Impedimetric Detection of Haemophilus Influenzae on Antibodies-Modified Boron-Doped Carbon Nanowall Electrodes

Abstract

Haemophilus influenzae (Hi) is a gram-negative pathogenic bacterium. Type b bacteria (Hib) and non-enveloped strains are of greatest clinical importance. Hib causes systemic infections by hematopoietic dissemination from the respiratory system to distant sites such as meninges.Nowadays the most reliable method for Hib infections diagnosis is isolation in microbiological culture. A cerebrospinal fluid sample from a patient suspected of having meningitis is stained and microbiologically cultured. Isolation efficiency and quality depend on many factors also in addition, carrying out the complete test procedure is time consuming and expensive. Thus it is crucial to develop techniques that allow detecting pathogenic bacteria in fast and qualitative way before the first symptoms appear.In the following work the electrochemical impedance methods were used to detect Hi bacteria and specific protein (protein D) appearing on its surface. Hi immunosensor was developed on boron-doped carbon nanowall (B:CNW) electrodes modified with anti-protein D antibodies.The B:CNW modification steps included electrochemical reduction of the aminobenzoic acid diazonium salt and antibody binding via EDC/NHS chemistry. Many scientists deal with the topic of use bovine serum albumin (BSA) for the blockage of unmodified electrode. Here this step was abandoned due to high resistance occurring on the sample after the electrografting stage, which indicates a very effective modification of the electrode surface. An additional argument confirming the correctness of skipping the BSA stage are the results of the sensor's cross-reactivity with other pathogens. For the determination of the sensor selectivity, three non-target pathogens were selected: S. pyogenes, S. pneumoniae, B. parapertussis. For each pathogen used for the selectivity study, the impedimetric responses were significantly lower than for Hi at similar concentrations.Electrochemical impedance spectroscopy measurements were performed on anti-protein D antibodies (ap. D) for protein D (Fig. 1a) and Hi. Then calibration curve was constructed as a relative change in the electron transfer resistance (Rct) (Fig. 1b). Since the calibration curve shows the logarithmic relationship of the antigen concentration to the impedimetric sensor response, linear regression cannot be used to calculate the LOD. It is therefore necessary to modify the calculation procedure so that it can be applied in the present case. This requires changing the equation's layout and then deriving the formula for the deviation of the logarithm of the concentration. The LOD value calculated in this way for Hi bacteria was 19.45 CFU/ml which is a reliable value.For the sensor’s selectivity studies, the electrodes were prepared identically to the electrodes intended for Hi detection. The response of sensor for S. pyogenes (64750 CFU/ml) and B. parapertussis (17200 CFU/ml) were less than 5%, which are almost unnoticeable in comparison to the response for Hi at level of 50% (47910 CFU/ml).This work was supported by the National Centre for Research and Development TECHMATSTRATEG 1/347324/12/NCBR/ 2017 Figure 1