Ultrasensitive and Label-Free Detection of the Measles Virus Using an N-Heterocyclic Carbene-Based Electrochemical Biosensor.

With the current intense need for rapid and accurate detection of viruses due to COVID-19, we report on a platform technology that is well suited for this purpose, using intact measles virus for a demonstration. Cases of infection due to the measles virus are rapidly increasing, yet current diagnostic tools used to monitor for the virus rely on slow (>1 h) technologies. Here, we demonstrate the first biosensor capable of detecting the measles virus in minutes with no preprocessing steps. The key sensing element is an electrode coated with a self-assembled monolayer containing the measles antibody, immobilized through an N-heterocyclic carbene (NHC). The intact virus is detected by changes in resistance, giving a linear response to 10-100 µg/mL of the intact measles virus without the need to label or process the sample. The limit of detection is 6 µg/mL, which is at the lower limit of concentrations that can cause infections in primates. The NHC-based biosensors are shown to be superior to thiol-based systems, producing an approximately 10× larger response and significantly greater stability toward repeated measurements and long-term storage. This NHC-based biosensor thus represents an important development for both the rapid detection of the measles virus and as a platform technology for the detection of other biological targets of interest.

Enhanced Signal Amplification in a Toll-like Receptor-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 h 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 alkanethiol into a mixed SAM containing hydroxyl- and carboxyl-terminated alkanethiols. 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 1 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 1 min and has enabled this sensor to be used with an inexpensive, portable, hand-held potentiostat that could be easily implemented in field settings.

An Inexpensive, Single Use Carbon-Based Sensor for the Rapid and Early Detection of Hydrocarbon Leaks.

A novel fabrication process for a single use, low-cost organic solvent sensor has been developed. The process is simple, and the materials are readily available. Carbon nanomaterials are self-assembled at a water/hexane interface, where the hexane phase contains dissolved paraffin wax. Upon the controlled evaporation of hexane, the paraffin wax precipitates, trapping the carbon nanoparticles at the surface in a paraffin wax backbone, realizing a carbon-nanoparticle-decorated film. The film is hydrophobic and highly electrically conductive. When exposed to hydrocarbons or a mixture of hydrocarbons, the conductive carbon network deteriorates and an increase in film resistivity is monitored. The rate of change in resistivity is proportional to the concentration and composition of organic molecules in contact with the film.

A multiplexed transcription activator-like effector system for detecting specific DNA sequences.

Transcription activator-like effectors (TALEs), originating from the Xanthomonas genus of bacteria, bind to specific DNA sequences based on amino acid sequence in the repeat-variable diresidue (RVD) positions of the protein. By altering these RVDs, it has been shown that a TALE protein can be engineered to bind virtually any DNA sequence of interest. The possibility of multiplexing TALEs for the purposes of identifying specific DNA sequences has yet to be explored. Here, we demonstrate a system in which a TALE protein bound to a nitrocellulose strip has been utilized to capture purified DNA, which is then detected using the binding of a second distinct TALE protein conjugated to a protein tag that is then detected by a dot blot. This system provides a signal only when both TALEs bind to their respective sequences, further demonstrating the specificity of the TALE binding.