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

对蛋白质结构及其对刺激或其他分子的动态反应的基本知识对于包括医学诊断和治疗在内的许多应用是重要的。本研究旨在开发一种高灵敏度的方法来研究人血清转铁蛋白(HSTF)，它是血液中一种重要的铁载体，具有临床意义。这项传感技术将使游离的HSTF蛋白与铁结合蛋白区分开来，并从非常少量的血液样本中评估铁缺乏或铁超载。这种传感器的成功开发还将使对广泛的其他蛋白质和生物分子的分析成为可能，例如DNA。该项目结合了生物化学、纳米工程、光子学和电子工程，为跨学科研究培训提供了极好的机会。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，K-12学校的外展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。堆叠式等离子体纳米传感器基于自诱导反作用(SIBA)驱动纳米孔电泳(SANE)传感概念。堆叠的纳米孔具有独特的设计，可以实现1)蛋白质或底物结合蛋白质复合体的受控捕获、释放和重新捕获，2)生物分子的瞬时变形，这可以由热效应或光学和电学技术的组合引起，以及3)它们的变形动力学研究。堆叠式纳米孔传感器中实现的SANE概念允许在浓度低于大块溶液中平衡解离常数1000倍的情况下研究蛋白质相互作用，使这项技术变得超灵敏。本研究的一个重要目的是利用SANE传感器研究游离HSTF蛋白和铁结合蛋白复合体的性质。为每个物种建立了光学特征轮廓，以使结合的复合体能够选择性地进入混合溶液中的未结合的蛋白质到下面的孔中。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、蛋白质松弛时间，以及电压诱导的蛋白质展开是否可逆。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.