Development of a nanobiosensor for the detection of Campylobacter fetus

Background: Campylobacter fetus is a gram-negative, fastidious microaerobic bacterium that resides mainly in the intestinal tract of cattle and sheep. The bacteria classified as a foodborne and opportunistic pathogen for humans, especially pregnant women, elderly and immunocompromised people. The detection of C. fetus is not routinely performed in many clinical laboratories. Nanoparticles-piezoelectric quartz crystal microbalance (QCM) combination, as a biosensor, has received extensive attention for pathogens detection. Our aim was to functionalize a QCM biosensor based on new generation superparamagnetic iron oxide nanoparticles (SPIONs) for the detection of C. fetus. Materials/methods: Synthetized SPIONs were treated by Rodamine B isothiocyanate and anti-C. fetus antibodies. The immunological interaction between immunomagnetic beads and C. fetus subsp. fetus, from the Campylobacter collection of the BCA Department, University of Padua, Italy, was conducted with five concentrations of the antibody-RITC-SPIONs complexes (5, 10, 50, 100 and 200 μg/ml) on the rotator for 10 min at RT. Bacterial cells were recognized by antibodyconjugated nanoparticles and isolated using an external magnet field. In order to confirm the successful recovery, supernatant and the magnetically separated bacteria were cultured on specific media and a PCR was also performed. Finally, magnetically recovered bacteria were evaluated by QCM system with the gold crystals modified by biotin, avidin and antibodies against Campylobacters. Piezoelectric measurements were done using the Elbatech instrument. Results: Campylobacter cultivation and PCR showed that the antibody-SPIONs complex enabled the easy and rapid separation of C. fetus avoiding cross reaction with other Campylobacters. According to results, the QCM biosensor, 50 and 100 μg/ml emerged as optimal concentrations for the purpose of the present study. Turnaround time of the total process was about 15 min. Conclusions: Due to fastidious growth of Campylobacters, detection by conventional approaches, mainly biological methods, is quite difficult and requires a number of biochemical tests. Our QCM system was developed by modifying gold crystals by covalently conjugated antibodies and integrated with antibodies-SPIONs complex. Designed biosensor applied for recognition of C. fetus provided robust, inexpensive and rapid detection with high specificity.