Collaborative Research: A Stacked Plasmonic Nanopore for Tether-Free Stretching and Label-Free Sensing of hSTf Dynamics and Complex Formation at Ultra-Low Concentrations

蛋白质结构及其对刺激或其他分子的动态响应的基础知识对于许多应用都很重要，包括医学诊断和治疗。 本研究的目的是建立一种高灵敏度的方法来研究人血清转铁蛋白（hSTf），这是一个重要的铁载体在血液中和临床重要性。 该传感技术将允许从铁结合蛋白中区分游离hSTf蛋白，并从非常小的血液样品中评估铁缺乏或铁过载。 这种传感器的成功开发也将使广泛的其他蛋白质和生物分子，例如，DNA. 该项目为跨学科研究培训提供了极好的机会，因为它结合了生物化学，纳米工程，光子学和电气工程。 通过南卫理公会大学和德克萨斯大学阿灵顿分校的各种项目，对K-12学校的推广工作有助于激励更多的学生攻读科学、技术、工程和数学（STEM）学位。堆叠的等离子体纳米传感器基于自诱导反作用（SIBA）驱动的纳米孔电泳（SANE）传感概念。 堆叠的纳米孔被独特地设计成能够实现1）蛋白质或基底结合的蛋白质复合物的受控捕获、释放和重新捕获，2）生物分子的瞬时变形，其可以由热效应或光学和电学技术的组合诱导，以及3）它们的变形动力学的研究。 在堆叠的纳米孔传感器中实施的SANE概念允许在本体溶液中低于平衡解离常数1000倍的浓度下研究蛋白质相互作用，使得该技术超灵敏。 本研究的一个重要目的是使用SANE传感器研究游离hSTf蛋白和铁结合蛋白复合物的性质。 光学签名配置文件建立的每一个物种，使选择性准入的结合复合物在未结合的蛋白质在混合溶液中的底层孔。它使用对称（VCapture = VRecapture），然后是不对称（VCapture VRecapture）电压条件，以便于研究与蛋白质-底物结合相关的强度和动力学参数，蛋白质弛豫时间，以及电压-该奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准。

Fundamental knowledge of protein structures and their dynamic responses to stimuli or other molecules is important for many applications, including medical diagnosis and therapy. This research aims to develop a highly sensitive approach for studying the human serum transferrin protein (hSTf), which is a vital iron carrier in blood and of clinical importance. The sensing technique would allow differentiation of the free hSTf protein from the iron-bound protein and evaluation of iron deficiency or iron overload from very small blood samples. Successful development of this sensor would also enable profiling of a wide range of other proteins and biological molecules, e.g., DNA. This project offers excellent opportunities for interdisciplinary research training as it combines biochemistry, nanoengineering, photonics, and electrical engineering. The outreach efforts to K-12 schools through various programs at the Southern Methodist University and the University of Texas at Arlington help to inspire more students to pursue science, technology, engineering and mathematics (STEM) degrees.The stacked plasmonic nanosensor is based on the self-induced back-action (SIBA) actuated nanopore electrophoresis (SANE) sensing concept. The stacked nanopores are uniquely designed to enable 1) controlled trapping, releasing, and recapturing of proteins or the substrate-bound protein complexes, 2) transient deformation of the biological molecules, which can be induced by thermal effect or a combination of optical and electrical techniques, and 3) study of their deformation dynamics. The SANE concept implemented in the stacked nanopore sensor allows investigation of protein interactions at concentrations 1000-fold below the equilibrium dissociation constant in bulk solution, making this technique ultra-sensitive. An important aim of this research is the study of the properties of free-hSTf protein and the iron-bound protein complex using the SANE sensor. Optical signature profiles are established for each of the species to enable selective admission of bound complexes over unbound proteins in a mixed solution to the underlying pore. It uses symmetric (VCapture = VRecapture), followed by asymmetric (VCapture ≠ VRecapture) voltage conditions to facilitate the investigation of the strength and kinetic parameters associated with protein-substrate binding, protein relaxation times, and whether voltage-induced protein unfolding is reversible or not.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.