Indirect determination of Escherichia coli based on β-D-glucuronidase activity and the voltammetric oxidation of phenolphthalein at graphite screen-printed electrodes

Abstract

Abstract Of the methods available for the determination of Escherichia coli (E. coli), an index of fecal contamination in aquatic systems, electrochemical methods based on the activity of β-D-glucuronidase (GLU) offer distinct advantages in terms of portability and instrumentation simplicity. GLU is an enzyme marker of E. coli and its activity can be measured by the voltammetric determination of various electroactive compounds released upon the action of GLU on specific substrates. In this work, we report for the first time on the development of graphite screen-printed electrodes (SPEs) and electrochemical 3-electrode cells (SPCs) that offer highly sensitive determination of phenolphthalein (Phe) by differential pulse (DP) voltammetry. DP voltammograms of Phe possess an oxidation peak at 0.69 V (SPE vs. Ag/AgCl reference electrode) or at 0.53 V (SPC vs. pseudoreference Ag printed electrode). Oxidation currents are related linearly with the concentration of Phe over the concentration range 10–1000 and 25–600 nM Phe, while for a signal-to-noise ratio of 3 the limits of detection were 5 and 10 nM Phe, respectively. SPCs were further used for the indirect determination of E. coli, via the electrochemical oxidation of the enzymatically liberated Phe upon the action of GLU on phenolphthalein β-D-glucuronide. Various experimental variables regarding the induction of GLU, the permeabilization of the cells, the preconcentration of GLU in the cell extract as well as the electrochemical determination of Phe were optimized, and the method was successfully used for the determination of 100 cfu/mL E. coli within 4 h.