Wash- and Separation-Free Electrochemical Detection of Proteases

Abstract

Proteases play crucial roles in biological systems, including digestion, catabolism, and cell signaling. Indirect detection of Porphyromonas gingivalis in saliva, based on proteolytic cleavage by an Arg-specific gingipain (Arg-gingipain) protease, has traditionally been used for simple, initial diagnosis of periodontitis. To accurately detect P. gingivalis using a point-of-care format, development of a simple biosensor that can measure the exact concentration of P. gingivalis is required. However, electrochemical detection in saliva is challenging due to the presence of various interfering electroactive species in different concentrations. Here, we report a washing- and separation-free electrochemical biosensor for sensitive detection of P. gingivalis in saliva. Glycine–proline–arginine conjugated with 4-aminophenol (AP) was used as an electrochemical substrate for a trypsin-like Arg-gingipain, and glycylglycine was used to increase the Arg-gingipain activity. The electrochemical signal of AP was increased using electrochemical–chemical (EC) redox cycling involving an electrode, AP, and tris(2-carboxyethyl)phosphine, and the electrochemical charge signal was corrected using the initial charge obtained before a 15 min incubation period. The EC redox cycling combined with the matrix-corrected signal facilitated a high and reproducible signal without requiring washing and separation steps. The proteolytic cleavage of the electrochemical substrate was specific to P. gingivalis. The calculated detection limit for P. gingivalis in artificial saliva was 5 × 105 colony-forming units/mL, and the concentration of P. gingivalis in human saliva could be measured. The developed biosensor can be used as an initial diagnosis method to distinguish between healthy people and patients with periodontal diseases.References S. Park, K. Park, H. S. Na, J. Jung, H. Yang, Anal. Chem. 93, 5644 (2021) S. Park, J. Shin, J. Kwon, W. Lee, J. Kim, G. Kim, J. M. Joo, H. Yang, ACS Sens. 6, 1305 (2021) S. Park, G. Kim, J. Seo, H. Yang, Anal. Chem. 88, 11995 (2016)S. Park, Y. M. Shin, J. Seo, J.-J. Song, H. Yang, Analyst 141, 2481 (2016)S. Park, Y. Shin, J.-J. Song, H. Yang, Biosens. Bioelectron. 72, 211 (2015)S. Park, H. Yang, Analyst, 139, 4051 (2014)