Nanostructured indium tin oxide electrodes immobilized with toll-like receptor proteins for label-free electrochemical detection of pathogen markers

We describe the development of 3D nanostructured indium-tin oxide (ITO) electrodes, immobilized with toll-like receptor proteins (TLRs) for label-free detection of pathogen-associated molecular patterns (PAMPs). Nanostructured ITO electrodes were fabricated on ITO-coated glass substrates using the glancing angle deposition (GLAD) technique. Two different TLRs (TLR4 and TLR5), which recognize different PAMPs, were immobilized onto different GLAD ITO electrodes using a simple, robust and direct methodology with an epoxysilane as the intermediate layer. The immobilization steps were characterized using microscopy, electrochemical techniques and x-ray photoelectron spectroscopy. The changes to electron charge transfer resistance in electrochemical impedance spectroscopy (EIS) of the TLR-based sensors triggered by independent exposures of the TLR4-modified sensor to E. coli O157:H7 endotoxin (lipopolysaccharide, LPS) and the TLR5-modified sensor to flagellin from Salmonella typhimurium were examined. These sensors showed a wide dynamic range (>104) and two-orders of magnitude lower detection limits and 30 times better sensitivity for LPS and flagellin compared to planar gold and ITO-based TLR sensors. Unlike other sensors that exploit specific pathogen recognition elements, TLR-based EIS biosensors have the potential to carry out broad-spectrum detection and classification of pathogens in near-real time.