Bioluminescence resonance energy transfer (BRET) is a sensitive optical detection method that can monitor changes in the relative orientation and the physical proximity of molecules in real-time. Since the light is generated internally by a bioluminescent protein, BRET does not rely on an external light source. The use of BRET simultaneously simplifies the hardware required for sensing and offers improved detection limits and sensitivity for applications targeting point-of-care bio-sensing. In this paper, we report a compact micro reactor integrating a thermostat with a re-useable glass-chip comprising a chaotic mixer, an incubation channel and optical detection chamber. The device was optimised to detect thrombin activities in serum, achieving a thrombin detection limit of 38 μU/μl in 10% (v/v) human serum in a 5 min assay time. This is a 90% assay time reduction, compared with previous BRET-based work or other technologies. It matches sensitivity levels achieved when the assay is deployed on a commercially available plate-reader. The device can be used continuously with low concentrations (3.4 μM) of luciferase substrate. The low cost associated with this approach, low interference from human serum and other proteases and good reproducibility (CV = 0.2-3.6%), establish new performance standards for point-of-care diagnostics with samples of human serum. Importantly, measuring protease activity levels, rather than concentrations, is the most informative approach for clinical diagnostics. Of the recently reported ultra-sensitive thrombin sensing techniques, this is the only one to measure thrombin activity in serum dilutions, rather than simply quantifying thrombin concentrations.

中文翻译：

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使用生物发光共振能量转移（BRET）的紧凑型设备的开发和表征，该设备可快速在芯片上实时实时检测人血清中的凝血酶活性。

生物发光共振能量转移（BRET）是一种灵敏的光学检测方法，可以实时监测分子的相对方向和物理接近度的变化。由于光是由生物发光蛋白在内部产生的，因此BRET不依赖外部光源。BRET的使用可同时简化感测所需的硬件，并为针对即时医疗生物感测的应用提供更高的检测极限和灵敏度。在本文中，我们报告了一个紧凑型微型反应器，该恒温器集成了恒温器和可重复使用的玻璃片，该玻璃片包括混沌混合器，孵育通道和光学检测室。该设备经过优化，可检测血清中的凝血酶活性，在5分钟的分析时间内，在10％（v / v）人血清中的凝血酶检测限达到38μU/μl。与以前的基于BRET的工作或其他技术相比，这节省了90％的分析时间。它与将分析物部署在市售酶标仪上所达到的灵敏度水平相匹配。该设备可与低浓度（3.4μM）的荧光素酶底物连续使用。与这种方法相关的低成本，来自人血清和其他蛋白酶的干扰少，重现性好（CV = 0.2-3.6％），为使用人血清样品进行即时诊断的性能确立了新的性能标准。重要的是，测量蛋白酶活性水平而不是浓度是临床诊断中最有用的方法。在最近报道的超敏感凝血酶传感技术中，这是唯一一种可测量血清稀释液中凝血酶活性的技术，