Comparison of Static and Microfluidic Protease Assays Using Modified Bioluminescence Resonance Energy Transfer Chemistry

Abstract

BackgroundFluorescence and bioluminescence resonance energy transfer (F/BRET) are two forms of Förster resonance energy transfer, which can be used for optical transduction of biosensors. BRET has several advantages over fluorescence-based technologies because it does not require an external light source. There would be benefits in combining BRET transduction with microfluidics but the low luminance of BRET has made this challenging until now.MethodologyWe used a thrombin bioprobe based on a form of BRET (BRETH), which uses the BRET1 substrate, native coelenterazine, with the typical BRET2 donor and acceptor proteins linked by a thrombin target peptide. The microfluidic assay was carried out in a Y-shaped microfluidic network. The dependence of the BRETH ratio on the measurement location, flow rate and bioprobe concentration was quantified. Results were compared with the same bioprobe in a static microwell plate assay.Principal FindingsThe BRETH thrombin bioprobe has a lower limit of detection (LOD) than previously reported for the equivalent BRET1–based version but it is substantially brighter than the BRET2 version. The normalised BRETH ratio of the bioprobe changed 32% following complete cleavage by thrombin and 31% in the microfluidic format. The LOD for thrombin in the microfluidic format was 27 pM, compared with an LOD of 310 pM, using the same bioprobe in a static microwell assay, and two orders of magnitude lower than reported for other microfluidic chip-based protease assays.ConclusionsThese data demonstrate that BRET based microfluidic assays are feasible and that BRETH provides a useful test bed for optimising BRET-based microfluidics. This approach may be convenient for a wide range of applications requiring sensitive detection and/or quantification of chemical or biological analytes.

Sub-nanomolar detection of thrombin activity on a microfluidic chip

“…3 We believe that the higher sensitivity observed here is probably due to the smaller detection volume (6.28 ll versus 100 ll in the microplate), which minimises self-absorption effects, and the constant refreshment of the thrombin biosensor and substrate with the continuous removal of potentially inhibiting reaction products. The LOD is also approximately 1.5 fold lower than that recently obtained using the BRET H system with a microscope 12 despite the lower luminance of the BRET 2 system and with the considerable …”

“…Two BRET systems were used in this study, namely, hybrid BRET (BRET H ) 12 and BRET 2 . The two systems have the same BRET construct.…”

“…[5][6][7][8][9][10] BRET 2 is ideally matched to the scale of many protein interactions and rearrangements due to its relatively long Förster distance but has relatively low luminance arising from the low quantum yield of the substrate oxidation. 11 Recently, we reported a microscope-based microfluidic system for BRET-based biosensing, 12 , we made two changes to the design of the detection system. First, we increased the volume of reaction that is optically sampled by incorporating a cylindrical chamber ("BRET reaction chamber"), perpendicular to the common direction of flow with a total volume of 2p ll.…”

“…[13][14][15][16][17] However, there are few reports of research into the use of optical fibres for bioluminescence resonance energy transfer detection on a microfluidic chip. The use of an optical fiber for light harvesting increases the photon collection efficiency compared to a microscopy 12 and markedly simplifies the optics, paving the way for multiplexed detection.…”

Progress in chemical luminescence-based biosensors: A critical review

“…This phenomenon has been exploited in a variety of bioanalytical formats including microtiter plate (96-and 384-well), microarrays, microfluidics, paper-based devices, and in vitro microscopy imaging (Marquette et al, 2012;Seidel and Niessner, 2014;Mirasoli et al, 2014b). Chemiluminescence labels can categorized as direct chemical or enzyme-based.…”