Development and characterisation of a compact device for rapid real-time-on-chip detection of thrombin activity in human serum using bioluminescence resonance energy transfer (BRET)

Resonance Energy Transfer-Based Biosensors for Point-of-Need Diagnosis—Progress and Perspectives

“…Their low optical signal output was particularly challenging for miniaturized applications, where low amounts of biosensors are used. However, considerable progress has been made in recent years in the development of brighter and more stable luciferaseluciferin combinations [44,45] coupled to superior acceptors [46,47] and in combination with sensitive BRET detection devices [48].…”

“…(c) CYBERTONGUE ® protease biosensors consist of the Renilla luciferase RLuc8 connected through a peptide linker, containing specific recognition sites for the target protease, to the Green Fluorescent Protein variant GFP 2 . Proteolytic activity exerted on the connecting peptide results in the dissociation of GFP 2 from RLuc8, leading to a profound change in BRET ratio [48,55,56]. (d) The CYBERTONGUE ® lactose biosensor consists of a lactose-binding protein tagged with RLuc8 and GFP 2 that undergoes a conformational change upon binding to lactose [57].…”

“…In order to shrink the device's footprint, micro photon multiplier tubes, instead of large and energy-intensive valve-based PMTs, were placed directly above and below the sample detection chamber. This was implemented within a controlled microenvironment enabled by a thermoelectric block bringing the concept of a compact table top device, termed the Cybertongue ® device, to fruition [48] (Figure 3a). The Cybertongue ® device is a microfluidics-based platform that can run a variety of homogeneous sensing applications tailored to different analyte types with assay times of 1-10 min.…”

“…Striking progress has been seen in the field of RET biosensors that do not require an external light source, especially for biosensors incorporating BRET as the transduction modality. The Cybertongue ® technology is approaching PON acceptance through a solid state BRET detection system within a microfluidics-and temperature-controlled reaction environment [48]. This system can be applied at the point-of-need, for instance in food production facilities, with minimum sample preparation and limited need for operator training while maintaining highly reproducible results.…”

“…Overview of the Cybertongue ® BRET analysis device: (a) functional schematic of the measurement device, (b) schematic design of a microfluidic chip used for protease assays and (c) image of compact microfluidics device with closed lid. Figure was taken from Weihs et al[48], with permission.…”