A Chairside Diagnostic to Reduce the Need for Endodontic Retreatment - The objective of this proposal is a chairside molecular diagnostic to detect Enterococcus faecalis in endodontic infections and improve root canal outcomes. There are over 15 million root canals in the United States each year, but this treatment has a failure rate of up to 14%, which requires a repeat (secondary) root canal with major negative impacts on patients, providers, and budgets. One common reason for root canal failure is secondary endodontic infection. The bacterium E. faecalis underlies up to 77% of these secondary infections, and thus tools to detect E. faecalis chairside to confirm site preparation before closing the root canal could improve outcomes. Unfortunately, E. faecalis is currently only quantified with lengthy lab-based methods (i.e., PCR, microbiological testing): These cannot be done quickly or chairside, and clinicians are thus limited to using visual inspection and clinical experience, making secondary treatment unnecessarily common. We propose here a rapid colorimetric test that uses this pathogen’s natural protease signature as a trigger to produce signal. The outcome of this work will be a rapid chairside colorimetric diagnostic that reports the presence of E. faecalis via its characteristic extracellular protease GelE. This assay will confirm the absence of the pathogen so the canal can be sealed with confidence. Infected canals would be directed to more aggressive irrigation or treatment prior to filling and sealing the space. This will also prevent excessive cleaning therefore decreasing the chance of perforation. The research workflow will include two parts. Aim 1 will build the reagents for colorimetric coccolysin detection. We will synthesize and screen peptides to optimize the substrate unique to coccolysin cleavage. We will then use this substrate to tune nanoparticle aggregation/disassembly (and thus a color change) in the presence of the E. faecalis GelE protease. We hypothesize that the colorimetric signal (i.e., color change) will increase linearly with protease concentration (p < 0.05). Our goal is to detecting sub-nanomolar concentrations in less than 10 minutes. Aim 2 will validate the sensor with cultured bacteria and clinical endodontic samples collected by Dr. Roges. E. faecalis levels in the samples will be measured independently using PCR by Dr. Chen, and GelE protease concentrations will be confirmed using ELISA. We hypothesize that the colorimetric signal will correlate to these gold standard metrics at R2 > 0.80. The clinical impact is a chairside diagnostic to detect E. faecalis and enhance clinician and patient experience by decreasing the likelihood of secondary endodontic infection. The innovation is grounded is novel diagnostics for an important clinical scenario as well as novel nano-chemistry approaches that use nanoparticle disassembly for a color change, thus minimizing the impact of the sample matrix.
