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.

Significance: The fundamental processes of life are organized and based on common basic principles. Molecular organizers, often interacting with the membrane, capitalize on cellular polarity to precisely orientate essential processes. The study of organisms lacking apparent polarity or known cellular organizers (e.g., the bacterium Staphylococcus aureus ) may enable the elucidation of the primal organizational drive in biology. How does a cell choose from infinite locations in its membrane? We have discovered a structure in the S. aureus membrane that organizes processes indispensable for life and can arise spontaneously from the geometric constraints of protein complexes on membranes. Building on this finding, the most basic cellular positioning system to optimize biological processes, known molecular coordinators could introduce further levels of complexity.

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.

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.