Development of high-throughput, high-sensitivity EPR sample handling capabilities for biomedical research

项目概要/摘要 电子顺磁共振（EPR）谱是生物医学研究中的一项重要技术 具有独特的能力来检测复杂生物学中天然存在或工程化的未配对电子， 环境. EPR在结构生物学、金属蛋白质研究、氧化还原生物学 合理的药物设计和临床诊断。样品体积要求的突破性进展 用于生物医学EPR光谱学应用是近四十年前随着 环隙谐振器（LGR），代表了突破性的优势，例如样品体积减少10倍 这可以满足更高的要求和更高的谐振器效率，以增加通常使用的空腔谐振器上的信号。到 适应甚至进一步增加信号强度的生物医学需求，并提供更广泛的 科学界的技术，我们建议利用我们最近的发展， 介电LGR（dLGR）概念并优化此谐振器技术以提高灵敏度 在引入LGR后实现。dLGR实际上是一个放置在内部的小型介电谐振器 LGR的回路，其中电介质的返回通量流过LGR的外部回路。解析理论 我们的高频结构模拟表明，dLGR使数量级 灵敏度优于LGR。为了充分利用极低的体积要求， dLGR，我们建议开发有效的样品处理技术，将dLGR与高- 通量样品处理仪器。我们的目标是开发两种变革性的生物医学技术， EPR应用：i）对于约0.2 µL样品体积，灵敏度提高10倍的纳米dLGR 使用定制的自动进样器; ii）微型dLGR，可显著提高~2 µL样品的灵敏度 使用优化的停流系统进行毫秒级时间尺度动力学测量。这些 具有杰出灵敏度的变革性和创新性原型将易于使用，并最终广泛使用 提供给科学界。与腔相比，LGR是一个变革性的进步 因此，dLGR有望成为LGR的又一次变革性飞跃。

Project Summary/Abstract Electron paramagnetic resonance (EPR) spectroscopy is a critically important technique in biomedical research with a unique ability to detect naturally occurring or engineered unpaired electrons in complex biological environments. EPR has wide-ranging applicability to structural biology, metalloprotein research, redox biology, rational drug design, and clinical diagnostics. Groundbreaking advancements in sample volume requirements for biomedical EPR spectroscopy applications were made nearly four decades ago with the development of the loop-gap resonator (LGR), which represented breakthrough benefits such as 10-fold lower sample volume requirements and higher resonator efficiencies to increase signals over the commonly used cavity resonator. To accommodate the biomedical needs of even further increased signal intensity and to provide the broader scientific community with accessible technology, we propose to capitalize on our recent development of the dielectric LGR (dLGR) concept and optimize this resonator technology to increase sensitivity beyond that achieved upon introduction of the LGR. A dLGR is effectively a small dielectric resonator placed inside the inner loop of an LGR where the return flux of the dielectric flows through the outer loops of the LGR. Analytic theory and our high-frequency structure simulations indicate that the dLGR enables an order-of-magnitude improvement in sensitivity over the LGR. To take full advantage of the extremely low volume requirements for the dLGR, we propose to develop efficient sample handling technologies that couple the dLGR to high- throughput sample handling instrumentation. We aim to develop two transformative technologies for biomedical EPR applications: i) a nano-dLGR with a 10-fold increase in sensitivity for ~0.2 µL sample volumes integrated with a customized autosampler and ii) a micro-dLGR for a dramatic increase in sensitivity for ~2 µL sample volumes with an optimized stopped-flow system for millisecond time scale kinetics measurements. These transformative and innovative prototypes with outstanding sensitivity will be easy to use and ultimately widely available to the scientific community. Where the LGR was a transformative advance compared with cavity resonators, the dLGR is expected to be another transformative leap from an LGR.