Developing a toll-like receptor biosensor for Gram-positive bacterial detection and its storage strategies.

The biorecognition ability of hybridized toll-like receptors (TLRs) 2 and 6 proteins on electrode surfaces has been studied. TLR biosensors have been designed to be non-specific to particular bacterial strains but rather to provide broad spectrum detection of cells and toxins containing relevant pathogen-associated molecular patterns (PAMPs). Our electrochemical TLR2/6 biosensors demonstrated selective detection towards Gram-positive bacterial whole-cells and a synthetic diacylated lipopeptide (Pam2CSK4), a PAMP. Responses towards Bacillus licheniformis (B. licheniformis) and Enterococcus hirae (E. hirae) were obtained. The biosensor was able to differentiate signals between B. licheniformis and a Gram-negative bacterial cell (control) as low as 100 CFU mL-1. One challenge in developing protein-based biosensors is to improve the shelf-life of the biosensor chips and preserve the detection activity of the protein molecules, therefore we did our first exploration into storage conditions. The activity of stored biosensors was found to be strongly dependent on storage medium, and that effective 'shelf-life' was obtained makes an important step towards creating robust sensors for real-life applications.