Washing- and Separation-Free Electrochemical Detection of Porphyromonas gingivalis in Saliva for Initial Diagnosis of Periodontitis

Abstract

Indirect detection of Porphyromonas gingivalis in saliva, based on proteolytic cleavage by an Arg-specific gingipain (Arg-gingipain), 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 × 10 5 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.

Implications of Porphyromonas gingivalis peptidyl arginine deiminase and gingipain R in human health and diseases

“…In this regard, a recent trend is directed towards fabrication of point-of-care biosensing devices, varying from electrochemical, optical, colorimetric, and immunosensors. These sensors mostly exploit gingipains activities or cell surface markers for P. gingivalis detection ( Nakka et al., 2015 ; Alhogail et al., 2018 ; Thonthat et al., 2019 ; Svärd et al., 2020 ; Park et al., 2021 ) but none of them is using PPAD. Hence, a huge scope exists for further development in this domain.…”

Advances in electrochemical biosensors for the detection of common oral diseases

“…Therefore, the indirect detection of P. gingivalis using Arg-gingipain in saliva has been employed for a simple, initial diagnosis of periodontitis (Liu et al, 2020). Park et al report a washing-and separation-free electrochemical biosensor for sensitive detection of P. gingivalis in saliva (Park et al, 2021). Glycine-prolinearginine 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.…”

A photoacoustic-fluorescent imaging probe for proteolytic gingipains expressed by Porphyromonas gingivalis

“…To date, reported strategies for measurement of gingipain activity have used in vitro detection methods, including a nanobody immunoassay [15a] , an electrochemical biosensor [27] , fluorogenic dipeptides [15b] , peptide-functionalized magnetic nanobeads [28] , and refractometry of protein-functionalized gold nanoparticles [16] . These have reported detection limits of 7.81E6 CFU/mL bacteria, 5E5 CFU/mL bacteria, 1E5 CFU/mL bacteria, 49 CFU/mL bacteria, and 4.3 nM Kgp (CFU/mL not reported), respectively.…”