发现目标分子量与报道值吻合良好。此外，使用电泳“连续注入”技术可将检测限降低 50 多倍（0.078 与 5 nM），在该技术中，样品材料连续加载 40 分钟。基于卵泡抑素的这一原理验证，PLE-IA 应该在细胞信号研究中找到应用，包括与衰老和再生相关的问题。
