Fast immunoassay for microfluidic western blotting by direct deposition of reagents onto capture membrane,Analytical Methods

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Fast immunoassay for microfluidic western blotting by direct deposition of reagents onto capture membrane
Analytical Methods ( IF 2.7 ) Pub Date : 2020/03/04 , DOI: 10.1039/d0ay00207k
Natalie E Arvin ₁ , Mohamed Dawod _{1,

2} , Don T Lamb ₃ , Jon P Anderson ₃ , Michael D Furtaw ₃ , Robert T Kennedy _{1,

4}

Affiliation

Western blotting is a widely used protein assay platform, but the technique requires long analysis times and multiple manual steps. Microfluidic systems are currently being explored for increased automation and reduction of analysis times, sample volumes, and reagent consumption for western blots. Previous work has demonstrated that proteins separated by microchip electrophoresis can be captured on membranes by dragging the microchip outlet across the membrane. This process reduces the separation and transfer time of a western blot to a few minutes. To further improve the speed and miniaturization of a complete western blot, a microscale immunoassay with direct deposition of immunoassay reagents has been developed. Flow deposition of antibodies is used to overcome diffusion limited binding kinetics so that the entire immunoassay can be completed in 1 h with detection sensitivity comparable to incubation steps requiring 20 h. The use of low microliter per min flow rates with antibody reagents applied directly and locally to the membrane where the target proteins have been captured, reduced antibody consumption ∼30-fold. The complete western blot was applied to the detection of GAPDH and β-tubulin from A431 cell lysate.

中文翻译：

通过将试剂直接沉积到捕获膜上进行微流控蛋白质印迹的快速免疫分析

蛋白质印迹是一种广泛使用的蛋白质检测平台，但该技术需要较长的分析时间和多个手动步骤。目前正在探索微流体系统，以提高自动化程度并减少蛋白质印迹的分析时间、样品量和试剂消耗。先前的工作已经证明，通过将微芯片出口拖过膜，可以将微芯片电泳分离的蛋白质捕获在膜上。此过程将蛋白质印迹的分离和转移时间缩短至几分钟。为了进一步提高完整蛋白质印迹的速度和小型化，开发了一种直接沉积免疫测定试剂的微型免疫测定法。抗体的流动沉积用于克服扩散限制的结合动力学，因此整个免疫测定可以在 1 小时内完成，检测灵敏度与需要 20 小时的孵育步骤相当。使用每分钟低微升流速，将抗体试剂直接局部施加到捕获目标蛋白的膜上，可将抗体消耗量减少约 30 倍。应用完整的蛋白质印迹法检测 A431 细胞裂解液中的 GAPDH 和 β-微管蛋白。

更新日期：2020-03-27

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