Voltammetric Measurement of Antioxidant Activity by Prevention of Cu(II)-Induced Oxidative Damage on DNA Bases Using a Modified Electrode

Abstract

The protective effect of antioxidants using electrochemical techniques can be evaluated by examining the oxidative changes in deoxyribonucleic acid (DNA) nucleobases. In this study, a gold nanoparticle (AuNP)-decorated and multiwalled carbon nanotube (MWCNT)-Nafion-modified glassy carbon electrode (GCE/AuNP/MWCNT-Nafion) was developed to evaluate the preventive ability of antioxidants on oxidative DNA damage. A modified working electrode was prepared and characterized by cyclic voltammetry, electrochemical impedance spectroscopy, and scanning electron microscopy. The developed electrochemical method relies on two phenomena: (i) reactive species (RS) produced by dissolved oxygen in the presence of copper(II) partially damage the DNA immobilized on the electrode surface and (ii) antioxidant compounds prevent this damage by scavenging the formed RS. Changes in guanine, adenine, and cytosine oxidation signals resulting from DNA damage were measured using differential pulse stripping voltammetry before/after the interaction of dsDNA with Cu(II) while antioxidants were absent or present. The DNA protective ability of antioxidants was assessed for a number of antioxidant compounds (i.e., ascorbic acid, gallic acid, epicatechin, catechin, epicatechin gallate, glutathione, chlorogenic acid, N-acetyl cysteine, rosmarinic acid, quercetin, and rutin). Quercetin was found to show the highest antioxidant effect, and its limit of detection was determined as 1 μM. The manufactured biosensor was put in an application for the determination of antioxidant activity of herbal teas.

Scheme 1. Schematic Diagram of GCE/AuNP/MWCNT-Nafion Biosensor Preparation

Scheme 2. Schematic Diagram of the Developed Electrochemical Method for the Determination of AO Ability in Preventing DNA Damage

Figure 1

Comparison of different modes of modification of the bare electrode with AuNP, MWCNT, and Nafion in the presence of 250 mg/L dsDNA [scan range: (+) 0.4 to (+) 1.4 V; step size: 5 mV; pulse size: 25 mV; sample period: 0.5 s; pulse time: 0.1 s; deposition time: 180 s; deposition potential: 0 V; and 50 mM pH 7.4 PBS].

Figure 2

Electrodeposition coating of AuNPs on the bare electrode surface (GCE) by the CV technique (2.5 mL of 0.04% (w/v) HAuCl₄ + 2.5 mL of 0.1 M H₂SO₄; scanning rate: 50 mV/s; cycle number: 30; and scanning range: −0.4 to 0.4 V).

Figure 3

Cyclic voltammetric behavior of GC electrodes in 0.1 M KCl containing 5 mM Fe(CN)₆^3−/4− (scan rate: 50 mV/s).

Figure 4

Nyquist plots of bare GCE, AuNP, and AuNP/MWCNT-Nafion-modified electrodes in a 0.1 M KCl solution containing 5 mM Fe(CN)₆^3–/4– (frequency range: 10⁶–10^–1 Hz and amplitude: 5 mV).

Figure 5

SEM images of modified electrodes: (a) GCE/AuNP, (b) GCE/AuNP/MWCNT-Nafion, and (c) GCE/AuNP/MWCNT-Nafion/DNA (magnification: 100,000×; AuNP sizes: 30–150 nm; and dsDNA: 250 mg/L immobilized).

Figure 6

UV–vis spectra of the dsDNA solution before and after interaction with an aerated Cu(II) solution (10-fold diluted).

Figure 7

DPS voltammograms of PBS, dsDNA, and Cu(II) + dsDNA solutions obtained with the modified electrode (GCE/AuNP/MWCNT-Nafion) (scan range: 0.4–1.4 V; 75 mM pH 7.4 PBS).

Figure 8

DPS voltammograms obtained with the modified electrode (GCE/AuNP/MWCNT-Nafion) (of 250 mg/L dsDNA; before interaction with a 1.5 mM Cu(II) solution, after 30 min interaction), in the presence of 0.005–0.05 mM CT (after 30 min interaction).

Figure 9

Comparison of ROS scavenging efficiencies (% AO effect) of various AO compounds (0.05 mM): QR—quercetin; CT—catechin; RT—rutin; GA—gallic acid; ECG—epigallocatechin; EC—epicatechin; CA—chlorogenic acid; NAC—N-acetyl cysteine; AA—ascorbic acid; GSH—glutathione; and RA—rosmarinic acid (n = 6).

Figure 10

Comparison of the radical scavenging efficiencies (AO effect, %) obtained by the developed electrochemical method for herbal tea extracts (0.05 mL) (n = 6).

Figure 11

HPLC-PDA chromatograms [250 mg/L dsDNA, after interaction with 1.5 mM Cu(II), after interaction with 1.5 mM Cu(II) in the presence of CT (a) and green tea (b)] (λ = 254 nm; n = 3).