Real-time, continuous detection of maltose using bioluminescence resonance energy transfer (BRET) on a microfluidic system.

We have previously shown that a genetically encoded bioluminescent resonance energy transfer (BRET) biosensor, comprising maltose binding protein (MBP) flanked by a green fluorescent protein (GFP(2)) at the N-terminus and a variant of Renilla luciferase (RLuc2) at the C-terminus, has superior sensitivity and limits of detection for maltose, compared with an equivalent fluorescent resonance energy transfer (FRET) biosensor. Here, we demonstrate that the same MBP biosensor can be combined with a microfluidic system for detection of maltose in water or beer. Using the BRET-based biosensor, maltose in water was detected on a microfluidic chip, either following a pre-incubation step or in real-time with similar sensitivity and dynamic range to those obtained using a commercial 96-well plate luminometer. The half-maximal effective concentrations (EC50) were 2.4×10(-7)M and 1.3×10(-7) M for maltose detected in pre-incubated and real-time reactions, respectively. To demonstrate real-time detection of maltose in a complex medium, we used it to estimate maltose concentration in a commercial beer sample in a real-time, continuous flow format. Our system demonstrates a promising approach to in-line monitoring for applications such as food and beverage processing. Crown