3.2. Differential Pulse Voltammetry (DPV)

In voltammetric techniques, the measured electrochemical signal is an algebraic sum of the undesirable capacitive current and the desired current related to the proper electrode reaction, the so-called Faraday current. DPV is an electrochemical technique which is widely used in chemical analysis and for studying the interactions of pharmaceuticals with DNA [41,43,45,51,60,63,65,94,95]. Its high sensitivity results from the pulsating change of potential applied to the working electrode. This type of signal modification effectively eliminates the capacitive current, thus facilitating the analysis of substances in a lower concentration range compared to the CV technique. The shape of the voltammetric curve is determined by a series of potential pulses applied at the right time to the working electrode. Following each pulse, the potential value returns to a slightly more negative value in the cathode part and to a more positive value in the anode part compared to that before the pulse. The pulse techniques work on the principle that with the step-change in potential, the values of both the currents increase sharply, while decreasing at different speeds. The capacitive current decreases rapidly compared to the Faraday current [84].

Buoro et al. used DPV for the electrochemical study of the interaction between gemcitabine (GEM) and DNA [43]. No GEM-associated redox process was observed under the experimental conditions. Two different approaches were used for studying the interactions: an unmodified GCE and a DNA electrochemical biosensor, prepared by successively covering the GCE surface with drops of the dsDNA solution. The DP voltammogram recorded immediately after the addition of GEM to the dsDNA solution displayed a decrease in the oxidation peak currents of dGuo and dAdo, compared with the control dsDNA solution. This effect was enhanced with an increase in the duration of the incubation of the sample and occurred under both experimental conditions (unmodified electrode and DNA electrochemical biosensor). The changes resulted from the aggregation of dsDNA, caused by the interaction with GEM, and were consistent with the spectrophotometric measurements. The formation of rigid DNA–GEM structures hindered the nucleoside residues from interacting and oxidizing at the GCE surface. The authors reported that the interaction between DNA and GEM caused modifications in the morphological structure of DNA. The mechanism of the DNA–GEM interaction occurred in two successive stages. The first stage was independent of the DNA sequence and led to the aggregation of dsDNA and the formation of the GEM–DNA rigid structure. The second stage favored the interaction between guanine hydrogen atoms in the CG base pair and fluorine atoms on the GEM ribose moiety, which induced the release of guanine residues on the electrode surface.

A similar approach was used by Diculescu et al. [45] in an experiment for analyzing the interaction between the anticancer drug danusertib and DNA. The studied drug was itself electrochemically active, which enabled the tracking of its individual signal changes. In addition, the experiment was carried out in incubated solutions, and DP voltammograms were recorded after different incubation periods. The voltammograms recorded after adding danusertib to the dsDNA solution displayed two oxidation peaks (D1 and D2) that were characteristic of the drug at lower potential values compared to the subsequent oxidation peaks of dGuo and dAdo. With a prolonged incubation period, a decrease in the peak current of the D2 signal was observed, while the intensity of peak D1 remained unchanged. An increase was observed in the intensity of the deoxyribonucleosides signals, which was in agreement with the conformational modification of the dsDNA. The second approach to the experiment involved the use of a prepared DNA electrochemical biosensor which had dsDNA immobilized on the GCE surface. The recorded voltammograms demonstrated the formation of a DNA–danusertib adduct (Figure 4A). The effect of drug concentration was also studied (Figure 4B). The binding of danusertib led to modifications in the morphological conformation of dsDNA, causing slight changes in the oxidation peak currents of dGuo and dAdo.

DP voltammograms, with no conditioning potential, in a 0.1 M acetate buffer with a pH of 4.5, with the dsDNA-electrochemical biosensor after: (A) (▬) 30 min in the buffer control experiment, and incubation in: 10 μM of danusertib during (•••) 15, (⁃⁃⁃) 30 and (▬) 60 min, and (B) incubation in (▬) 5 μM and (▬) 25 μM of danusertib during 30 min. Figure adapted from the reference [45] with permission from Elsevier.

The studies determined the interaction of dsDNA–danusertib that occurred in two successive stages. The first stage involved the electrostatic interaction of the positively charged piperazine ring with the DNA phosphate backbone. In the second step, the formation of a DNA–drug complex involving the pyranopyrazole moiety occurred, resulting in morphological modifications in the DNA double helix.

The changes in the current signals recorded using the DPV technique can also be used to calculate the value of the drug–DNA binding constant. Dindar et al. [47] studied Citalopram (CIT) and its S-enantiomer—escitalopram (ESC), which are antidepressants belonging to the selective serotonin reuptake inhibitors class. The experiment was conducted by adding increasing concentrations of drugs (from 2 to 10 µg/mL) to the 100 µg/mL ctDNA in an acetate buffer solution with a pH of 4.7 and recording the oxidation signals of dGuo and dAdo using GCE. Based on the reduction in the intensity of the current response (I) caused by the binding of DNA to the CIT and ESC molecules, the plots of log IcomplexIDNA−Icomplex vs. log C_drug were determined. Based on the slope and the intercept of the plot values, the binding constants for CIT and ESC were calculated (K_CIT-DNA = 5.6 × 10⁴ M⁻¹ and K_ESC-DNA = 8.5 × 10⁴ M⁻¹), using the following equation:

where I_DNA and I_complex are dAdo are the peak currents in the absence and presence of different drug concentrations, respectively. Slightly lower values of the binding constants compared to typical intercalators suggest a groove or an electrostatic binding mode rather than an interaction; however, it did not exclude it.

Bayraktepe [54] used DPV to describe the interaction of DNA with dasatinib (DSB) and to determine the adduct binding constant value. In her experiment, a 10.0 μM DSB solution and an acetate buffer solution with a pH of 4.8 was used, and dsDNA was added (from 2 to 70 μM). DPV voltammograms showed that the peak current of DSB decreased with increasing DNA concentrations up to 30.0 μM and then remained constant (Figure 5). Moreover, the peak potential of DPV voltammograms changed to more positive values. The binding constant of the DSB–DNA complex was calculated as K = 2.51 × 10⁴ M⁻¹. Moreover, the Gibbs free energy (ΔG°) of the adduct was estimated as −25.10 kJ/mol, using the following equation:

DPV voltammograms of 10.0 μM DSB with increasing concentrations of DNA in an acetate buffer solution with a pH of 4.8. Insets: (A) C_DNA−i_pDSB; (B) C_DNA−E_pDSB; (C) logidrug−DNAidrug−idrug−DNA−log1DNA. Figure adapted from the reference [54] with permission from Elsevier.

The negative value proves the DSB and DNA interaction and indicates that binding occurred spontaneously. All the obtained results indicate that DSB interactions with DNA may have an intercalation mode. Thermodynamic parameters found from voltammetric measurements are comparable to those obtained by the UV spectroscopic method.

Ponkarpagam et al. [49] studied the interactions between ctDNA and rosiglitazone (RG)—a thiazolidinedione anti-diabetic drug—in a 0.05 M Tris-HCl buffer solution (pH 7.3) in the absence and presence of increasing concentrations of ctDNA, using GCE. The decrease in the peak current suggested an interaction of RG with ctDNA by the forming of an electrochemically non-active adduct. This is due to the low diffusion coefficient resulting from the Stokes-Einstein equation and, consequently, from low or negligible currents. The shift of the peak to a more negative potential indicated the groove binding mode of the interaction, which was also confirmed by molecular docking. The binding constant has been determined as K = 3.4 × 10³ M⁻¹.

Due to their ease of use, the construction of disposable measurement systems is an interesting trend in electrochemical approaches. In particular, modified surface electrodes are designed to improve the sensitivity or selectivity of measurements. Single-use modified biosensors are sensitive, time-saving, and practical tools for detecting the analyte. Such systems have several main advantages: a large surface area, effective mass transport, controllability, and their ability to study interactions in solution. Eksin et al. [51] studied the interaction between daunorubicin (DNR) and ctDNA at the surface of disposable carbon quantum dot-modified PGEs (cQD-PGE). For monitoring the surface-confined interaction, ctDNA was first immobilized onto the electrode surface, and then the electrochemical detection of the interaction between DNA and DNR was carried out. The study aimed to optimize the experimental conditions, such as the concentrations of both ctDNA and DNR, as well as determine the effect of interaction time (from 3 to 15 min) on the changes in the oxidation signals of guanine and DNR. Under optimal conditions, very low values of the detection limits were obtained for DNR and ctDNA—0.02 μg/mL and 0.89 μg/mL, respectively.

Other examples of interesting modifications were presented by Findik et al., who have modified pencil graphite electrodes (NFs-PGE; Figure 6) as sensitive electrochemical biosensors for the anticancer drugs daunorubicin (DNR) [53] and mitomycin C (MC) [52]. In these studies, newly designed and different organic–inorganic hybrid nanoflowers were used.

(A) Schematic illustration of the formation of amino acids-Cu₃(PO₄)₂ hybrid NFs, (B) The representative scheme of the pretreatment of PGE (i), modification of NFs (ii), immobilization of DNA (iii) and DNR (iv), surface-confined interaction of DNR and ctdsDNA (vi). Figure adapted from the reference [53] with permission from Elsevier.

In the case of the development of disposable voltammetric sensors for the electrochemical analysis of ctDNA, DNR, and the interaction between them, the L-glutamic acid nanoflowers (ga-NFs) and L-cysteine nanoflowers (c-NFs) were applied. Amino acid-Cu₃(PO₄)₂ hybrid NFs were modified at the surface of single use PGE. The c-NFs-PGE electrode turned out to be very sensitive for the detection of both DNA (0.93 µg/mL) and DNR (2.93 µM). In the case of the DNR–DNA interaction, which was the main purpose of Findik’s study, it was determined that both the DNR oxidation peak and the guanine peak decreased at all interaction times. The highest decrease in a short time of 1 min showed that c-NFs-PGE is a very useful sensor for DNR studies.

The sensors developed to determine MC and its interactions with DNA used glycine and lysine nanoflowers, and were labeled as GNFs and LNFs, respectively. Nanoflowers formed the mono-dispersed 3D hierarchical superstructures (Figure 7). The average diameter of these hybrid NFs with excellent monodispersity was determined to be 3 μm and they were obtained in a homogeneous structure.

SEM image of (A) GNFs; (B) LNFs; (C) PGE; (D) GNFs-PGE; (E) LNFs-PGE ((a–c)—different resolutions) and EDX pattern (d). Figure adapted from the reference [52] with permission from Elsevier.

The detection limit of the biosensor was determined (1.09 μg/mL for ctDNA) and the biointeraction between MC and ctDNA was investigated.

In order to develop a sensitive tool for DNA detection and to elucidate its structural changes after the interaction with drugs, Bolat [55] constructed a DNA biosensor based on electrodeposited cetyl trimethylammonium bromide-multiwalled carbon nanotubes (poly(CTAB-MWCNTs)) composite on single-use PGE. The DPV and UV–Vis techniques were used to study the interaction of dsDNA with the anticancer drug irinotecan (CPT-11). Voltammetric measurements were based on the changes at the guanine oxidation peak. A high sensitivity was obtained for DNA and DNA–anticancer drug interaction with detection limits of 3.06 μg/mL and 1.03 μg/mL, respectively. Moreover, the binding constant value was determined as K = 6.84 × 10⁴ M⁻¹. The experiment showed that the interaction between CPT-11 and DNA leads to a condensation of the DNA double helix and indicated a groove binding mechanism.

Janiszek et al. [48] in their experiment compared two prospective anticancer drugs, 6-(1H-imidazo [4,5-b]phenasine-2-yl)benzene-1,3-diol (IPBD) and its -Cl derivative (Cl-IPBD) with doxorubicin, a widely used anthracycline anticancer agent. For the comparison of the DNA interactions with the drugs, plasmid modified GCEs were used. The aim of the modification of the electrode with supercoiled plasmid instead of typically chromosomal DNA was to minimize the interference of the DNA oxidation. Plasmid (scpUC19) accumulation resulted in the formation of well defined, reproducible plasmid DNA layers on a typical, easily available GCE. In this experiment DPV, square wave voltammetry (SWV) and the less frequently used alternating current voltammetry (ACV) with phase detection 0°, ACV (0°), as well as 90°, ACV (90°) techniques were used in a specific combination. The correlation of the redox signals of IPBD and Cl-IPBD, with their biological effect on cancer cells were shown. Moreover, the effect of Vitamin C on the redox signals of Cl-IPBD that resemble the reduction in Pt(IV) anticancer prodrugs to Pt(II) compounds was observed.

Moreover, biopolymers are used for electrode modification as they offer stable, biocompatible, and large surface areas for the immobilization of biomolecules. Congur et al. [56] modified PGEs with Levan (LVN), a fructan homopolysaccharide comprised of β-d-fructofuranose residues linked by β-(2→6) glycosidic bonds (Figure 8). The aim of the experiment was to develop disposable electrochemical biosensors for the detection of DNA, daunorubicin (DNR), and the biomolecular interaction of DNR with DNA. The interaction of 20 μM DNR with DNA at the DNA-LVN-PGE modified electrode was evaluated between 3 and 10 min and a decrease in guanine and DNR signals (increasing with the interaction time) was observed. This was caused due to the intercalation of DNR into double stranded DNA resulting in strand breaks.

The experimental steps of the modification of LVN at the PGE surface, voltammetric determination of fsDNA and DNR using LVN-PGE and the voltammetric analysis of the biomolecular interaction between fsDNA and DNR at the LVN-PGE surface. Figure adapted from the reference [56] with permission from Elsevier.

Javar et al. [58] developed an electrochemical DNA biosensor based on modified CPEs (Eu³⁺-doped NiO/CPEs) for the determination of the anti-cancer drug amsacrine. The powder XRD technique was used to examine the crystal structure of the synthesized nanocomposite and cyclic voltammograms of Fe[CN]₆^3-/4- redox couple were recorded at the surface of the bare CPE. NiO NPs/CPE, Eu³⁺-doped NiO/CPE, and dsDNA/Eu³⁺-doped NiO/CPE were used as the indicators for modification. The effect of the amsacrine–guanine interaction has been electrochemically investigated in comparison to the alterations in the guanine oxidation peak in the absence and presence of amsacrine.

The DPV technique can also be used to study the interaction between metal–ligand complexes and the DNA chain. In the experiment carried out by Kumar et al. [60], the results of voltammetric and spectroscopic studies confirmed that tetraazamacrocyclic complexes interacted with DNA through the same type of binding. Voltammograms obtained for each macrocyclic complex displayed a significant decrease in the current intensity in the presence of ctDNA, which indicates that these metal ions stabilize the ctDNA duplex by the intercalation mode. It was found that the macrocyclic cobalt (II) ion complex interacts most strongly with ctDNA.