Abstract
The activity of a peroxidase-mimicking DNAzyme was optimized to be used as a catalytic label in a stem-loop genosensor construction for quantifying the gene sequence Shiga-like toxin I of verotoxigenic E. coli. Experimental conditions such as pH, buffer composition, potassium ion concentration, and hemin-to-oligonucleotides ratio, were analyzed to maximize optical and electrochemical responses using microvolumes. Different stem-loop constructions were evaluated to obtain the optimum response against the target concentration. Linear ranges of 0.05-0.5 µM and limits of detection of 174 nM and 144 nM were estimated for the optical and electrochemical measurements, respectively. Selectivity was proved by assaying other verotoxigenic, enterotoxigenic and enteroinvasive sequences. The results show that, if a combination of small-volume electrochemical cells and low-cost untreated screen-printed electrodes with a relatively high geometric area is used, electrochemical measurements present similar sensitivity and limits of detection to the more usual optical ones, allowing the development of low-cost electrochemical biosensors based on the use of soluble DNAzymes as labels.
Highlights
Peroxidase-mimicking DNAzymes are complexes formed by oligonucleotides containing guanine quadruplexes and hemin as prosthetic group, which catalyze the decomposition of hydrogen peroxide with the consequent oxidation of a substrate
3.1 Optical and electrochemical determination of the catalytic activity of DZ5T3-hemin DNAzyme Peroxidase-mimicking DNAzymes catalyze the reduction of hydrogen peroxide by ABTS, which is in turn oxidized to ABTS+
The catalytic activity of a peroxidase-mimicking DNAzyme depends on factors such as pH, buffer composition, potassium ions concentration, and the hemin-to-oligonucleotides ratio
Summary
Peroxidase-mimicking DNAzymes are complexes formed by oligonucleotides containing guanine quadruplexes and hemin as prosthetic group, which catalyze the decomposition of hydrogen peroxide with the consequent oxidation of a substrate. Based on strategies similar to those reported previously [4,5,6,7,8,9,10,11,17], the aim of the present work was to prove the hypothesis that, by employing small-volume electrochemical cells in combination with inexpensive untreated printed electrodes, a sensitive detection of specific sequences of verotoxigenic E. coli could be carried out using DNAzymes as soluble electrochemical labels, yielding similar results to those obtained with the more usual optical detection.
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