Studies on the Mechanism of Electrochemical Detection of Haemophilus Influenzae at Diamond-Based Electrodes

Haemophilus influenzae (HI) is a Gram-negative pathogenic bacterium whose infections occur worldwide and mainly affect unvaccinated children and those who have not completed the basic vaccination course. Type B of this bacterium (Hib) can cause sepsis, and other invasive diseases as dermatitis or arthritis but the most dangerous is pneumonia meningitis. The most reliable method for recognizing Hib infections is isolation in microbiological culture. Serological tests are also performed to diagnose Hib infections. They rely on the detection of the presence of bacterial antigens or antibodies which have the ability to bind to specific antigens. Detection of antibodies doesn’t mean disease but only indicate contact with a given bacterium in the past. Recognition of bacteria using presently known methods takes a long time and requires expensive equipment although fast and sensitive detection of Haemophilus influenzae is crucial l to start treatment early enough. In this work, we present electrochemical detection of HI on diamond-based pastes electrodes using binding specific antibodies (ap. D) with proteins occurring on the surface of bacteria (protein D). The measuring system consisted of a specially prepared printed circuit board (PCB) and diamond-based electrode, which was prepared by screen printing in special conditions. Places printed on it, served as a working electrode, counter electrode and reference. Ceramics was first used as a substrate, but it was quickly changed to Teflon, which showed better adhesion to diamond pastes. Better sample strength was also obtained, so it could be washed in an ultrasonic cleaner without fear of damage. Before detection, the electrode had to be modified by electrochemical reduction of the aminobenzoic acid diazonium salt, antibodies deposition via EDC / NHS and blocking the unmodified electrode sites by incubating in solution of Bovine Serum Albumin (BSA). The last step of modification prevents unspecific adsorption on electrode surface which would lead to false detection results. Electrochemical Impedance Spectroscopy Fig. 1) were used to bacteria detection. The electron transfer resistance rises with each step of electrode modification and increased bacterial concentration in solution. Then a calibration curve was constructed. Three pathogens were selected to check the biosensor selectivity: S. pyogenes, S. pneumoniae, B. parapertussis. For each pathogen responses were significantly lower than for HI at similar concentrations. For example, the response of sensor for S. pyogenes (67474,28 CFU/ml) and B. parapertussis (17819,27 CFU/ml) were less than 5%, which are much smaller in comparison to response at level of 30% for HI (47003,48 CFU/ml). Acknowledgments The authors gratefully acknowledge financial support from the National Centre for Science and Development Grant Techmatstrateg No. 347324 2015/16/T/ST7/00469. Figure 1