Micro and Nanofluidic Methods for Studying Supramolecular Assembly

本项目重点发展用于研究超分子蛋白质组装的微流体和纳米流体方法。我们的目标是通过利用电泳、静电和水动力将蛋白质和蛋白质复合物捕获在使用软光刻技术制造的微流体装置的纳米结处，从而识别复杂混合物中的蛋白质和蛋白质复合物。这种方法将允许对感兴趣的物种进行选择性富集，从而使我们即使使用稀释的分析物溶液也能达到非常高的灵敏度，从而允许识别少数错误折叠的蛋白质和错误组装的蛋白质复合物。作为该项目的一部分，我们将开发纳米和微流控元件在芯片上有效集成的方法。这一目标将通过使用双光子光刻技术来克服非线性光学的衍射极限，并在用于传统微加工的光刻胶中定义纳米级特征来实现。在软光刻工艺中，光刻图纹基底将用于压印聚合物，从而产生可扩展的途径来制造集成纳米和微尺度特征的器件。我们将通过深紫外荧光检测蛋白质，这将使我们能够与完全未标记的物种一起工作。这些结果将为在其天然环境中以高分辨率定量表征蛋白质自组装开辟道路。

This project focuses on the development of micro and nanofluidic methods for the study of supramolecular protein assembly. We will aim to identify within a complex mixture proteins and protein complexes by trapping them, using electrophoretic, electrostatic and hydrodynamic forces, at nanojunctions on microfluidic devices fabricated using soft lithographic techniques. This approach will allow the selective enriching of species of interest and will thus allow us to reach very high sensitivities even with dilute analyte solutions, allowing minority populations of misfolded proteins and incorrectly assembled protein complexes to be identified. As part of the project we will develop approaches for effective integration of nano and microfluidic elements on chip. This objective will be achieved through the use of two-photon lithography to overcome the diffraction limit through non-linear optics and to define nanoscale features in the same photoresist used for conventional microfabrication. The lithographically patterned substrate will then be used to imprint polymers in a soft lithography process to yield a scalable avenue to fabricate devices integrating nano and micro scale features. We will detect proteins through deep UV fluorescence, which will allow us to work with fully unlabelled species. These results will open up the path towards quantitative charaterisation of protein self-assembly at high resolution in their native environment.