Improved Voltammetric Determination of Kynurenine at the Nafion Covered Glassy Carbon Electrode - Application in Samples Delivered from Human Cancer Cells

Ilona Sadok; Katarzyna Tyszczuk-Rotko; Robert Mroczka; Jędrzej Kozak; Magdalena Staniszewska

doi:10.1177/11786469211023468

Improved Voltammetric Determination of Kynurenine at the Nafion Covered Glassy Carbon Electrode - Application in Samples Delivered from Human Cancer Cells

Int J Tryptophan Res. 2021 Jun 30:14:11786469211023468. doi: 10.1177/11786469211023468. eCollection 2021.

Authors

Ilona Sadok¹, Katarzyna Tyszczuk-Rotko², Robert Mroczka³, Jędrzej Kozak², Magdalena Staniszewska¹

Affiliations

¹ Laboratory of Separation and Spectroscopic Method Applications, Centre for Interdisciplinary Research, Faculty of Science and Health, The John Paul II Catholic University of Lublin, Lublin, Poland.
² Institute of Chemical Sciences, Faculty of Chemistry, Maria Curie-Skłodowska University in Lublin, Lublin, Poland.
³ Laboratory of X-ray Optics, Centre for Interdisciplinary Research, Faculty of Science and Health, The John Paul II Catholic University of Lublin, Lublin, Poland.

Abstract

Nowadays, development of analytical methods responding to a need for rapid and accurate determination of human metabolites is highly desirable. Herein, an electrochemical method employing a Nafion-coated glassy carbon electrode (Nafion/GCE) has been developed for reliable determination of kynurenine (a key tryptophan metabolite) using a differential pulse adsorptive stripping voltammetry. To our knowledge, this is the first analytical method to allow for kynurenine determination at the Nafion-coated electrode. The methodology involves kynurenine pre-concentration in 0.1 M H₂SO₄ in the Nafion film at the potential of +0.5 V and subsequent stripping from the electrode by differential pulse voltammetry. Under optimal conditions, the sensor can detect 5 nM kynurenine (for the accumulation time of 60 seconds), but the limit of detection can be easily lowered to 0.6 nM by prolonging the accumulation time to 600 seconds. The sensor shows sensitivity of 36.25 μAμM^-1cm^-2 and 185.50 μAμM^-1cm^-2 for the accumulation time of 60 and 600 seconds, respectively. The great advantage of the proposed method is easy sensor preparation, employing drop coating method, high sensitivity, short total analysis time, and no need for sample preparation. The method was validated for linearity, precision, accuracy (using a high-performance liquid chromatography), selectivity (towards tryptophan metabolites and different amino acids), and recovery. The comprehensive microscopic and electrochemical characterization of the Nafion/GCE was also conducted with different methods including atomic force microscopy (AFM), optical profilometry, time-of-flight secondary ion mass spectrometry (TOF-SIMS), electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV). The method has been applied with satisfactory results for determination of kynurenine concentration in a culture medium collected from the human ovarian carcinoma cells SK-OV-3 and to measure IDO enzyme activity in the cancer cell extracts.

Keywords: Kynurenine; Nafion modified electrodes; adsorptive stripping voltammetry; cancer cells; kynurenine pathway; tryptophan metabolites analysis.