Immunobinding-induced alteration in the electrophoretic mobility of proteins: An approach to studying the preconcentration of an acidic protein under cationic isotachophoresis
Immunobinding-induced alteration in the electrophoretic mobility of proteins: An approach to studying the preconcentration of an acidic protein under cationic isotachophoresis
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DOI：
10.1002/elps.201800441
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发表时间：
2019-05-01
期刊：
影响因子：
2.9
通讯作者：
Dong, Wen-Ji
中科院分区：
文献类型：
--
作者：
Guo, Shuang;Jacroux, Thomas;Dong, Wen-Ji
本研究的目的是探索一种利用纸基阳离子ITP分析带负电荷蛋白质的方法。在不利的阳离子ITP条件下电泳聚焦具有负电荷的靶蛋白的基本原理是通过抗原-抗体免疫结合来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纸基分析装置上进行。用（3-氨丙基）三甲氧基硅烷对纸基质进行改性，以最大限度地减少样品对阳离子ITP表面的吸引。使用带负电荷的BSA作为阳离子ITP实验的模型靶蛋白。在阳离子ITP实验条件下，未观察到仅BSA样品的电泳迁移率。然而，BSA的一抗的存在显著改善了靶蛋白的电动行为。将与富含胺的量子点缀合的二抗添加到样品中进一步促进了ITP的浓缩效果，减少了实验时间，并提高了堆积比。在我们优化的实验条件下，阳离子ITP为基础的纸设备在不到7分钟的时间内堆叠了94%的加载BSA。我们的研究结果表明，该技术具有广泛的潜力，用于快速和具有成本效益的等速电泳分析的多重蛋白质生物标志物的血清样品在护理点。
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.