Strategies to develop amperometric bioelectrodes for infection diagnosis

BELLUZO, M. S.; RIBONE, M. E.; STELLA, E.; DE LA IGLESIA, A.; MARCIPAR, I. S.; MORBIDONI, H. R.; LAGIER, C. M.

Praga, República Checa

Congreso; XII International Conference on Electroanalysis, ESEAC 2008; 2008

Most of infection diagnostic tests rely on detecting either the infecting agent (whole microorganism or selected molecules) or specific antibodies raised by the host.1 We developed amperometric systems to detect both of these infection markers. In one approach, we detected antibodies to Trypanosoma cruzi parasite, by using a classical indirect immunoassay with electrochemical detection. We used recombinant proteins specifically designed to achieve three goals: i) to avoid cross-reactions with antibodies generated by the host during other diseases, ii) to link these molecules to the electrode in an oriented manner so as to enhance sensitivity and iii) to attach them covalently, so that the device could be reused after rigorous treatments. This strategy allowed us not only to efficiently and specifically detect the infection marker but also to regenerate the device to be reused in consecutive sample analysis, envisaging automatization.2 The results obtained showed 100% specificity for the entire positive and negative samples assayed. The sensitivity was in the same order as that obtained using a commercial ELISA kit following an indirect immunoassay format with spectrophotometric detection. The biosensors could be regenerated and then reused to analyze 10 different samples consecutively before showing a signal loss of 50% (p < 0.05), with a cut-off value corresponding to a 20% of the original signal measured. In another approach, to verify the presence of pathogenic bacteria, we detected by amperometry the consumption of catechol, a model substrate of bacterial enzymes. Considering that several bacteria have enzymatic systems that can degrade catechol, we enhanced the assay specificity by biologically releasing exclusively the enzymatic content of the target bacteria. Amperometries were performed in a classical three-electrode cell where a carbon paste electrode was the working electrode, using PBS as bathing electrolyte. The current difference obtained between the background and the tested sample was used to monitor the presence of catechol-degrading enzymatic system. Results obtained with samples of intact bacteria and specifically lysated showed significant differences (p < 0.05). REFERENCES 1. Belluzo M.S., Ribone M.E. and Lagier C.M. Sensors 8, 1366 (2008). 2. Lagier C.M., Marcipar I.S., Ribone M.E., Belluzo M.S., Camussone C. INPI, Inscription N° P060103687.