A Bioluminescent Sensor for Rapid Detection of PPEP-1, a Clostridioides difficile Biomarker

Abstract

Current assays for Clostridioides difficile in nonhospital settings are outsourced and time-intensive, resulting in both delayed diagnosis and quarantining of infected individuals. We designed a more rapid point-of-care assay featuring a “turn-on” bioluminescent readout of a C. difficile-specific protease, PPEP-1. NanoLuc, a bright and stable luciferase, was “caged” with a PPEP-1-responsive peptide tail that inhibited luminescence. Upon proteolytic cleavage, the peptide was released and NanoLuc activity was restored, providing a visible readout. The bioluminescent sensor detected PPEP-1 concentrations as low as 10 nM. Sensor uncaging was achieved within minutes, and signal was captured using a digital camera. Importantly, the sensor was also functional at ambient temperature and compatible with fecal material, suggesting that it can be readily deployed in a variety of settings.

Development of Luminescent Biosensors for Calprotectin

“…To establish a user-friendly calprotectin detection method, we focused on adapting the Fab binders with the NanoBiT-based technology, in which the two binding modules are fused to LgBiT or SmBiT (VTGY­RLFE­EIL), two fragments of split NanoLuc luciferase (NanoLuc) and reconstitute an active luciferase enzyme upon dual binding to calprotectin, due to the close proximity of split NanoLuc fragments . NanoBiT-based technology has recently been developed with various synthetic scaffolds and applied for different tests, including but not limited to SARS-CoV-2 (COVID-19), , Clostridium difficile infections, , and botulinum neurotoxins . While efforts have been reported in combining NanoBiT with full-length antibodies, − the most common and versatile protein-based binder so far, such antibody engineering usually relies on chemical conjugation of antibody with LgBiT or SmBiT, which necessitates not only the costly production of antibodies, but also extensive purifications due to the heterogeneity of products.…”

Bioluminescence Goes Dark: Boosting the Performance of Bioluminescent Sensor Proteins Using Complementation Inhibitors

“…The use of autoinhibited split-NanoLuc systems introduced here is likely to be more broadly applicable, both to improve the performance of existing sensors and the construction of sensors for novel targets, provided they can be designed to undergo a sufficiently large conformational change to disrupt the LgBiT-DarkBiT interaction. For instance, intensiometric sensors such as NanoLuc-based protease sensors 32 or NanoBiT-based genetically encoded Ca 2+ sensors 33 could be improved by reducing the bioluminescent background signal in the OFF-state, resulting in higher dynamic ranges. Intramolecular inhibition of LgBiT activation could also further improve the performance of sensors based on intermolecular complexation, providing an alternative caging strategy for the one used in the lucCage system that requires sophisticated protein engineering.…”

Therapeutic drug monitoring of immunotherapies with novel Affimer–NanoBiT sensor construct

“…Binding of the recognition elements co-localises the two inactive reporter fragments, increasing their effective concentration and prompting re-assembly of the active enzyme, the activity of which can be measured. 17,18,22,27…”

“…Binding of the recognition elements colocalises the two inactive reporter fragments, increasing their effective concentration and prompting re-assembly of the active enzyme, the activity of which can be measured. 17,18,22,27 Here, we describe the development of a homogenous, split enzyme assay for the detection of four routinely used TmAb treatments (adalimumab, ipilimumab, rituximab and trastuzumab). The sensor combines Affimer proteins as recognition elements with an established split-luciferase, NanoLuc® Binary Technology (NanoBiT).…”