Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA, USA.
School of Mechanical and Materials Engineering, Washington State University, Pullman, WA, USA.
Electrophoresis. 2019 May;40(9):1314-1321. doi: 10.1002/elps.201800441. Epub 2019 Feb 7.
The objective of this study is to explore an approach for analyzing negatively charged proteins using paper-based cationic ITP. The rationale of electrophoretic focusing the target protein with negative charges under unfavorable cationic ITP condition is to modify the electrophoretic mobility of the target protein through antigen-antibody immunobinding. Cationic ITP was performed on a paper-based analytical device that was fabricated using fiberglass paper. The paper matrix was modified with (3-aminopropyl)trimethoxysilane to minimize sample attraction to the surface for cationic ITP. Negatively charged BSA was used as the model target protein for the cationic ITP experiments. No electrophoretic mobility was observed for BSA-only samples during cationic ITP experimental condition. However, the presence of a primary antibody to BSA significantly improved the electrokinetic behavior of the target protein. Adding a secondary antibody conjugated with amine-rich quantum dots to the sample further facilitated the concentrating effect of ITP, reduced experiment time, and elevated the stacking ratio. Under our optimized experimental conditions, the cationic ITP-based paper device electrophoretically stacked 94% of loaded BSA in less than 7 min. Our results demonstrate that the technique has a broad potential for rapid and cost-effective isotachphoretic analysis of multiplex protein biomarkers in serum samples at the point of care.
本研究旨在探索一种利用基于纸张的阳离子型 ITP 分析带负电荷蛋白的方法。在不利的阳离子型 ITP 条件下，通过抗原-抗体免疫结合使带负电荷的靶蛋白聚焦电泳的原理是改变靶蛋白的电泳迁移率。阳离子 ITP 在使用玻璃纤维纸制造的基于纸张的分析装置上进行。用(3-氨丙基)三甲氧基硅烷对纸基质进行修饰，以最大程度地减少样品对阳离子 ITP 的表面吸引力。将带负电荷的 BSA 用作阳离子 ITP 实验的模型靶蛋白。在阳离子 ITP 实验条件下，仅 BSA 样品没有观察到电泳迁移率。然而，存在针对 BSA 的一抗显著改善了靶蛋白的动电行为。将与富含胺的量子点偶联的二抗添加到样品中，进一步促进了 ITP 的浓缩效应，缩短了实验时间，并提高了堆积比。在我们优化的实验条件下，基于阳离子 ITP 的纸装置在不到 7 分钟的时间内电泳堆积了 94%负载的 BSA。我们的结果表明，该技术具有在即时护理点快速、经济有效地分析血清样品中多重蛋白质生物标志物的广泛潜力。
