Enhanced Signal Amplification in a TLR-4 Biosensor Utilizing Ferrocene-Terminated Mixed Monolayers.

A major challenge in effectively treating infections is to provide timely diagnosis of a bacterial or viral agent. Current cell culture methods require >24 hours to identify the cause of infection. The Toll-Like Receptor (TLR) family of proteins can identify classes of pathogens and has been shown to work well in an impedance-based biosensor, where the protein is attached to an electrode via a self-assembled monolayer (SAM). While the sensitivity of these sensors has been good, they contain a high resistance (>1 kΩ) SAM, generating relatively small signals and requiring longer data collection, which is ill-suited to implementation outside of a laboratory. Here, we describe a novel approach to increase the signal magnitude and decrease the measurement time of a TLR-4 biosensor by inserting a redox-active ferrocenyl-terminated alkane thiol into a mixed SAM containing hydroxyl- and carboxyl-terminated alkane thiols. The SAM formation and modification was confirmed via contact angle and X-ray photoelectron spectroscopy measurements, with TLR-4 immobilization demonstrated through a modified immunosorbent assay. It is shown that these TLR-4 biosensors respond selectively to their intended target, Gram-negative bacteria at levels between 100 and 105 lysed cells/mL, while remaining insensitive to Gram-positive bacteria or viral particles at up to 105 particles/mL. Furthermore, the signal enhancement due to the addition of ferrocene decreased the measurement time to less than one minute and has enabled this sensor to be used with an inexpensive, portable, handheld potentiostat that could be easily implemented in field settings.