Measurement of antioxidant capacity represents an analytical major challenge in terms of accuracy, efficiency, rapid response, or low cost of detection methods. Quantification of antioxidant capacity of food samples using disposable screen‐printed microelectrodes (SPMEs) was based on cyclic voltammetry versus open-circuit potential (CV vs OCP) and differential pulse voltammetry (DPV) as compared with spectrophotometric measurement of the CUPRAC reaction with 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox). The SPMEs are organic‐resistant electrodes and thus compatible with food samples and organic solvents used to dissolve trolox. A micropipette was used to release a drop of 50
Antioxidants are compounds capable of counteracting the effects of oxidative processes in cells or exogenous systems, reacting in particular with reactive oxygen or nitrogen species [
The search for a simple and accurate rapid method of measuring the antioxidant capacity of a biological sample is a matter of major concern both for fundamental and applicative research as well as for the biotechnological industry. Standard methods employed to evaluate antioxidants from various biological samples are based on colorimetric methods. Different methods have been used to estimate antioxidant capacity, involving 2,2-azino-bis-3-ethylbenzthiazoline-6-sulphonicacid (ABTS) scavenging assays, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical assays, and ferric-reducing antioxidant power (FRAP) assays [
In the last decade, many electrochemical methods were developed as complementary assays thus allowing the antioxidants evaluation based on their net electric charge measurement [
Screen-printed electrodes (SPEs) are novel devices developed in the last five years, fact explaining why the electrochemical approaches using microsensors are less explored. Electroanalytical methods, using SPE, present many advantages such as speed, low cost, simplicity, and low consumption of reagents when compared to other methods.
The objective of the present work was to develop the electrochemical CUPRAC method by using a screen-printed electrochemical sensor with carbon as the working and counterelectrode, as a novel device for the investigation of the antioxidant capacity. An important advantage of the SPE with carbon as the working and counterelectrode is that the thin layers of carbon-dispersed particles acting as main components of working electrode (WE) and auxiliary electrode (AE) can oxidize electroactive species at low potential that minimizes the background current and hence favour the signal-to-noise ratio. Also, the working technique was selected to minimize the adsorption process of reduced/oxidized species on the surface of electrodes.
There are many indirect methods measuring the antioxidant activity of tea extracts [
Scheme of the redox reaction between copper-neocuproine complex and trolox.
For the preparation of both standard solutions and samples, the following chemicals and reagents were used: ammonium acetate (solid); neocuproine (solid); copper chloride, CuCl2 × 2H2O (solid); and ethanol (p.a.), as well as trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (solid).
All supporting solutions were prepared using analytical grade reagents and purified water from a Millipore Milli-Q system (conductivity ≤ 0.1
A freshly prepared buffer solution of ammonium acetate (1.2 mol·L−1) was tested with a Hanna HI 9125 pH meter with HI 1230B electrode and adjusted to pH = 7 by adding small amounts of 1 mol·L−1 NaOH. The aqueous solution of copper chloride (0.012 mol·L−1) was prepared with deionized water, while neocuproine (0.001 mol·L−1) and trolox (0.001 mol·L−1) were alcoholic solutions.
The standards for the calibration curve were prepared with 0.2, 0.4, 0.6, 0.8, and 1.0 mL, respectively, of the 0.001 mol·L−1 trolox solution, diluted with deionized water up to 4 mL, then completed to 10 mL total mixture with 2 mL from each of the following solutions: ammonium acetate buffer (1.2 mol·L−1), copper chloride (0.012 mol·L−1), and neocuproine (0.001 mol·L−1). The standard mixtures were maintained for 30 minutes to react; then, the absorbance of each trolox standard versus blank was measured at 450 nm wavelength.
The absorbance measurements were recorded with a T90 + UV/VIS Spectrometer (PG Instruments Ltd.).
Electrochemical experiments were performed with a potentiostat/galvanostat/EIS analyzer Palmsense 4® integrated with the PS Trace 5® software, Version 5.3.1127 Build 198586t. A Palm Speholder assured connection with the BVT-AC1.W4 R1 (BVT Technologies) screen-printed electrodes (of 7 by 25 mm dimensions and ceramic alumina as support material) with carbon as the working and counterelectrode (Ø of the disc WE = 1 mm, WE geometric area = 0.79 mm2) and Ag/AgCl as the reference electrode.
The spectrophotometric measurements were recorded at room temperature (21 ± 1°C), by using quartz cells of a 1 cm optic path to measure the absorbance of the sample versus blank at 450 nm wavelength. For the spectrum recording, each trolox standard and sample were tested separately using a one-cell measurement at the abovementioned same spectrophotometer.
The experimental conditions for cyclic voltammetry versus open-circuit potential (CV vs OCP) were as follows:
For DPV, the pretreatment settings
For both CV vs OCP and DPV, the corresponding current of the oxidation peak was used to quantify the concentration of [Cu(Nc)2]1+.
CV vs OCP curves have been modified by multiplying (30 times) and very high smoothing (25 times).
DPV curves were processed through baseline subtraction, respectively, and linear baseline followed by moving average baseline (windows size = 2 points; max. number of sweeps = 1001).
The practical application was focused on the analysis of the antioxidant capacity of various tea infusion samples (black and green) that involved simple preliminary preparation.
Five of each black (B1–B5) and green (G1–G5) tea samples of different brands were purchased from the town’s commercial network. The infusions were made by adding 0.5 g of packed tea into 10 ml of deionized water at 95°C and maintained there for 5 minutes. After removing the satchel, the resulting infusion was let to cool down at room temperature before the preparation of the CUPRAC samples mixture, as follows: 4 ml infusion plus 2 ml of each buffer, CuCl2 and neocuproine solutions, to a total of 10 ml final volume.
The electrochemical measurements were performed individually for each reagent by cyclic voltammetry versus open-circuit potential tests (Figure
Voltammetric behaviour of each reagent of the reaction system: (a) cyclic voltammetry versus open circuit potential tests; (b) differential pulse voltammetry tests.
Figure
Voltammetric behaviour of the reaction system’s mixture of reagents, evolution according to the working protocol: (a) cyclic voltammetry versus open circuit potential tests; (b) differential pulse voltammetry tests.
The cyclic voltammograms (Figure
The voltammograms obtained through cyclic voltammetry versus open-circuit potential for the trolox standards were processed by 30 times multiplying and smoothing with PS Trace 5 software. CV vs OCP voltammograms of cyclic voltammetry versus open-circuit potential tests (Figure
Voltammograms of trolox standards: (a) cyclic voltammetry versus open circuit potential tests; (b) differential pulse voltammetry tests.
The voltammograms obtained through cyclic voltammetry versus open-circuit potential and differential pulse voltammetry for the trolox standards (Figures
Calibration curve for trolox standards: (a) cyclic voltammetry versus open circuit potential tests; (b) differential pulse voltammetry tests; (c) spectrophotometric tests.
Both cyclic voltammetry versus open-circuit potential results as well as differential pulse voltammetry results were fitted to a linear function (Figures
Both DPV and spectrophotometric methods were investigated for precision, limit of detection, and limit of quantification.
Precision, as a measure of statistical variability, was investigated for the standard solution corresponding to the 12 · 10−4 mol·L−1 trolox concentration under two aspects: repeatability and intermediate precision. Repeatability results (expressing the consistency of the measurements under identical experimental conditions at short time intervals, in the same day/intra-assay), as well as intermediate precision (expressing the fidelity of the measurement at large intervals of time, in different days/interassays), are presented in Table
Precision measurements for 12 · 10−4 mol·L−1 trolox standard solution.
Statistical parameter | Intra-assay | Interassay | ||||||
---|---|---|---|---|---|---|---|---|
DPV | Spectrophotometry | DPV | Spectrophotometry | |||||
Day 1 | Day 2 | Day 3 | Day 1 | Day 2 | Day 3 | |||
Average | 12.244 | 11.890 | 12.110 | 12.439 | 11.969 | 12.204 | 12.081 | 12.204 |
Standard deviation (SD) | 0.130 | 0.128 | 0.042 | 0.189 | 0.0334 | 0.170 | 0.179 | 0.235 |
Relative standard deviation (RSD) | 1.061 | 1.073 | 0.348 | 1.520 | 0.284 | 1.391 | 1.482 | 1.928 |
To confirm the precision of the proposed methods, trolox standard addition was applied to a tea sample. The resulted voltammograms of trolox standard additions to a tea sample measured by DPV are presented in Figure
Voltammograms of trolox standard additions to a tea sample.
Spectrophotometric calibration curve of trolox standards (red points) and the corresponding trolox standard addition to a tea sample (blue points) are shown in Figure
Spectrophotometric calibration curve of trolox standards + trolox standard addition to a tea sample.
As Table
Recovery rates for trolox standard addition to a tea sample.
Sample | Trolox added (×10−4 mol·L−1) | Trolox found (×10−4 mol·L−1) | Recovery (%) | |||
---|---|---|---|---|---|---|
DPV | Spectrophotometry | DPV | Spectrophotometry | DPV | Spectrophotometry | |
Addition 1 | 2 | 2 | 1.954 | 2.059 | 97.692 | 102.941 |
Addition 2 | 3 | 3 | 3.023 | 2.864 | 100.769 | 95.466 |
Addition 3 | 4 | 4 | 4.077 | 3.882 | 101.925 | 97.059 |
Addition 4 | 5 | 5 | 4.958 | 5.118 | 99.160 | 102.353 |
Addition 5 | 6 | 6 | 6.130 | 6.235 | 101.166 | 103.922 |
To evaluate the limit of detection (LOD), the lowest trolox standard concentration was diluted serially and analysed six times with each technique as well. Then, the mean and the standard deviation and relative standard deviations were calculated (Table
Limit of detection (LOD) and limit of quantification (LOQ) in DPV and spectrophotometry.
Method | DPV | Spectrophotometry | DPV | Spectrophotometry |
---|---|---|---|---|
Statistical parameter | Limit of detection (LOD) | Limit of quantification (LOQ) | ||
Trolox concentration | 2 · 10−4 mol·L−1 | 4 · 10−4 mol·L−1 | ||
Average (mean of 6 repetitions) | 1.996 | 4.047 | 4.015 | 2.037 |
SD | 0.175 | 0.363 | 0.276 | 0.176 |
RSD | 8.742 | 8.960 | 6.863 | 8.646 |
The tea sample voltammograms (Figure
Voltammograms of tea samples by differential pulse voltammetry tests.
The voltammograms of the tea samples were used for the calculus of the antioxidant capacity expressed in trolox equivalents, as presented in Table
Current values (
Method | DPV | Spectrophotometry | ||
---|---|---|---|---|
Sample | Current ( |
Concentration (×10−4 mol·L−1) | Absorbance ( |
Concentration (×10−4 mol·L−1) |
G1 | 0.024351 | 8.624014 | 0.261 | 8.517647 |
G2 | 0.021224 | 11.230403 | 0.305 | 11.10588 |
G3 | 0.247403 | 8.299720 | 0.258 | 8.341176 |
G4 | 0.029478 | 4.351879 | 0.192 | 4.458824 |
G5 | 0.032166 | 2.111386 | 0.151 | 2.047059 |
|
||||
B1 | 0.022960 | 9.783305 | 0.279 | 9.576471 |
B2 | 0.020059 | 12.200286 | 0.319 | 11.92941 |
B3 | 0.025903 | 7.330995 | 0.238 | 7.164706 |
B4 | 0.023679 | 9.183913 | 0.270 | 9.047059 |
B5 | 0.018433 | 13.555708 | 0.348 | 13.63529 |
The results from Table
To evaluate the efficiency of the voltammetric method used in quantifying the concentrations of the samples, correlation between differential pulse voltammetry and spectrophotometry data was made (Figure
Correlations of tea sample concentrations between differential pulse voltammetry and spectrophotometry tests.
However, the antioxidant capacity (trolox equivalents) of the tea infusion samples ranged between 2.04 and 11.23 · 10−4 mol·L−1 for green tea samples and 7.16–13.63 · 10−4 mol·L−1 for black tea samples (Figure
Comparative results for the tea infusion samples.
However, the statistic tests (Table
Test |
|
Statistical results |
---|---|---|
|
|
|
|
||
F-test |
|
|
The analysis from the present study were employed to find an alternative to classical CUPRAC spectrophotometric or electrometric methods and to quantify the antioxidant capacity by using voltammetric technique with screen-printed microelectrodes. The screen-printed microelectrodes response was linearly correlated with Trolox content of the standards. Analytical results of the antioxidant capacity (expressed as mol·L−1 Trolox equivalents) of the tea samples showed a better agreement in the case of spectrophotometry and differential pulse voltammetry (
The raw data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that they have no conflicts of interest.