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

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.

Electrochemical Determination of Kynurenine Pathway Metabolites—Challenges and Perspectives

“…Advantages of the GCE and BDDE surface modification have also been emphasized by our group during the design of voltammetric sensors for Kyn [41,42]. Electrochemical deposition of Bi film onto the BDDE surface presents an easy and rapid way to improve the sensor sensitivity toward Kyn measured by DPV, and it reaches a low LOD (30 nM) [42].…”

“…Electrochemical deposition of Bi film onto the BDDE surface presents an easy and rapid way to improve the sensor sensitivity toward Kyn measured by DPV, and it reaches a low LOD (30 nM) [42]. The coating of the GCE surface with a thin layer of Nafion polymer allows for detection of lower contents of Kyn (Table 1), as the cationic form of this molecule can be pre-concentrated onto the electrode surface before the stripping step [41]. Nafion-perfluorinated polymer containing functional sulphonic acid end groups-is highly conductive to protons and acts as an exchanger membrane [91], causing the repulsion of anions and attraction of positively charged species.…”

“…The Nafion layer can be formed by the drop-coating method (without the need for sophisticated apparatus) and easily removed by polishing using alumina slurries. Furthermore, Kyn can be effectively accumulated onto the Nafioncoated GCE at the potential of +0.5 V in 0.1 M H 2 SO 4 , before being stripped by scanning potential toward more positive values [41].This strategy allows for working with diluted samples and decreases some interferences delivered from the sample matrix components. However, these two sensors are not specific to Kyn, and their accuracy should be verified using comparative approaches with respect to the biological matrix of interest.…”

“…Influence of Trp on Kyn signal Trp signal tends to overlap with Kyn peak in supporting electrolytes at pH lower than 4.6 [42]. In the analysis of samples with similar concentration of Trp and Kyn, the selectivity problem can be resolved by working in the supporting electrolyte at pH 6.5 [42] or in an acidic medium using a Nafion-coated electrode [41]. In case of biological samples containing much higher amounts of Trp in relation to Kyn, e.g., serum, further developments are required;  Overlapping signals from 3HKyn and 3HAA Voltammetric signals of these two KP metabolites appear at similar potentials (Table 4).…”

“…Importantly, they can influence signals of KP metabolites. For instance, an enhancement or decrease in Kyn peak current during voltammetric measurements has been reported [41];  Effect of amino acids…”