Abstract
Porphyromonas gingivalis, known as an anaerobic gram-negative bacteria, is associated with the progress of a periodontitis. Since gingipain (gp), a specific protease secreted from its bacteria, is its main pathogenicity, gp has been utilized as a marker of the periodontitis. Here, an electrochemical protease assay using a ferrocenylpeptide probes was applied to the detection of gp activity; the probe was cleaved with its protease activity and current peak decreased by the released ferrocene part to balk solution. A series of ferrocenylpeptides as specific substrates of Arg-gp (Rgp) and Lys-gp (Kgp) was synthesized: FRC and FKC having cysteine residue; FRSS and FKSS having lipoic acid; FRpra and FKpra having propargylglycine; FRDE and FRDK having cysteine and D-type amino acid. The electron transfer rate constant of all of ferrocenylpeptide immobilized on a gold electrode were estimated by Lavirons analysis. The obtained electron transfer rate constant were 400-3500 s−1, where fastest one of 3500 s−1 was obtained from FRC immobilized electrode. Peak currents of these electrodes were decreased after the treatment of a sample containing Porphyromonas gingivalis. The detection limits of Porphyromonas gingivalis were 1.0 × 107, 5.0 × 106, 3.3 × 106, 5.0 × 106, and 5.0 × 106 cells, for FRPra- FKPra-, FRC-, FRDE-, and FRDK-immobilized electrodes, respectively. FRDE- and FRDK-immobilized electrodes were applied for patients suffering from periodontitis. It was successfully classified between the patients and healthy peoples using the FRDK-immobilized electrode. These results suggest that gp activity is connected with a clinical condition of periodontitis, and it is expected to be applied to a periodontal disease screening.
