DEVELOPMENT OF NUCLEIC ACID-BASED BIOSENSORS TO DETECT GENES INVOLVED IN XENOBIOTIC DEGRADATION IN CONTAMINATED ENVIRONMENTS.

CARDILLO MARIA EUGENIA; SACCO NATALIA J; RAIGER IUSTMAN LAURA J

Los Cocos, Cordoba

Congreso; XVII Congreso Argentino de Microbiología General; 2022

samige

A biosensor is an integrated device capable of providing quantitative or semi-quantitative analytical information. It consists of a biological recognition element (BRE) where the signal displayed by the interaction between the BRE and the analyte is transformed into information by the transducer element (TE) . Several BREs have been described like enzymes, bacteria, ssDNA, cells, etc. Genosensors, a type of biosensor in which the BRE is a DNA, are useful to analyze specific DNA sequences allowing us to detect and quantify the presence of certain microbial species or metabolic pathways in a complex sample like soil or water. Since nowadays this kind of experiments are conducted by qPCR, a replacement of this technique by a cheapest and fastest one like a biosensor could be useful to decrease costs and working times.In this work, we present the design and development of a genosensor to detect the presence of the alkB gene, which is involved in alkane degradation. Through different tests, we have studied and optimized the surface modification of screen-printed electrodes with oxidized multi-wall carbon nanotubes (MWNTs ox) and chitosan (Qui). The nanostructuring of the surface of the electrodes obtained makes it possible to increase its sensitivity and broaden its field of application. Different conditions were tested for the binding of the ssDNA sequence to the surface of the working electrode, including condensation reactions between the 5´-PO43- of the ssDNA and the functional groups established on the surface (using glutaraldehyde, GA), or modification reactions performed at the -NH2 terminus of the ssDNA. After a process of optimization of the test conditions, we have managed to obtain a modified electrode with MWCNTs ox, QUI, and GA where the ssDNA probe is covalently bound to the surface. For testing, alkB gene amplicon and chromosomal DNA of P. extremaustralis were obtained and used as positive controls while a chromosome DNA of Pseudomonas sp KA-08, a alkB- strain was selected as negative control.