Development and characterisation of a compact device for rapid real-time-on-chip detection of thrombin activity in...

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.