LAUSR

Periodontitis (gum disease) is a common biofilm-mediated oral condition, with around 7% of the adult population suffering from severe disease with risk for tooth loss. Moreover, periodontitis virulence markers have been found in atherosclerotic plaque and brain tissue, suggesting a link to cardiovascular and Alzheimer’s diseases. The lack of accurate, fast, and sensitive clinical methods to identify patients at risk leads, on the one hand, to patients being undiagnosed until the onset of severe disease and, on the other hand, to overtreatment of individuals with mild disease, diverting resources from those patients most in need. The periodontitis-associated bacterium, Porphyromonas gingivalis, secrete gingipains which are highly active proteases recognized as key virulence factors during disease progression. This makes them interesting candidates as predictive biomarkers, but currently, there are no methods in clinical use for monitoring them. Quantifying the levels or proteolytic activity of gingipains in the periodontal pocket surrounding the teeth could enable early-stage disease diagnosis. Here, we report on a monitoring approach based on high-affinity microcontact imprinted polymer-based receptors for the Arg and Lys specific gingipains Rgp and Kgp and their combination with surface plasmon resonance (SPR)-based biosensor technology for quantifying gingipain levels in biofluids and patient samples. Therefore, Rgp and Kgp were immobilized on glass coverslips followed by microcontact imprinting of poly-acrylamide based films anchored to gold sensor chips. The monomers selected were N-isopropyl acrylamide (NIPAM), N-hydroxyethyl acrylamide (HEAA) and N-methacryloyl-4-aminobenzamidine hydrochloride (BAM), with N,N′-methylene bis(acrylamide) (BIS) as the crosslinker. This resulted in imprinted surfaces exhibiting selectivity towards their templates high affinity and selectivity for the templated proteins with dissociation constants (Kd) of 159 and 299 nM for the Rgp- and Kgp-imprinted, surfaces respectively. The former surface displayed even higher affinity (Kd = 71 nM) when tested in dilute cell culture supernatants. Calculated limits of detection for the sensors were 110 and 90 nM corresponding to levels below clinically relevant concentrations.