Quantitative Assessment of Periodontal Bacteria Using a Cell-Based Immunoassay with Functionalized Quartz Crystal Microbalance

Abstract

Periodontal disease is an inflammatory disorder that is triggered by bacterial plaque and causes the destruction of the tooth-supporting tissues leading to tooth loss. Several bacteria species, including Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, are considered to be associated with severe periodontal conditions. In this study, we demonstrated a quartz crystal microbalance (QCM) immunoassay for quantitative assessment of the periodontal bacteria, A. actinomycetemcomitans. An immunosensor was constructed using a self-assembled monolayer of 11-mercaptoundecanoic acid (11-MUA) on the gold surface of a QCM chip. The 11-MUA layer was evaluated using a cyclic voltammetry technique to determine its mass and packing density. Next, a monoclonal antibody was covalently linked to 11-MUA using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide to act as the biorecognition element. The specificity of the monoclonal antibody was confirmed by an enzyme-linked immunosorbent assay. A calibration curve, for the relationship between the frequency shifts and number of bacteria, was used to calculate the number of A. actinomycetemcomitans bacteria in a test sample. Based on a regression equation, the lower detection limit was 800 cells, with a dynamic range up to 2.32 × 106 cells. Thus, the QCM biosensor in this study provides a sensitive and label-free method for quantitative analysis of periodontal bacteria. The method can be used in various biosensing assays for practical application and routine detection of periodontitis pathogens.