Multiplex Biosensor for Pathogen Detection at Point of Care Facilities

Objectives: To combat pathogens more effectively, it is advantageous to identify the etiologic agents. Recently, many effective molecular techniques, including antibody-based and nucleic acid-based systems, have been developed to complement the traditional microbiological identification of pathogens. Unfortunately, most of these techniques require extended time, extensive sample preparation, costly instruments, and highly trained personnel making them unsuitable for point of care facilities. To address this shortcoming, we developed a biosensor system for real time detection of bacterial pathogens that is cost effective, highly resistant to environmental insults, and does not require extensive sample preparation or highly trained personnel. Our label-free biosensor system is composed of magnetoelastic sensor particles (MEP) coated with phage-displayed oligopeptide probes to capture pathogens. Binding of pathogens increases the mass of the MEP biosensor and changes the resonant frequency that can be measured in real time.
Methods: In our continuing effort to improve our system, we developed approaches at the molecular probe side to enhance specificity and sensitivity.
Results: We improved the "biopanning" technique to isolate only highly selective probes with minimal cross-reactivity. Using this approach, we isolated highly selective probes for Salmonella enterica serovar Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes. In addition, we developed a "parallel biopanning" scheme to isolate probes that recognize a wide variety of S. enterica serovars. Furthermore, we developed a scheme to efficiently tag every copy of pIX of M13 for enhanced immobilization of probes on the sensor platforms.
Conclusions: By constructing unique combinations of molecular probes with different sized MEPs, we can convert our singleplex biosensors into a multiplex system for simultaneously detecting multiple pathogens. Our MEP biosensor system can be readily adapted for detection of oral bacteria including periodontal pathogens such as Porphyromonas gingivalis, Treponema denticola, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans that can be used at point of care dental facilities.