Protease‐Triggered Chromogenic Release From Peptide‐Modified Calcium Carbonate Microspheres Enables Colorimetric Detection of <i>Porphyromonas gingivalis</i> Activity

Porphyromonas (P.) gingivalis is a Gram-negative anaerobic bacterium with exceptionally high pathogenicity among periodontal pathogens. It is associated not only with periodontal disease but also with various systemic diseases, including Alzheimer's disease, rheumatoid arthritis, and atherosclerosis. Therefore, early detection of this bacterium is crucial for enabling subsequent treatment and prevention of such conditions. Herein, we report a chromogenic release-based assay system utilizing calcium carbonate microspheres and a dye-labeled peptide that is selectively cleaved by an Arg-specific protease called gingipain (RgpB) that is secreted by P. gingivalis. By utilizing the reactive anhydride functional groups of poly(isobutylene-alt-maleic anhydride), we simultaneously achieved the conjugation of the dye-labeled peptide and its immobilization onto calcium carbonate microspheres. Through optimization of the peptide sequence and the surface modification density on calcium carbonate microspheres, the assay of RgpB detection with the absorbance of supernatant exhibited a low limit of detection of 0.25 nM, as well as high storage stability and selectivity for P. gingivalis. The measurements obtained with this assay system for clinical gingival crevicular fluid samples showed a high correlation (Pearson's r = 0.79) with qPCR results. This assay system offers an on-site, visual means to monitor P. gingivalis activity without requiring specialized instrumentation.