Intermittent pulse amperometry as an effective electrochemical assay of 4-nitrophenol

Abstract

4-nitrophenol (4-NP) is a precursor of many industrial products and drugs, an environmental pollutant, and is used as a reporter molecule in many enzyme assays and disease biomarker screens. Accordingly, 4-NP analysis is a cross-disciplinary requirement, and we propose intermittent pulse amperometry (IPA) as an alternative to spectrophotometric methods. The sensor signal is acquired by applying 0.5 s pulses of 0.95 V once every 99.5 s during continuous amperometric recordings at 0 V resting potential to drive intermittent 4-NP detection at a diffusion-limited rate. With IPA, sensor surface fouling by polymerization of radical intermediates formed in the anodic oxidation process, which is a severe problem in constant-potential amperometry and voltammetry of phenols, is kept at acceptable minimum by restriction of the time at the potential that produces contamination. Complex potential profiles with electrochemical activation steps and/or electrolyte supplementation with anti-fouling agents are not required. Calibration plots are linear up to 500 μM with a sensitivity of 35 nA μM−1 and a practical detection limit of 10 μM. Model samples of 100 μM 4-NP were assessed with suitable recovery rates, and in a proof-of-principle test as electrochemical readout of an enzyme assay, IPA accurately reported the time course of enzymic release of 4-NP from GlcNAc-4NP, a synthetic substrate of glucosaminidases. Simplicity and high performance are the major features of the proposed electrochemical 4-NP testing, and unless the analytical target is present only at trace levels, the technique is a promising alternative for the evaluation of 4-NP, either as a solution component or as a reporter molecule in enzyme assays.