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

The demand for point-of-need (PON) diagnostics for clinical and other applications is continuing to grow. Much of this demand is currently serviced by biosensors, which combine a bioanalytical sensing element with a transducing device that reports results to the user. Ideally, such devices are easy to use and do not require special skills of the end user. Application-dependent, PON devices may need to be capable of measuring low levels of analytes very rapidly, and it is often helpful if they are also portable. To date, only two transduction modalities, colorimetric lateral flow immunoassays (LFIs) and electrochemical assays, fully meet these requirements and have been widely adopted at the point-of-need. These modalities are either non-quantitative (LFIs) or highly analyte-specific (electrochemical glucose meters), therefore requiring considerable modification if they are to be co-opted for measuring other biomarkers. Förster Resonance Energy Transfer (RET)-based biosensors incorporate a quantitative and highly versatile transduction modality that has been extensively used in biomedical research laboratories. RET-biosensors have not yet been applied at the point-of-need despite its advantages over other established techniques. In this review, we explore and discuss recent developments in the translation of RET-biosensors for PON diagnoses, including their potential benefits and drawbacks.

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).

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.

A red-shifted Bioluminescence Resonance Energy Transfer (BRET) biosensing system for rapid measurement of plasmin activity in human plasma.

Proteases are key signalling molecules for many physiological processes and their dysregulation is implicated in the progression of a range of diseases. Sensitive methods to measure protease activities in complex biological samples are critical for rapid disease diagnoses. The proteolytic activity of plasmin reflects the fibrinolysis state of blood and its deregulation can indicate pathologies such as bleeding events. While Bioluminescence Resonance Energy Transfer (BRET) is a powerful and sensitive method for the detection of protease activity, the commonly applied blue-shifted BRET2 system, consisting of the Renilla luciferase Rluc2 and the large-stokes shift fluorescent protein GFP2, suffers from light absorption and light scattering in human plasma samples. To address this challenge, we developed a red-shifted BRET-based plasmin sensor by substituting BRET2 with the BRET6 system. BRET6 is composed of the red-shifted RLuc8.6 luciferase linked to the red light emitting fluorescent protein TurboFP635. The BRET6 biosensor exhibited 3-fold less light absorption in plasma samples compared to the BRET2 sensor leading to an up to a 5-fold increase in sensitivity for plasmin detection in plasma. The limits of detection for plasmin were determined to be 11.90 nM in 7.5% (v/v) plasma with a 10 min assay which enables biologically relevant plasmin activities of thrombolytic therapies to be detected. While a colorigenic plasmin activity assay achieved a similar detection limit of 10.91 nM in 7.5% (v/v) human plasma, it required a 2 h incubation period. The BRET6 sensor described here is faster and more specific than the colorigenic assay as it did not respond to unspiked human plasma samples.

Experimental determination of the bioluminescence resonance energy transfer (BRET) Förster distances of NanoBRET and red-shifted BRET pairs.

Bioluminescence Resonance Energy Transfer (BRET) is widely applied to study protein-protein interactions, as well as increasingly to monitor both ligand binding and molecular rearrangements. The Förster distance (R0) describes the physical distance between the two chromophores at which 50% of the maximal energy transfer occurs and it depends on the choice of RET components. R0 can be experimentally determined using flexible peptide linkers of known lengths to separate the two chromophores. Knowledge of the R0 helps to inform on the choice of BRET system. For example, we have previously shown that BRET2 exhibits the largest R0 to date for any genetically encoded RET pair, which may be advantageous for investigating large macromolecular complexes if its issues of low and fast-decaying bioluminescence signal can be accommodated. In this study we have determined R0 for a range of bright and red-shifted BRET pairs, including NanoBRET with tetramethylrhodamine (TMR), non-chloro TOM (NCT), mCherry or Venus as acceptor, and BRET6, a red-shifted BRET2-like system. This study revealed R0 values of 6.15 nm and 6.94 nm for NanoBRET using TMR or NCT as acceptor ligands, respectively. R0 was 5.43 nm for NanoLuc-mCherry, 5.59 nm for NanoLuc-Venus and 5.47 nm for BRET6. This extends the palette of available BRET Förster distances, to give researchers a better-informed choice when considering BRET systems and points towards NanoBRET with NCT as a good alternative to BRET2 as an analysis tool for large macromolecular complexes.

Heterogeneous localisation of membrane proteins in Staphylococcus aureus.

The bacterial cytoplasmic membrane is the interface between the cell and its environment, with multiple membrane proteins serving its many functions. However, how these proteins are organised to permit optimal physiological processes is largely unknown. Based on our initial findings that 2 phospholipid biosynthetic enzymes (PlsY and CdsA) localise heterogeneously in the membrane of the bacterium Staphylococcus aureus, we have analysed the localisation of other key membrane proteins. A range of protein fusions were constructed and used in conjunction with quantitative image analysis. Enzymes involved in phospholipid biosynthesis as well as the lipid raft marker FloT exhibited a heterogeneous localisation pattern. However, the secretion associated SecY protein, was more homogeneously distributed in the membrane. A FRET-based system also identified novel colocalisation between phospholipid biosynthesis enzymes and the respiratory protein CydB revealing a likely larger network of partners. PlsY localisation was found to be dose dependent but not to be affected by membrane lipid composition. Disruption of the activity of the essential cell division organiser FtsZ, using the inhibitor PC190723 led to loss of PlsY localisation, revealing a link to cell division and a possible role for FtsZ in functions not strictly associated with septum formation.

Supramolecular structure in the membrane of Staphylococcus aureus.

All life demands the temporal and spatial control of essential biological functions. In bacteria, the recent discovery of coordinating elements provides a framework to begin to explain cell growth and division. Here we present the discovery of a supramolecular structure in the membrane of the coccal bacterium Staphylococcus aureus, which leads to the formation of a large-scale pattern across the entire cell body; this has been unveiled by studying the distribution of essential proteins involved in lipid metabolism (PlsY and CdsA). The organization is found to require MreD, which determines morphology in rod-shaped cells. The distribution of protein complexes can be explained as a spontaneous pattern formation arising from the competition between the energy cost of bending that they impose on the membrane, their entropy of mixing, and the geometric constraints in the system. Our results provide evidence for the existence of a self-organized and nonpercolating molecular scaffold involving MreD as an organizer for optimal cell function and growth based on the intrinsic self-assembling properties of biological molecules.

Intracellular monitoring of target protein production in Staphylococcus aureus by peptide tag-induced reporter fluorescence.

An intracellular approach for monitoring protein production in Staphylococcus aureus is described. mCherry, fused to the dodecapeptide Tip, was capable of inducing tetracycline repressor (TetR). Time- and concentration-dependent production of mCherry could be correlated to TetR-controlled GFPmut2 activity. This approach can potentially be extended to native S. aureus proteins.