Integrated microstructures for label-free interrogation of protein conformational dynamics by plasmon-enhanced THz spectroscopy

蛋白质的构象组织和动力学至关重要地决定了它们的生物学功能和它们在疾病中的功能障碍。当前的结构生物学技术在高度动态或非结构化的情况下基本上是失败的。太赫兹光谱作为一种补充方法，对蛋白质的集体振动模式、电荷分布和水合作用高度敏感，有望成为研究结构柔性蛋白质的新方法。虽然太赫兹光谱学在蛋白质构象和构象动力学的无标记分析方面的独特潜力被广泛接受，但传统太赫兹测量所需的非常高的数量和浓度仍然严重限制了其在生物学和医学相关靶蛋白上的应用。我们的项目旨在通过全面的太赫兹传感器设计来克服这一限制，该传感器专门用于低量和低浓度蛋白质的光谱分析。通过膜生物学（UOS）、计算物理学（UKS）和硅工程学（IHP）的跨学科方法，该项目旨在建立一个基于硅CMOS兼容、谐振太赫兹近场光学的高性能、低成本的太赫兹蛋白质传感器平台。在项目的前24个月，我们通过英国和IHP之间的密切合作，成功地设计和制造了带有太赫兹微谐振器的Ge/Si微结构，这些微结构在材料特性和太赫兹共振方面具有特征。IHP和UOS共同开发了Ge/Si微结构的材料和几何特异性表面修饰，可以直接从细胞中捕获特定位点的蛋白质和在共振热点中进行样品浓缩。这些努力将导致使用Ge微谐振器演示太赫兹感应蛋白质的概念验证。在项目的第二阶段，我们将重点优化材料性能和传感器设计。通过加入金属纳米粒子和欺骗等离子体结构，将实现进一步的场增强和灵敏度。为了进一步提高信噪比，我们将使用光聚合的功能化水凝胶，用蛋白质样品和包含金属纳米颗粒的混合表面结构覆盖整个传感器热点。先进的表面功能化将与传感器设计相结合，方便通过微流体处理样品。使用一组具有代表性的模型蛋白质覆盖结构定义良好，灵活和内在无序的蛋白质，我们将探索太赫兹微器件的功能和局限性。

The conformational organization and dynamics of proteins critical determine their biological function and their malfunction in diseases. Current structural biology techniques largely fail when it comes to highly dynamic or largely unstructured cases. As a complementary approach, THz spectroscopy holds tremendous promises as a new approach to study structurally flexible proteins as it is highly sensitive to collective vibrational modes, charge distribution and hydration of proteins. While the unique potential of THz spectroscopy for label-free interrogation of protein conformations and conformational dynamics is broadly accepted, the application to biologically and medically relevant target proteins is still severely limited by the very high quantities and concentrations required for traditional THz measurements. Our project aims to overcome this limitation by a comprehensive THz sensor design dedicated for spectroscopic analysis of proteins available in low amounts and concentrations. By an interdisciplinary approach between membrane biology (UOS), computational physics (UKS) and Si engineering (IHP), the project targets to set up a high performance, cost-effective THz protein sensor platform based on Si CMOS compatible, resonant THz near field optics. During the first 24 month of the project, we have by close collaboration between UKS and IHP successfully designed and fabricated Ge/Si microstructures with THz microresonators that were characterized with respect to material properties and THz resonance. IHP together with UOS developed material- and geometry-specific surface modification of Ge/Si microstructures that allowed site-specific protein capturing and sample concentration in resonance hotspots directly from cells. These efforts will lead to the proof-of-concept to demonstrate THz sensing of proteins using Ge microresonators. In the second phase of the project, we will focus on optimizing material properties and sensor design. By including metallic nanoparticles and spoof plasmonic structures, further field enhancement and sensitivity will be achieved. To further increase signal to noise, we will use functionalized hydrogels by photopolymerization to cover the entire sensor hots with protein samples and hybrid surface architectures incorporating metallic nanoparticles. Advanced surface functionalization will be combined with sensor designs that facilitate sample handling via microfluidics. Using a set of representative model proteins cover structurally well defined, flexible and intrinsically disordered proteins, we will explore capabilities and limitations of our THz microdevices.