Presenting Author Metropolitan State University of Denver Northglenn, Colorado
Whooping cough caused by Bordetella pertussis can cause serious and prolonged health effects and is especially deadly in infants. Early treatment can significantly reduce the duration of illness and lead to better outcomes, but current diagnostic procedures need a relatively large patient sample and have an extended wait period for testing results. There is thus a pressing need for quick and accurate diagnosis. In response, we are developing an electrochemical DNA-based (E-DNA) biosensor for the rapid detection of B. pertussis. Our biosensor utilizes a DNA aptamer that targets P.69 pertactin, a well-known adhesion factor in the outer membrane of B. pertussis. To generate this aptamer, we first obtained purified P.69 pertactin and biotinylated it. Biotinylation extent was measured via HABA assay and showed ~ 42 biotin molecules per P.69 pertactin molecule. This biotinylated protein was used for selection in a modified SELEX (systematic evolution of ligands by exponential enrichment) process, followed by high-throughput sequencing. That analysis discovered six aptamer sequences with potential strong binding for P.69 pertactin. Potential aptamers were synthesized, and binding was confirmed via fluorescence anisotropy and gel shift assays. These techniques identified one aptamer sequence as having strong, low-nanomolar binding affinity for P.69 pertactin. This aptamer was then computationally analyzed for likely secondary and tertiary structures, allowing identification of the minimum binding unit, and then was modified to generate a two-state binding system. This modified aptamer sequence serves as the basis for an electrode-tethered E-DNA biosensor. This design will be verified against purified P.69 pertactin standards as well as B. pertussis isolates to demonstrate its potential as a rapid and accurate diagnostic biosensor for whooping cough infections.
