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

蛋白质的构象结构和动力学对其生物学功能和疾病中的功能障碍起着至关重要的作用。目前的结构生物学技术在处理高度动态或基本上非结构化的病例时很大程度上失败了。作为一种补充手段，太赫兹光谱因其对蛋白质的集体振动模式、电荷分布和水合作用高度敏感而成为研究结构柔性蛋白质的一种新方法。虽然太赫兹光谱在无标记询问蛋白质构象和构象动力学方面的独特潜力已经被广泛接受，但由于传统的太赫兹测量所需的非常高的量和浓度，其在生物和医学上相关的目标蛋白的应用仍然受到严重的限制。我们的项目旨在通过一种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数量和低浓度的可用蛋白质的光谱分析。通过膜生物学(UOS)、计算物理(UKS)和硅工程(IHP)之间的交叉学科方法，该项目的目标是建立一个高性能、高性价比的基于硅CMOS兼容的共振THz近场光学的THz蛋白质传感器平台。在该项目的头24个月里，我们通过英国和国际水文计划的密切合作，成功地设计和制造了具有THz微谐振器的Ge/Si微结构，并对材料特性和THz共振进行了表征。IHP与UOS一起开发了Ge/Si微结构的材料和几何特定表面修饰，允许直接从细胞的共振热点捕获特定位置的蛋白质和样品浓缩。这些努力将导致概念验证，以演示使用GE微谐振器对蛋白质进行太赫兹传感。在该项目的第二阶段，我们将重点优化材料性能和传感器设计。通过包括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，我们将使用光聚合功能化水凝胶，在整个传感器表面覆盖蛋白质样品和含有金属纳米颗粒的混合表面结构。先进的表面功能化将与传感器设计相结合，便于通过微流体处理样品。使用一组具有代表性的模型蛋白质，涵盖结构定义良好、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.