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决定了它们的生物学功能及其在疾病中的功能障碍。目前的结构生物学技术在高度动态或大部分非结构化的情况下基本上失败了。作为一种补充方法，太赫兹光谱作为一种新的方法来研究结构灵活的蛋白质，因为它是高度敏感的集体振动模式，电荷分布和蛋白质的水化具有巨大的希望。虽然THz光谱用于蛋白质构象和构象动力学的无标记询问的独特潜力被广泛接受，但传统THz测量所需的非常高的数量和浓度仍然严重限制了生物和医学相关目标蛋白的应用。我们的项目旨在通过一种全面的THz传感器设计来克服这一限制，该传感器专门用于对低含量和低浓度的蛋白质进行光谱分析。该项目通过膜生物学（UOS）、计算物理学（UKS）和硅工程学（IHP）之间的跨学科方法，目标是建立一个基于硅CMOS兼容的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UKS和IHP之间的密切合作，成功地设计和制造了具有THz微谐振器的Ge/Si微结构，其特征在于材料特性和THz谐振。IHP与UOS一起开发了Ge/Si微结构的材料和几何结构特异性表面改性，允许直接从细胞中在共振热点中进行位点特异性蛋白质捕获和样品浓缩。这些努力将导致概念验证，以证明使用Ge微谐振器的蛋白质THz传感。在项目的第二阶段，我们将专注于优化材料性能和传感器设计。通过包括金属纳米颗粒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，我们将使用功能化的水凝胶通过光聚合来覆盖整个传感器热与蛋白质样品和混合表面架构，包括金属纳米粒子。先进的表面功能化将与通过微流体促进样品处理的传感器设计相结合。使用一组代表性的模型蛋白质涵盖结构良好定义，灵活和内在无序的蛋白质，我们将探索我们的太赫兹微器件的能力和局限性。

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.