Investigation of binding characteristics of immobilized toll-like receptor 3 with poly(I:C) for potential biosensor application.

Toll-like receptor 3 (TLR3), a pathogen recognition receptor of the innate immune response, recognizes and is activated by double-stranded RNA (dsRNA), which is indicative of viral exposure. A sensor design exercise was conducted, using surface plasmon resonance detection, through the examination of several immobilization approaches for TLR3 as a biorecognition element (BRE) onto a modified gold surface. To examine the TLR3-dsRNA interaction a synthetic analogue mimic, poly (I:C), was used. The interaction binding characteristics were determined and compared to literature data to establish the optimal immobilization method for the TLR3 BRE. A preliminary evaluation of the efficacy of the selected TLR3 surface as a broad-spectrum viral biosensor was also performed. Amine-coupling was found to be the most reliable method for manufacturing repeatable and consistent TLR3 BRE sensor surfaces, although this immobilization schema is not tailored to place the receptor in a spatially-specific orientation. The equilibrium dissociation constant (KD) measured for this immobilized TLR3-poly (I:C) interaction was 117 ±â€¯3.30 pM. This evaluation included a cross-reactivity study using a selection of purified E. coli and synthetic double- and single-stranded nucleic acids. The results of this design exercise and ligand binding study will inform future work towards the development of a broad-spectrum viral sensor device.

Detection of the Lipopeptide Pam3CSK4 Using a Hybridized Toll-like Receptor Electrochemical Sensor.

Electrochemical detection of Pam3CSK4, a synthetic triacylated lipopeptide that mimics the structural moieties of its natural Gram negative bacterial pathogen-associated molecular pattern (PAMP) counterpart, has been achieved using hybridized toll-like receptors (TLR) combining TLR1 and TLR2 onto a single sensor surface. These sensors represent the first hybridized TLR sensors. The limit of detection for Pam3CSK4 attained was 7.5 µg/mL, which is within the same order of magnitude for that of the more labor-intensive and time-consuming cell-assay technique, 2.0 µg/mL. The results gathered in these electrochemical experiments show that sensors fabricated by immobilizing a mixture of cooperative TLR1 and -2 generate higher responses when exposed to the analyte in comparison to the control sensors fabricated using pure TLR1 or -2 standalone. A PAMP selectivity test was carried out in line with our inspiration from the mammalian innate immune response. TLRs1-5 as standalone biorecognition elements and the hybridized "TLR1 and 2" sensor surface were investigated, understanding the known TLR-PAMP interactions, through the exploitation of this electrochemical sensor fabrication technique. The experimental result is consistent with observations from previously published in vivo and in vitro studies, and it is the first demonstration of the simultaneous evaluation of electrochemical responses from multiple, unique fabricated TLR sensor surfaces against the same analyte.

An unexpected use of ferrocene. A scanning electrochemical microscopy study of a toll-like receptor array and its interaction with E. coli.

Imaging of toll-like receptor microarrays was achieved using scanning electrochemical microscopy with the successful integration of two ferrocene derivatives in order to enhance the background contrast. This investigation has resulted in the novel fabrication of a tuneable, multiplex, broad-spectrum bacterial sensor for the interrogation of conserved microbial stimuli.

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.

An initial investigation of the fabrication of a novel biosensor utilizing toll-like receptor 5 (TLR5) has been conducted. The detection assay using this sensor platform has been carried out using two complementary electrochemical techniques. The electrochemical properties of the modified bare gold surface following TLR5 immobilization were characterized. The electrochemical response to changes in the sensor film resistance and electron charge-transfer permittivity triggered by independent exposures to flagellins from Salmonella typhimurium (S. typhimurium) and Bacillus subtilis (B. subtilis) were examined and observed. The quantified film resistance data gathered using electrochemical impedance spectroscopy (EIS) over a macroscopic scale are in significant agreement with the corresponding electron charge-transfer permittivity measured locally by scanning electrochemical microscopy (SECM). Unlike other sensors that exploit pathogen recognition elements, TLR5 biosensors have the potential to carry out broad-spectrum detection of flagellated bacterial pathogens in near real time. This broad-spectrum detection platform is a significant step toward the development of fast, inexpensive clinical tools for early warning diagnoses and immediate on-site treatment.