The rapid and accurate detection of bacteria is of great concern in both clinical and defence settings. However, to date, the gold standard in the detection of bacteria remains the method of culturing bacteria in a laboratory setting. This process is slow and laborious, often taking multiple days before confirmation is given. This delay between presentation and identification is detrimental in both settings, as patients could go untreated or, in the case of biological terrorism or warfare, many potential victims could be exposed to a pathogen. As such, there is a need for the development of a rapid and sensitive detection system capable of identifying the presence of bacteria outside of a clinical laboratory.

In the human body, this function is fulfilled by the innate immune system, which responds to pathogen-associated molecular patterns (PAMPs) that are present on broad classifications of biological threats. For example, Toll-Like Receptor-4 (TLR-4), a pattern recognition receptor in the innate immune system, responds to the presence of lipopolysaccharide, the PAMP displayed on Gram-negative bacteria. As Gram-negative and Gram-positive bacteria can be treated with different antibiotics, even this broad distinction can be medically relevant.

TLR-4 and the other proteins in the Toll-Like Receptor family have recently been studied for their use in biosensors^1–5, and have shown great promise in being able to rapidly differentiate classes of bacteria and other biological threat agents. One major advantage over aptamer or antibody based sensors is that the user does not need to know what the biological agent is. Instead, the sensor can be used as a broad screening tool. This is especially true for the use of electrochemically based Toll-Like Receptor sensors, where the protein is attached to a surface via a linker, such as a self-assembled monolayer (SAM). However, these sensors have relied almost exclusively on the presence of relatively high resistance (> 1 kΩ) systems that provide small signals that are ill-suited to implementation outside of a laboratory¹.

To address this deficiency, we sought to optimize a TLR-4/SAM-based biosensor. Towards this aim, we have built upon previous work in which TLR-4 is oriented in a bio-mimicking fashion on a Au electrode surface¹. The protein was tethered to a Ni²⁺-nitrilitriacetic acid (NTA) functional group, covalently attached to the end of a carboxyl-terminated thiol-based SAM. Various SAMs were explored in this work, ranging from long-chain single-component monolayers to tripartite SAMs containing multiple functional groups. An optimal ratio of thiol components was determined, based on the TLR-4 sensor response to the presence of Gram-negative bacteria, while remaining insensitive to the presence of Gram-positive bacteria or viral particles, with all samples presenting ≤1 kΩ of interfacial resistance to the rectification reaction. This represents a marked decrease in resistance compared to the current state-of-the-art for TLR-based sensors, providing a convenient avenue for future studies.

1. Mayall, R. M., Renaud-Young, M., Chan, N. W. C. & Birss, V. I. An electrochemical lipopolysaccharide sensor based on an immobilized Toll-Like Receptor-4. Biosens. Bioelectron. 87, 794–801 (2017).

2. Amini, K., Chan, N. W. C. & Kraatz, H.-B. Toll-like receptor 3 modified Au electrodes: an investigation into the interaction of TLR3 immobilized on Au surfaces with poly(I:C). Anal. Methods 6, 3322 (2014).

3. She, Z. et al. Investigation of the utility of complementary electrochemical detection techniques to examine the in vitro affinity of bacterial flagellins for a toll-like receptor 5 biosensor. Anal. Chem. 150222112048003 (2015). doi:10.1021/ac5042439

4. Amini, K. & Kraatz, H.-B. Toll-like receptors for pathogen detection in water: challenges and benefits. Int. J. Environ. Anal. Chem. 7319, 1–9 (2016).

5. Yeo, T. Y. et al. Electrochemical endotoxin sensors based on TLR4/MD-2 complexes immobilized on gold electrodes. Biosens. Bioelectron. 28, 139–145 (2011).