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

项目摘要/摘要 电子顺磁共振(EPR)谱在生物医学研究中是一项至关重要的技术 具有独特的能力来探测复杂生物中自然产生的或工程制造的未配对电子 环境。EPR在结构生物学、金属蛋白研究、氧化还原生物学、 合理的药物设计和临床诊断。样本量要求方面的突破性进展 近40年前，随着电子顺磁共振波谱技术的发展，生物医学电子顺磁共振波谱得到了应用。 环隙谐振器(LGR)，具有突破性优势，例如样本量降低10倍 要求和更高的谐振器效率，以增加通常使用的腔谐振器的信号。至 适应生物医学的需要，进一步增加信号强度，并提供更广泛的 对于拥有可获得技术的科学界，我们建议利用我们最近的发展 介质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质谐振器 LGR的一种回路，其中介质的回流磁通流经LGR的外部回路。分析理论 我们的高频结构模拟表明，dLGR能够实现数量级 比LGR的灵敏度更高。要充分利用极低的容量要求， DLGR，我们建议开发高效的样品处理技术，将DLGR耦合到高- 吞吐量样品处理仪器。我们的目标是为生物医学开发两项变革性的技术 电子顺磁共振应用：i)具有10倍灵敏度的纳米dLGR，集成了约0.2µm的L样品体积 具有定制的自动进样器和ii)微型dLGR，可大幅提高~2微米L样品的灵敏度 体积与优化的停流系统，用于毫秒时间尺度的动力学测量。这些 具有卓越灵敏度的变革性和创新性原型将易于使用，并最终得到广泛应用 可供科学界使用。与空腔相比，LGR是一种变革性的进步 对于谐振器，dLGR预计将是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.