Enzyme-Catalyzed O₂ Removal System for Electrochemical Analysis under Ambient Air: Application in an Amperometric Nitrate Biosensor

Abstract

Electroanalytical procedures are often subjected to oxygen interferences. However, achieving anaerobic conditions in field analytical chemistry is difficult. In this work, novel enzymatic systems were designed to maintain oxygen-free solutions in open, small volume electrochemical cells and implemented under field conditions. The oxygen removal system consists of an oxidase enzyme, an oxidase-specific substrate, and catalase for dismutation of hydrogen peroxide generated in the enzyme catalyzed oxygen removal reaction. Using cyclic voltammetry, three oxidase enzyme/substrate combinations with catalase were analyzed: glucose oxidase with glucose, galactose oxidase with galactose, and pyranose 2-oxidase with glucose. Each system completely removed oxygen for 1 h or more in unstirred open vessels. Reagents, catalysts, reaction intermediates, and products involved in the oxygen reduction reaction were not detected electrochemically. To evaluate the oxygen removal systems in a field sensing device, a model nitrate biosensor based on recombinant eukaryotic nitrate reductase was implemented in commercial screen-printed electrochemical cells with 200 μL volumes. The products of the aldohexose oxidation catalyzed by glucose oxidase and galactose oxidase deactivate nitrate reductase and must be quenched for biosensor applications. For general application, the optimum catalyst is pyranose 2-oxidase since the oxidation product does not interfere with the biorecognition element.

Construction of effective disposable biosensors for point of care testing of nitrite

“…The enzymatic scavenger system was adapted from reference [22] and was tested only with the SPE modified with the ccNiR/carbon conductive ink. It comprised a mix of glucose oxidase, catalase and glucose (GO x /Cat/glucose) in the following concentrations: 15 U mL -1 , 2000 U mL -1 and 50 mM, respectively.…”

“…However, this method requires laboratory-based equipment that is not suited for on-site analysis. In general, a good practical oxygen removal system must: (i) be able to sustain anaerobic conditions throughout the measurement's duration; (ii) do not interfere in any way with the electrochemical process, biorecognition element or mediators involved in the analyte quantification; (iii) present a fast oxygen reduction kinetics, to minimize the waiting time necessary to achieve anaerobic conditions; (iv) be autonomous from any specialized equipment [22].…”

“…Afterwards, because none of the oxygen scavenging system components should interfere with the electroanalytical measurements [22], the catalytic activity was assessed in the presence of GOx/Cat/glucose and compared with the response obtained when using the argon purging method. The bioelectrode's sensitivity decreased 27% e and the I cat @ 2 mM was 47% lower, when employing the scavenging system.…”

“…Since the ultimate goal is to create a POCT for nitrite, we decided on employing an oxygen scavenger system based on glucose oxidase, catalase and glucose [22]. This allows avoiding the inconvenient degassing process before and while performing analysis in real samples, stepping towards the establishment of a novel disposable methodology for on-site nitrite monitoring.…”

Novel immobilization process of a thermophilic catalase: efficient purification by heat treatment and subsequent immobilization at high temperature

“…Catalase is often applied to decompose hydrogen peroxide, which is a dangerous byproduct for the biocatalysts and products in enzymatic conversion [2][3][4], and an oxidant pollute in the wastewater in many industries, including the textile, pharmaceutical, and environmental protection industries [5][6][7]. In our previous work, a catalase gene from Bacillus sp.…”