Biosensing

We have used and developed enzyme-switch assays for quick, easy, and sensitive analyte measurement. Traditional immunoassays require a number of wash steps, necessitating manual handling or complex automation systems. Our developed enzyme-switches are wash free, enabling simple analyte measurement for use in a wide range of settings, including low-resource settings and at the point-of-care.

The "BLA-BLIP" assay was first developed in 2013 by Maarten Merkx group (ACS Chem. Biol. 2013, 8, 10, 2127–2132). It utilises TEM-1β-lactamase (BLA) and its inhibitor β-lactamase inhibitor protein (BLIP), which are tethered together with a long flexible linker containing two biorecognition elements. In the absence of any analyte, BLIP inhibits BLA activity resulting in no turnover of the nitrocefin substrate, resulting in no signal generated. In the presence of the target analyte, the two biorecognition elements bind to the target and pull BLA and BLIP apart. This allows BLA to hydrolyse nitrocefin, resulting in a colour change from yellow to red (Figure 1). We have incorporated Affimers as biorecognition elements in the BLA-BLIP system for the detection of a number of analytes. Affimers are non-immunoglobulin-binding proteins and offer the benefit of being small, stable, and easily expressed as recombinant proteins when fused genetically to split-enzyme fragments.

Figure 1 - Schematic of BLA-BLIP enzyme switch utilising Affimers as biorecognition elements (ACS Sens. 2019, 4, 11, 3014–3022).

The NanoBit enzyme-switch sensor combines Affimer proteins as biorecognition elements with a split-luciferase, NanoLuc® Binary Technology (NanoBiT, Promega). The NanoBit system relies on the NanoLuc split-luciferase enzyme (ACS Chem. Biol. 2016, 11, 2, 400–408). The NanoLuc enzyme is comprised of the LgBit, which is the majority of the enzyme, and the SmBit, which is a 13-amino acid peptide cleaved off the rest of the enzyme. Without the SmBit, the LgBit has no enzymatic activity and cannot turn over its furimazine substrate to generate a signal. We have recombinantly fused Affimer biorecognition elements to the LgBit and the SmBit, respectively, via a semi-flexible peptide linker (Figure 2). In the presence of the target analyte, the LgBit and SmBit are brought together allowing recomplementation of the NanoLuc enzyme and generation of a bioluminescent signal.

Figure 2 - Schematic of the NanoBit split-luciferase assay.

Therapeutic drug monitoring (TDM) is the practice of measuring drug concentrations in patient samples and adjusting dosing towards an optimal concentration, to personalise care. TDM is a complicated intervention that requires taking blood samples at specified points across the therapy, accurately measuring the active drug in the sample, calculating the amount of drug to give the patient in the next dose, and reporting results and recommendations to clinicians. Currently, TDM is challenging as standard techniques for measuring concentrations, particularly for small molecule drugs, can be prohibitive for TDM. Liquid chromatography coupled mass spectrometry (HPLC-MS/MS) is the gold-standard for small molecule measurement. HPLC-MS/MS requires specialised facilities and staff, it is expensive and introduces more logistical challenges in sending samples to centralised laboratories.

We are working towards the implementation of our biosensor technology for quick and accessible TDM for a wide range of clinical settings, including at the point-of-care. One area we are currently focusing on is the personalisation of antibiotics in critical care. Antimicrobial resistance (AMR) is driven, in part, by inappropriate dosing of antibiotics. Too little antibiotic can select for resistant bacterial populations, exacerbating the emergence of resistance. Too much antibiotic can result in toxicities including neurotoxicity and renal toxicity. β-lactams are the most widely used group of antibiotics that include penicillins, cephalosporins, and carbapenems. Most patients receive a standard dose of β-lactam antibiotics, however evidence shows that most patients receive inappropriate amounts of antibiotic due to variable pharmacologies between patients, making it not possible to predict β-lactam concentrations from the amount that is given. Some patients get rid of the drug very quickly, in others it accumulates. A rapid biosensor that can be used in any clinical laboratory or at the point-of-care could help guide clinician dosing to improve care and reduce the emergence of AMR.