Development of competitive QCM biosensor system to detect a pathogenic bacterium causing periodontitis, Actinobacillus actiomycetemcomitans
S-R Hong, S. Hong
Kangnung National University, KR
Keywords: competitive QCM, periodontitis, Actinobacillus actiomycetemcomitans
Abstract:Bacterial plaque is considered the principle etiological factor in the onset and progression of periodontitis. Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythensis, Prevotella intermedia, and Micromonas micros are strong markers of periodontitis in adults. These bacteria can be commonly detected and quantified in subgingival plaque using the anaerobic culture technique. The traditional microbiological diagnosis is time consuming and needs lots of efforts. Thus researchers are seeking rapid and simple methods to detect these bacteria. In this study a QCM biosensor was developed to detect Actinobacillus actiomycetemcomitans, one of periodontitis causing bacteria, using a specific antibody. A. actinomycetemcomitans is a gram-negative, nonmotile coccobacillus that colonizes the human oral cavity. A. actinomycetemcomitans can also enter the submucosa and cause infective endocarditis and other nonoral infections. This bacterium has a limitation in using conventional QCM system which immobilize antibody on gold surface of crystal due to the non specific affinity of A. actinomycetemcomitans to gold surface. It is known that A. actinomycetemcomitans form tenacious biofilms on surfaces such as glass, plastic, and saliva-coated hydroxyapatite during a culture in broth. Tight adherence is due to long, bundled pili (fimbriae) which have been known to play an important role in the ability of A. actinomycetemcomitans to colonize the mouths of rats. We found a decreased frequency after incubating formalin killed A. actinomycetemcomitans on sensor chip without antibody in QCM system. Thus this study introduced a new QCM analysis methodology, named competitive QCM system. This competitive QCM system recruits antigen coated sensor chip instead of antibody coated ones. Hypothesis is simple that amount of antibody which can bind to antigen on sensor chip will be reduced by incubating with the bacteria before applying to the antigen coated sensor chip and the reduced antibody binding can be monitored in real time. The methodology can be summarised briefly below. The antigen coated sensor chip was prepared by immobilising sonicated bacterial cells on carboxyl chip. To produce carboxyl chip, self assembled monolayer (SAM) was conformed on gold surface of crystal quartz and activated by EDC/NHS. To detect bacterial cells in sample, whole bacterial cells containing sample was pre-incubated with antibody solution at 37oC for 30 min and the reaction was applied on to the antigen coated sensor chip. Antibody solution without incubating with bacterial cells was applied as positive control. The frequency shifts were measure in each case and the relative ratios of the frequency shift of sample (Fs) to the frequency shift of positive (Fp) were calculated. Consequently, it was proved that considerable amount of sonicated antigen was immobilized and the relative ratio of the Fs to the Fp was changed in dose dependent manner. The newly developed sensor system was also available for regeneration.