Applications of a DNA-electrochemical biosensorVictor Constantin Diculescu, Ana-Maria Chiorcea-Paquim, Ana Maria Oliveira-Brett *Department of Chemistry, University of Coimbra, Portugal

A R T I C L E I N F O

Keywords:DNA-electrochemical biosensorVoltammetryG-quadruplexDNA oxidative damageProteinDrugMetal ionPollutantFree radicalRadiation

A B S T R A C T

As carrier of genetic information, DNA is one of the most important intracellular targets that undergomodification and damage upon interaction with endogenous and exogenous factors. DNA is an excel-lent biomaterial for the construction of new devices, in nanotechnology and biosensor technology, forevaluation of DNA interaction with a broad range of chemical compounds and biomolecules, essentialfrom a biological and a medical point of view.

This review discusses recent advances on the design and applications of DNA-electrochemicalbiosensors that use DNA direct electrochemistry as a detection platform. AFM and voltammetriccharacterization of new bottom up immobilisation procedures of self-assembled nanostructures basedon DNA single- and double-stranded, G-quadruplex, and i-motif configurations are presented, relevantfor the development of new DNA-electrochemical biosensor devices. The applications of DNA-electrochemical biosensors, for the label-free detection of interactions with proteins, pharmaceuticalcompounds, metal ions and metal complexes, pollutants, free radicals, and electromagnetic radiation,were revisited.

© 2016 Elsevier B.V. All rights reserved.

Contents

1. Introduction ........................................................................................................................................................................................................................................................... 232. Development of DNA-electrochemical biosensors .................................................................................................................................................................................. 243. Applications of DNA-electrochemical biosensors .................................................................................................................................................................................... 27

3.1. Proteins ...................................................................................................................................................................................................................................................... 273.2. Drugs .......................................................................................................................................................................................................................................................... 273.3. Metal ions and their complexes ....................................................................................................................................................................................................... 313.4. Pollutants .................................................................................................................................................................................................................................................. 323.5. Free radicals ............................................................................................................................................................................................................................................. 333.6. Radiation ................................................................................................................................................................................................................................................... 34

4. Conclusion .............................................................................................................................................................................................................................................................. 34Acknowledgements ............................................................................................................................................................................................................................................. 35References .............................................................................................................................................................................................................................................................. 35

1. Introduction

DNA is a stable, low-cost and easily adaptable molecule, beingan excellent building block for the construction of new devices innanotechnology and biosensor technology. DNA-based biosensorshave been successfully used in numerous applications, such as, in-vestigation and evaluation of DNA-drug interaction mechanisms,detection of DNA base damage in clinical diagnosis, rapid moni-toring of metals or pollutant agents in the environment, direct

monitoring hybridization processes or label-free detection of spe-cific DNA sequences and proteins.

A DNA-electrochemical biosensor is formed by an electrode (theelectrochemical transducer) with a DNA probe immobilized on itssurface (the biological recognition element) and is used to detectDNA-binding molecules (the analyte) that interact and inducechanges in the DNA structure and electrochemical properties, whichare further translated into an electrical signal (Scheme 1). Electro-chemical methods offer rapid detection, great sensitivity, and lowcost. A DNA-electrochemical biosensor build in this way can eitherbe label-free, by directly monitoring the changes in the DNA basesoxidation peaks before and after the interaction with the analyte,or use different amplification strategies [1–3].

* Corresponding author. Tel.: 00351239854487; Fax: 00351239827703.E-mail address: brett@ci.uc.pt (A.M. Oliveira-Brett).

http://dx.doi.org/10.1016/j.trac.2016.01.0190165-9936/© 2016 Elsevier B.V. All rights reserved.

Trends in Analytical Chemistry 79 (2016) 23–36

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In this review, we will discuss recent advances on the design andapplications of DNA-electrochemical biosensors that use DNA directelectrochemistry as a detection platform. This type of DNA-electrochemical based biosensors are highly sensitive, up tofemtomoles of analyte, label-free and allowed the use of differentelectrode substrates, which made them suitable for inexpensiveminiaturization for clinical diagnosis and on-site environmentalmonitoring.

2. Development of DNA-electrochemical biosensors

Understanding the redox behaviour of the DNA probe is criti-cal for the design and successful application of label-free DNA-electrochemical biosensors. Electrochemical studies of DNA bases,purines: adenine (A) and guanine (G), and pyrimidines: thymine (T)and cytosine (C) (Scheme 2A), their nucleosides, and nucleotides,as well as of native or denaturated DNA probes were performed atmercury and carbon electrodes [5–8].

Cyclic voltammograms of nucleic acids at dropping mercuryelectrode showed one cathodic peak, due to irreversible reductionof C and A residues, while the reduction of G residues occurredat very negative potentials, and only the oxidation peak of the Gresidues reduction product was detected in the reverse scan[5,6].

At carbon electrodes, the voltammetric studies showed that DNAbases, nucleosides and nucleotides are all electroactive (Fig. 1), andtheir oxidation is pH dependent [7,8]. The voltammetric detectionof the G and A oxidation products, 8-oxoguanine (8-oxoG) and 2,8-dihydroxyadenine (2,8-DHA), biomarkers of DNA oxidative stress,allowed direct detection of DNA oxidative damage after interac-tion with the analyte. Differential pulse (DP) voltammogramsrecorded at a glassy carbon electrode (GCE) in DNA solutions showedtwo anodic peaks, corresponding to the oxidation of guanosine(dGuo) and adenosine (dAdo) residues in dsDNA (Fig. 2A) [9–11].The difference obtained for single- (ssDNA) versus double- (dsDNA)stranded DNA oxidation peak currents was correlated with thegreater difficulty for the transfer of electrons from the inside of thedouble-helix to the electrode surface, when compared with a single-helix with residues in closer proximity to the electrode surface(Fig. 2). These results represented the sensing strategy of many label-free DNA-electrochemical sensors.

The development of DNA-electrochemical biosensors involvedthe immobilization of DNA at the electrode surface. This processinfluences the characteristics of the DNA probe, the accessibilityof the chemical compounds to the DNA, the sensor responseand its performance. The development of new immobilisationmethodologies, based on controlled bottom-up self-assembling ofnucleic acid nanostructures, starting with either long chain nucleic

acid or custom synthetic short oligodeoxynucleotide (ODN)sequences is a current concern in DNA-electrochemical biosensortechnology.

Studies of DNA adsorption were first conducted on mercury[5,11,14] and later on carbon electrodes [9–11], and a smalleradsorption was always observed for dsDNA compared with forssDNA. Ellipsometry and spectroscopic techniques, such as surfaceenhanced Raman spectroscopy, have been used to investigate theadsorption of DNA onto electrode surfaces [11]. More recently, atomicforce microscopy (AFM) was employed to resolve at nanoscale thesurface morphological structure of nucleic acid molecules and tounderstand the nature of the DNA-electrode surface interactions[12,13,15–23]. Both dsDNA and ssDNA showed tendency to spon-taneously self-assemble onto carbon electrodes, forming thin, two-dimensional network films, whose characteristics depended on DNAconcentration, pH and immobilization procedure (Fig. 2B-E).Adsorption under low positive applied potential, not sufficient tooxidise the DNA bases, leaded to more robust and stable DNA films(Fig. 2C, E). The knowledge of the morphology of adsorbed DNA onelectrode surfaces explained the non-specific adsorption on the DNA-electrochemical biosensor surface. DNA-electrochemical biosensorswith a low degree of non-specific binding required deposition ofmultilayers of DNA probe which can be achieved for a higher DNAconcentration.

Many DNA biophysical properties, such as its conformationalflexibility and ability to self-assemble through hydrogen bonds, areinfluenced by the DNA base sequence, length, concentration, pH andionic strength. In order to determine the optimum conditions forthe nucleic acid immobilization on carbon electrodes, the influ-ence of the sequence composition on the DNA self-assembly wasinvestigated. The adsorption and redox behaviour of homo-ODNsd(A)10, d(T)10 and d(C)10, were studied, by AFM at a highly orientedpyrolytic graphite (HOPG) surface and by DP voltammetry at a GCE[4]. The combination of AFM and voltammetry revealed strongcorrelations between the degree of surface coverage, the base com-position of the ODN molecules, and the ODN secondary structurewhich is directly influenced by the solution concentration and pH.Homo-ODNs can adopt different configurations, ranging from singleto quadruple helical structures (Scheme 2D), since bases of the sametype self-associate in a variety of arrangements (Scheme 2C),different from the Watson Crick base-pairs (Scheme 2B). Underphysiological pH, d(A)10, d(C)10 and d(T)10 self-assembled at thesurface of carbon electrodes as network films with knobbyappearance, due to the aggregation and coiling of the single-strands. In mild acid pH solutions, d(A)10 double-helicalconformations (Scheme 2D-left) were observed, by AFM as networkfilms with lower surface coverage, and detected by DP voltammetrythrough the decrease of the A oxidation peak current. In the same

Scheme 1. - DNA electrochemical biosensor principle of operation.

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conditions, d(C)10 formed i-motifs (Scheme 2D-right), observed byAFM as spherical aggregates.

G-rich DNA can formG-quadruplex (GQ) structures and have beenthe subject of numerous applications, covering areas from struc-tural biology to medical chemistry, supra-molecular chemistry,nanotechnology and biosensor technology. They are consideredcancer-specific molecular targets for anticancer drugs, since the GQstabilisation by small organic molecules can lead to telomeraseinhibition and telomere dysfunction in cancer cells.

The redox behaviour of DNA sequences able to self-assemble intoGQ configurations was studied only recently [24–27]. The first reporton the electrochemical oxidation of GQs concerned the investiga-tion of two, different length, thrombin-binding aptamer (TBA)sequences, d(G2T2G2TGTG2T2G2) and d(G3T2G3TGT3T2G3), using DP

voltammetry at a GCE and AFM at a HOPG surface [24]. In Na+

containing solutions, the oxidation of both TBA sequences showedone anodic peak corresponding to the oxidation of G residues in theTBA single-strands. Upon addition of K+ ions, both sequences foldinto GQs, causing the decrease of G oxidation peak and occurrenceof a new GQ peak at a higher potential, due to the oxidation of Gresidues in the GQs.

The process of GQ formation is directly influenced by the ODNsequence and concentration, pH and presence of monovalentcations (Na+ vs. K+). This was determined using 10-mer ODNs thatcontain only one block of 8–10 guanines, d(G)10, d(TG9) andd(TG8T) [4,26–28], and expected to form parallel tetra-molecularGQ structures (Scheme 2D-middle). DP voltammetry allowedthe detection of the association of single-strands into GQs and

Scheme 2. - (A) Chemical structure of DNA bases, (B) Watson-Crick base-pairing (C) Homo-ODNs base-pairing and (D) Schematic representation of the DNA double-strand, G-quadruplex and i-motif configurations. [Adapted from Ref. [4] with permission].

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G-based nanostructures, in freshly prepared solutions, atconcentrations 10 times lower than usually detected using othertechniques currently employed to study the formation of GQs.Single-stranded ODNs were detected only in Na+ ions containingsolutions for short incubation times. The GQ structures wereformed slowly in Na+ ions, after a long incubation time, and fasterin K+ ions, after a short incubation time. The formation of higher-order nanostructures, due to the presence of a long contiguous Gregion, and the influence of the T residues at the 5’ and 3’molecular ends was clarified. For increased d(G)10 concentrations,long G-nanowires were formed, demonstrating the potential ofG-rich DNA sequences as a scaffold for nanotechnologicalapplications.

The d(TG4T) telomeric repeat sequence of the free-living ciliateprotozoa Tetrahymena forms tetra-molecular GQ structures, and isconsidered a simpler model of biologically relevant GQs, being usedto obtain high resolution data on drug-DNA interactions. The well-known conformation of the d(TG4T)-GQ and its extraordinary stiffnesshave made the d(TG4T) sequence a good candidate for thedevelopment of novel devices, with medical and nanotechnologicalapplications. AFM and DP voltammetry showed d(TG4T) single-strands self-assembling into GQs, very fast in K+ and slowly in Na+

ions containing solution (Scheme 3 and Fig. 3) [29]. The optimumK+ ions concentration for the formation of d(TG4T)-GQs was similarto the healthy cells intracellular K+ ions concentration. In the pres-ence of Na+ ions, d(TG4T) also formed short nanowires andnanostructured films that were never observed in K+ ions contain-ing solution, suggesting that rapid formation of stable GQs in thepresence of K+ is relevant for the good function of cells.

Synthetic polynucleotides poly(dG) and poly(G) are widelyprevalent in the human and other genomes at both DNA and RNAlevels, andwere used asmodels to determine the interaction of drugswith G-rich segments of DNA. AFM and DP voltammetric studiesshowed the poly(G) single-strands self-assembling into short GQregions at low incubation time, while large poly(G)-GQ aggregateswith low adsorptionwere formed after high incubation times in thepresence of monovalent Na+ or K+ ions [30]. The DP voltammetry infreshly preparedpoly(G) solutions showedonly theGoxidationpeak,due to the oxidation of G residues in the poly(G) single-strand.Increasing the incubation time, the G oxidation peak decreased anddisappeared, and a GQ residues oxidation peak in the poly(G)-GQmorphology appeared, at a higher oxidation potential, dependenton the incubation time, presenting a maximum after 10 daysincubation, and reaching a steady value after ~ 17 days incubation.

The recent advances in theDNA electrochemical characterisationand immobilisation of short and long DNA sequences have deter-mined in further detail the key factors in controlling the distribution,size and shape of highly ordered two- and three-dimensional DNAnanostructures on the electrode surface. Thebottomup self-assemble

Fig. 1. - DP voltammograms baseline corrected recorded in a 20 μM equimolarmixture of guanine (G), adenine (A), thymine (T) and cytosine (C) in pH = 7.4 with:(a) 1.5 mm, (b) 7 μm diameter GCE. [From Ref. [8] with permission].

Fig. 2. - (A) DP voltammograms base line corrected obtained with the GCE in solutions of 60 μg mL−1 (••••) ssDNA and ( ) dsDNA. (B-E) AFM images of an HOPG elec-trode modified by free adsorption and adsorption at + 300 mV, vs. AgQRE, from solutions of (B) 60 μg mL−1 ssDNA, (C) 1.0 μg mL−1 ssDNA, and (D, E) 60 μg mL−1 dsDNA[Adapted from Refs. [12,13] with permission].

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of diverseDNAnanostructures allowsdifferent applications. The longchains dsDNA, ssDNA, and the purine homo-polynucleotide poly(A)and poly(G), as well as ODNs in single- and double-stranded andGQ configurations, can be used for screening cancer therapeuticagents. The perfectly aligned G-nanowires may represent buildingblocks of molecular nanowires for nanoelectronics, and the G-basedsuper-structures and frayed G-nanowires with slipped-strands canwork as a nucleation platform for the addition of subsequent strandsand the formation of larger structures.

3. Applications of DNA-electrochemical biosensors

The DNA-electrochemical biosensors applications for the label-free detection of proteins and detection of DNA damage inducedby drugs, metal ions and their complexes, pollutant agents, free radi-cals and radiation, will be discussed.

3.1. Proteins

The detection and quantification of proteins play an essential rolein fundamental research and clinical applications.

Aptamers are small nucleic acid sequences (DNA or RNA) selectedin vitro from large combinatorial pools to bind to specific targets.Due to their high affinity for a series of biomolecules, they are largelyused in biosensor development. The structure-activity relationshipof the complex formed between two different length TBA aptamersequences, d(G2T2G2TGTG2T2G2) and d(G3T2G3TGT3T2G3), and theserine protease thrombin was evaluated, by DP voltammetry at aGCE and AFM at a HOPG surface, and the interaction mechanismwas established [25,26]. The effects on the interaction with thrombinof TBA primary and secondary structures, as well as of its foldingproperties in the presence of alkaline metals were investigated.Single-stranded TBA sequences coiled around thrombin, leading tothe formation of a robust TBA-thrombin complex that maintainedthe thrombin symmetry and conformation, therefore, the thrombinoxidation peaks within the TBA-thrombin complex occurred at morepositive potentials, than in the case of free thrombin. In the presenceof K+ ions, the aptamers folded into GQs that facilitated theinteraction with thrombin. TBA-thrombin complexes adsorbed onthe carbon electrode with the TBA in contact with the surface andthe thrombin on top, far from the surface, thus thrombin being lessaccessible to oxidation and also leading to the occurrence ofthrombin oxidation peaks at more positive potentials.

Monoclonal antibodies (mAb) have earned special attention dueto their specific and effective therapeutic properties and have becomeone of themost promising strategies for cancer treatment. The studyof the interaction between mAbs and dsDNA has great impor-tance to predict its actionmechanism as a genotoxic anticancer drugand to understand its biological activity and toxicity in vivo.

Bevacizumab (BEVA) is a recombinant humanized IgG1 mono-clonal antibody that targets vascular endothelial growth factor A,being effective in treatment of several types of cancer, such as colon,lung, kidney, ovarian and brain cancers. BEVA interactionwith dsDNAwas studied by voltammetry and gel-electrophoresis in incubatedsamples and using a dsDNA-electrochemical biosensor [31]. Thevoltammetric results at a DNA electrochemical biosensor revealeda decrease and disappearance of the dsDNA oxidation peaks withincreasing incubation time, showing that BEVA binds to dsDNA butno DNA oxidative damagewas detected (Fig. 4). BEVA also undergoesstructural modification upon binding to dsDNA, and BEVAelectroactive amino acid residues oxidation peaks were identified.Non denaturing agarose gel-electrophoresis experiments were inagreement with the DP voltammetric results showing the formationof compact BEVA-dsDNA adducts.

Rituximab (RTX) is a chimeric human/mouse mAb that targetsspecifically the CD20 antigen, a receptor expressed on the major-ity of malignant B-cells (more than 80%) and on normal differentiatedB-lymphocytes (pre-B andmature B-lymphocytes). RTXwas the firstFood and Drug Administration (FDA) approved genetically engineeredmAb for use in indolent B-cell non-Hodgkin’s lymphoma (b-NHL),and is currently used in both indolent and aggressive B-NHLs, B-cellchronic lymphocytic leukemia and some autoimmune disease. RTXinteraction with dsDNA was investigated by DP voltammetry, inincubated samples and using a multilayer DNA-electrochemicalbiosensor, and gel electrophoresis [32]. The dsDNA-RTX interactionpromoted a strong condensation of the dsDNA helical structure,followed in DP voltammetry by the dGuo oxidation peak currentdecrease, dAdo oxidation peak disappearance, and the occurrenceof the oxidation peaks of free G and A bases released from the DNA,but no DNA base oxidative damage was detected.

3.2. Drugs

Protein kinases are enzymes responsible for phosphorylationprocesses. Several classes of kinases have been recognised as targetsfor the development of small inhibiting molecules for anticancer

Scheme 3. - Schematic representation of d(TG4T) single-stranded and quadruplex electrochemical detection. [From Ref. [29] with permission].

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therapy. In vitro studies demonstrated that kinase inhibitors and/or their metabolites can increase the amount of DNA damage [33]and showed that some of these compounds retained a genotoxicactivity either through intercalation into the DNA, or formation ofalkali-labile sites and/or DNA strand breaks.

Danusertib is a kinase inhibitor and anti-cancer drug, and itsinteraction with dsDNA was investigated in bulk solution and usinga dsDNA-electrochemical biosensor [34]. It has been shown that the

dsDNA-danusertib interaction occurs in two sequential steps. First,danusertib binds electrostatically to dsDNA phosphate backbonethrough the positively charged piperazine moiety. The second stepinvolves the pyrrolo-pyrazole moiety and leads to small morpho-logical modifications in the dsDNA double helix which wereelectrochemically characterised through the changes of dGuo anddAdo oxidation peaks and confirmed by electrophoretic and spec-trophotometric measurements. The nitrenium cation radical productof the danusertib amino group oxidation was electrochemicallygenerated in situ on the dsDNA-electrochemical biosensor surface.The danusertib nitrenium cation radical redox product wascovalently attached to the C8 of G residues, protecting them and

Fig. 3. - AFM images of d(TG4T) spontaneous adsorbed onto HOPG from 0.3 μMd(TG4T) in sodium phosphate buffer pH = 7.0, in the presence of 100 mM K+ ions,after (A) 0 h, (B) 48 h and (C) 7 days incubation. [From Ref. [29] with permission].

Fig. 4. - DP voltammograms baseline corrected, in 0.1 M phosphate buffer pH 7.0and 100 μgmL−1 dsDNA ( ) before and (A) after incubation in solution with 100 μgmL−1 BEVA during ( ) 0 and (•••) 48 h, and (B, C) after incubation in solution with(•••) 10 and ( ) 500 μg mL−1 BEVA during (B) 0 and (C) 48 h. [From Ref. [31] withpermission].

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preventing their oxidation (Scheme 4). A dsDNA-danusertib inter-actionmechanismwas proposed and the formation of the danusertibredox nitrenium radical product-guanine adduct explained.

The kinase inhibitor lapatinib (LPT) is a new active drug for breastcancer and other solid tumours. The electro-oxidation mechanismof LPT at a GCE was studied using various voltammetric techniques,and the effect of pH and scan rate on lapatinib signal was investi-gated [35]. LPT exhibited three charge transfer reactions, eachinvolving the transfer of two electrons. The LPT oxidation wascompared with model compounds which contained aromatic aminestructures, and an electrooxidation pathway was proposed. ThedsDNA-LPT interaction using a DNA-electrochemical biosensor andby spectroscopic techniques was studied. The binding constant (K)between LPT and DNA was calculated as 6.03 × 105 M−1 by electro-chemistry, 4.20 × 105 M−1 by UV-vis spectrophotometry, and3.50 × 104M−1 by fluorescence spectroscopy. Based on electrochemical

and spectroscopic methods, it was confirmed that LPT interca-lated into the dsDNA helix.

Methotrexate (MTX) is an antimetabolite of folic acid that targetsthe enzyme dehydrofolate reductase and plays a supporting, butessential, role for the synthesis of thymine nucleotide. Recently, itwas demonstrated that MTX interfered with the JAK/STAT (JanusKinase / Signal Transducer and Activator of Transcription) pathwayalthough the action mechanism was not fully understood.Nevertheless, it has been shown that the MTX treatment caused theaccumulation of 8-oxoG in cells. The in situ evaluation of the dsDNA-MTX interaction was performed by DP voltammetry using a DNA-electrochemical biosensor and characterized by AFM at HOPG [36].The electrochemical experiments in incubated solutions showed thatthe interaction of MTX with dsDNA leads to modifications to thedsDNA structure in a time-dependent manner (Fig. 5), and AFMimages showed a reorganization of the DNA self-assembled networkupon MTX binding. The intercalation of MTX in dsDNA leaded todsDNA unwinding, detected by the increase of the purine residuesoxidation peaks. The dsDNA-electrochemical biosensor, and thepurine homo-polynucleotide sequences poly(G) and poly(A)-electrochemical biosensors, were used to investigate and understandthe interaction between MTX and dsDNA.

Telomeres and their associated proteins act by protecting the DNAfrom recombination, degradation and end-to end fusion. Theformation of GQ structures at the end of telomeric DNA increasesgenomic instability. Another class of anticancer drugs, small ligandsable to induce and stabilize GQ configurations, with broad thera-peutic selectivity, target the telomerase inhibition, and telomeredysfunction in cancer cells. The GQ-targeting acridine derivativeBRACO-19 has been an important tool for studying the antitumoractivity of acridine heterocyclic compounds. However, BRACO-19was relatively non GQ-selective, having also significant bindingaffinity for dsDNA. For this reason, more recently, a series of newtriazole-linked acridine ligands, e.g. GL15 and GL7, with enhancedselectivity for human telomeric GQs binding versus dsDNA bindinghave been designed, synthetized and evaluated. The redox propertiesof GL15 and GL7 were investigated using cyclic, DP, and square wavevoltammetry at a GCE [37], showing a complex, pH-dependent,adsorption-controlled irreversible mechanism. The interactionbetween dsDNA and GL15 or GL7 was investigated in situ, inincubated solutions and using dsDNA-, poly(G)-, and poly(A)-electrochemical biosensors [37]. It was demonstrated that theinteraction is time-dependent, both GL15 and GL7 binding to dsDNA

Scheme 4. - Proposed electrochemical mechanism of danusertib redox metabolite-guanine adduct formation. [From Ref. [34] with permission].

Fig. 5. - DP voltammograms, base line corrected, in pH 4.5 0.1M acetate buffer withdsDNA-electrochemical biosensors ( ) before and after incubation during ( )5, (Ë Ë Ë) 10 and (•••) 20 min in a solution of 100 μM MTX. [From Ref. [36] withpermission].

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and causing condensation of dsDNA morphological structure, butno oxidative damage was detected.

The interaction of the GQ-targeting triazole-linked acridine ligandGL15 with the Tetrahymena telomeric DNA repeat sequence d(TG4T)and with the poly(G) sequence have been investigated at the single-molecule level, using AFM and voltammetry [38]. GL15 interactedwith both sequences, in a time dependent manner, and the GQformation was detected by AFM via the adsorption of GL15-d(TG4T)-GQ and GL15-poly(G)-GQ small spherical aggregates (Fig. 6A-E) andlarge GL15-poly(G)-GQ assemblies, and by DP voltammetry via GL15and G oxidation peak current decrease and disappearance, and theoccurrence of a GQ oxidation peak (Fig. 6F, G). The small-moleculecomplex with the d(TG4T) quadruplex is discrete and approximatelyglobular, whereas the GQ complex with poly(G) is formed at a

number of points along the length of the polynucleotide, analo-gous to beads on a string. These results are consistent with theinteraction of triazole-linked acridine derivatives with terminalG-quartets in an individual GQ. The GL15 stabilized and acceleratedGQ formation, in both Na+ and K+ ion-containing solutions,although only K+ promoted the formation of perfectly alignedtetra-molecular GQs.

Anthracyclines are among the most effective antibiotics used forthe treatment of several types of cancers, including leukemia,lymphomas, breast, uterine, ovarian, bladder or lung cancers, buttheir use is considerably limited by their cardiotoxicity [39,40].Adriamycin, doxorubicin hydrochloride, is an antibiotic of the familyof anthracyclines with a wide spectrum of chemotherapeuticapplications and antineoplasic action, but causes very highcardiotoxicity that ranges from a delayed and insidiouscardiomyopathy to irreversible heart failure [40–42]. In order tounderstand the adriamycin in vivo mechanism of action, theoxidation and reduction of adriamycin on GCE was studied.Adriamycin intercalation and in situ interaction with dsDNA wereinvestigated using a DNA-electrochemical biosensor [40,41]. ThedsDNA-adriamycin interaction mechanism showed that adriamycinintercalated onto DNA, disrupted the double helix, and G and 8-oxoGwere detected, showing the occurrence of DNA oxidative damage.

Idarubicin (IDA), 4-demethoxydaunorubicin, is an anthracyclinederivative and widely used in the treatment of leukemia. Theelectrochemical behaviour of IDA was examined at GCE in differentaqueous supporting electrolyte solutions, using cyclic and DPvoltammetry [43]. The oxidation process of IDA was found to be apH dependent, irreversible diffusion controlled mechanism thatoccurred with the transfer of one proton and one electron. Theelectroactive center is the hydroxyl group on the aromatic ring whichproduces a final quinonic product. The diffusion coefficient of IDAwas calculated to be DIDA = 7.47 × 10−6 cm2 s−1 in pH = 4.3. Theinteraction of IDA with dsDNA was investigated using dsDNA-electrochemical biosensors and incubated solutions [43], showingthat IDA interacted with DNA causing changes in the DNAmorpho-logical structure. DNA damage was detected following the changesin the oxidation peaks of dGuo and dAdo. In addition, poly(G)- andpoly(A)-electrochemical biosensors were also used to confirm theinteraction between dsDNA and IDA. However, no oxidation peaksof the purine residues oxidation products, 8-oxoG and 2,8-DHA, wereobserved.

Apart from anticancer drug investigation, DNA-electrochemicalbiosensors were widely used for studying other pharmaceuticalcompounds.

Thalidomide (TD) was developed as a sedative and anti-emeticdrug to combat morning sickness during pregnancy, but it wasremoved from market due to its teratogenic side effects. Morerecently, TD regained interest due to its potential for treating anumber of otherwise intractable inflammatory skin diseases, suchas erythema nodosum leprosum, graft versus host disease, weightloss in tuberculosis, aphthous ulcers and human immunodeficiencyvirus replication in acquired immune deficiency syndrome. The TDinteraction with dsDNA was studied by AFM and DP voltammetryat GCE, UV-vis spectrophotometry and electrophoresis [44]. Afterincubation of dsDNA with TD, AFM and voltammetry showedmodifications of the TD-DNA adsorption and redox behaviour,depending on the TD concentration and incubation time. A modelwas proposed for the TD-DNA interaction, considering that TDintercalates into the dsDNA, causing unwinding and defects in thedsDNA secondary structure. Moreover, DNA condensation oxidativedamage was detected electrochemically by the appearance of the8-oxoG and/or 2,8-DHA oxidation peaks.

Aripiprazole (ARP) is an atypical antipsychotic agent used to treatcognitive deficit symptoms in patients with schizophrenia, whichacts through dopamine D2 partial agonism, serotonin 5-HT1A partial

Fig. 6. - GL15-d(TG4T) complex after 42 days incubation. AFM images and cross-section profiles through the white dotted lines: (A, B, C) d(TG4T) control and (D, E)GL15-d(TG4T), in the presence of (A, B, D) Na+ and (C, E) K+ ions. DP voltammogramsbaseline corrected: ( ) d(TG4T) control, ( ) GL15 control and ( ) GL15-d(TG4T), in the presence of (G) Na+ and (F) K+ ions. [From Ref. [38] with permission].

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agonism and 5-HT2A antagonism. Neurobehavioral effects andgenotoxic/mutagenic activities of the ARP were investigated anddemonstrated increased DNA strain-break damage in peripheralblood but not in the brain, suggesting a direct effect of ARP on thelong-term genomic stability. The dsDNA-ARP interaction wasinvestigated by DP voltammetry and UV-vis spectrophotometry inincubated solutions and using a dsDNA-electrochemical biosensor[45]. The binding constant between dsDNA and ARP in pH = 4.7,K ∼ 3 × 105 M−1, was obtained spectrophotometrically. Moreover, thedsDNA-ARP association was confirmed by voltammetry andspectrophotometry in mixed solutions of either poly(G) or poly(A)with ARP. The interaction between ARP and ultraviolet C (UVC)radiation-damaged dsDNA was also investigated using DPvoltammetry. UVC is the shortest and highest energy UVwith wave-lengths less than 290 nm, the most damaging type of UV radiation.However, it is completely filtered by the atmosphere and does notreach the earth’s surface. When the UVC radiation was applied tothe dsDNA-electrochemical biosensor, the dGuo response increaseddue to helix structure opening. The damaged dsDNA-electrochemicalbiosensor was incubated in ARP and a greater decrease in dGuo signalwas observed when compared to the dsDNA-electrochemicalbiosensor control.

Nitrofurantoin (NFT) is an antibacterial drug that acts at theinflammation site produced by various gram-negative and gram-positive bacteria. The dsDNA-NFT interaction was monitored by DPvoltammetry at a DNA-electrochemical biosensor fabricated bymodifying a GCE with poly(5-amino-2-mercapto-1,3,4-thiadiazole)(PAMT) [46]. The GCE/PAMT/dsDNA electrode was prepared byadsorption of dsDNA upon deposition of PAMT at the GCE surface,and the decrease of the G oxidation peak current was used as anindicator of the NFT-dsDNA binding. The reproducibility, repeatability,stability and applicability of the analysis to pharmaceutical dosageforms in human serum samples, were examined, demonstrating thatthe GCE/PAMT/dsDNA-electrochemical biosensor could be used forthe sensitive, accurate and precise determination of NFT-dsDNAinteraction with a detection limit of 0.65 mg L−1.

Natural products are the greater contributors to the productionof activeproducts, andmanyareused asdrugs. Biflorin is a prenylatedortho-naphthoquinone, isolated from the roots of Capraria biflora, aperennial shrub distributed in North and South Americas, whichdemonstrated cytotoxic activity against several tumour cell linesindicating an antitumor therapeutic potential. However, the exactmechanisms underlying its activity still remain unclear. Thepharmacoelectrochemistry of biflorin was evaluated byelectrochemistry and spectrophotometry in the presence of ssDNA,dsDNA and isolated DNA bases [47]. DNA-biflorin bindingconstants were obtained by DP voltammetry and fluorimetry.Spectroscopic studies and thermodynamic data had shown thatbiflorin can intercalate with dsDNA through van der Waalsinteractions and hydrogen bonds. The effects of biflorin-dsDNAinteractionwereaddressed throughamolecular cytogenetic approach,using the comet assay and the chromosome aberration inductionevaluation. Biflorin, compared to the negative control, presentedapproximately 4- and 6-fold increase in DNA damage. However,biflorin did not significantly induced chromosome aberrations,suggesting that it does not possess clastogenic potential, but cytotoxicpotential. The absence of either clastogenic or aneuploidogenicactivity of the compound reinforced its safety.

3.3. Metal ions and their complexes

Epidemiological studies have shown that occupational andenvironmental exposure to specific metals is associated with anincreased risk of different cancers and adverse health effects.Transition and post-transition metal ions can interact specificallywith DNA inducing partial disordering of the B-form DNA and

reduction of base stacking and base pairing. However, consensusover the transition and post-transition metal ions’ direct interac-tion with dsDNA is still needed.

The in situ evaluation of the direct interaction of chromiumspecies with dsDNA-electrochemical biosensors was studied usingDP voltammetry at a GCE [48]. The DNA damage was electrochem-ically detected following the changes in the dGuo and dAdo oxidationpeaks. The results obtained revealed the interaction with dsDNA ofthe Cr(IV) and Cr(V) reactive intermediates of Cr(III) oxidation byO2 dissolved in the solution bound to dsDNA. This interaction leadsto different modifications and causes oxidative damage in the DNAstructure. Using poly(A) and poly(G)-electrochemical biosensors, ithas been shown that the interaction between reactive intermediatesCr(IV) and Cr(V) with DNA causes oxidative damage, and takes placepreferentially at G-rich segments, leading to the formation of 8-oxoG,the oxidation product of G residues and a biomarker of DNA oxidativedamage. The interaction of Cr(VI) with dsDNA caused breaking ofhydrogen bonds, conformational changes, and unfolding of thedouble helix, which enabled easier access of other oxidative agentsto interact with DNA, and the occurrence of DNA oxidative damage.

DNA damage by Cr(V) and/or Cr(IV) intermediates of Cr(VI)electrochemical reduction was also evaluated using a supercoiledDNA-modified mercury electrode [49]. The AC voltammetric signalsensitive to the formation of DNA strand breaks increased afterincubation of the DNA-modified electrode in micromolar solutionsof Cr(VI) at potentials sufficiently negative for the Cr(VI) reduction.Damage to DNA in solutions containing Cr(VI) and a chemicalreductant (ascorbic acid) was observed only at relatively highchromium concentrations (hundreds of μM). To eliminateinterferences of excess Cr(VI) in the measurements of the Gelectrochemical signals, a magnetoseparation double surfaceelectrochemical technique was introduced. Using this approach, DNAdamage in solution was detected for 50–250 μM Cr(VI) uponaddition of 1mM ascorbic acid. The results suggested amore efficientDNA damage at the electrode surface due to continuous productionof the reactive chromium species, compared to DNA exposure tochromium being reduced chemically in solution.

The evaluation of the interaction of lead, cadmium, nickel andpalladium divalent cations with dsDNA, forming a metal-DNAcomplex, was studied by AFM on HOPG and DP voltammetry at GCE[50,51]. The electrochemical behaviour of these metal-DNAcomplexes was related to the different adsorption patterns andconformational changes. The dsDNA interaction was specific witheach metal cation, inducing structural changes in the B-DNAstructure, local denaturation of the double helix and oxidativedamage. The AFM images showed an increase of the electrode surfacecoverage by lead-, cadmium- and nickel-DNA complexes (Fig. 7A,B, D). For cadmium- and nickel-DNA complexes oxidative damageto DNAwas electrochemically detected for the concentrations studied(Fig. 7C). Palladium interaction with dsDNA induced condensationof the dsDNA secondary structure, which led to helicesbx;1aggregation. The voltammetric data for the palladium-DNAcomplex showed a sharp decrease of the dGuo and dAdo oxidationpeak currents, consistent with the AFM results for DNA condensationin the presence of palladium, but no DNA oxidative damage wasdetected, for the range of concentrations investigated.

Themechanism of interaction of a lipoic acid-palladium complex(LAPd) with dsDNA, as well as the adsorption process and the redoxbehaviour of LAPd, of its ligand lipoic acid (LA), and of the LAPd-containing dietary supplement, Poly-MVATM, were studied using AFMand voltammetry [52]. In the presence of small concentrations ofLAPd, the dsDNA appeared less knotted and bended, and moreextended onto HOPG, when compared with the dsDNA adsorbedcontrol. The voltammetric results demonstrated the interaction ofboth LAPd and Poly-MVATM with dsDNA, but no oxidative damagecaused to dsDNA was detected. The LA, LAPd and Poly-MVATM

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adsorption patterns depended on the chemical structures, thedimensions, the solution concentration and the applied potential.The LAPd molecules interacted and adsorbed strongly on HOPG, incomparison with LA, due to the incorporation of palladium into theligand structure. The application of a negative potential caused thedissociation of the LAPd complex and Pd(0) nanoparticle deposition,whereas the application of a positive potential induced the oxidationof the LAPd complex and the formation of a mixed layer of LA andpalladium oxides.

The development of new chemotherapeutic agents led to thesynthesis of polynuclear metal complexes, a new class of thirdgeneration anticancer agents with specific chemical and biologicalproperties designed as alternatives to first-generation agents suchas cisplatin. The biogenic polyamines spermidine (Spd) and spermine(Spm) were addressed in several studies of polynuclear metalcomplexes as potential antineoplastic drugs, due to their importantbiological activity and affinity for DNA. In this context, the interactionof dsDNA with two polynuclear Pd(II) chelates with Spd and Spm,Pd(II)-Spd and Pd(II)-Spm, as well as with the free ligands Spd andSpm, was studied using AFM, voltammetry, and gel electrophoresis[53]. The AFM and voltammetric results showed that the interactionof Spd and Spm with DNA occurred even for a low concentrationof polyamines and caused no oxidative damage to DNA. The Pd(II)-Spd and Pd(II)-Spm complexes were found to induce greatermorphological changes in the dsDNA conformation, when comparedwith their ligands. The interaction was specific, inducing distortionand local denaturation of the DNA structure with release of someG bases. The DNA strands partially opened give rise to palladiumintra- and inter-strand cross-links, leading to the formation of DNAadducts and aggregates, particularly in the case of the Pd(II)-Spdcomplex.

Metal complexes of fungal and plant secondary metabolites arein the centre of interest, especially due to their biological properties

including cytotoxicity. The electrochemical behaviour of seventernary copper(II) complexes of lawsone (2-hydroxy-1,4-naphthoquinone) with additional O-donor (water) and N-donorligands (pyridine, 2-, 3-, and 4-aminopyridine, 3-hydroxypyridine,and 3,5-dimethylpyrazole) using cyclic and DP voltammetry wasstudied [54]. The ability of these complexes to interact with DNAwas also tested using the DNA-electrochemical biosensor on carbonpaste electrode. The results indicated that the most simple complexCu(lawsone)2(H2O)2•0.5 H2O showed significant prooxidantproperties, which contributed to its cytotoxicity. In addition, allcomplexes evidenced ability to interact with dsDNA, and theinteraction mechanisms were discussed.

Polyphenols exhibit well-known chemoprotective effects, as wellas prooxidant activity, via interactions with metal ions. Currentresearch focuses not only on natural polyphenols but also onsynthetically prepared analogues with promising biological activities.Quercetin, in the presence of transition metals, acts as a prooxidant,has mutagenic activity and intercalates into the dsDNA. Quercetininteraction with dsDNA was investigated electrochemically inincubated solutions [55] and using two types of DNA-electrochemicalbiosensors [56], in order to evaluate the occurrence of DNA damagecaused by oxidized quercetin. The results showed that quercetinbinds to dsDNAwhere it can undergo oxidation. The radicals formedduring quercetin oxidation caused hydrogen bond breaks in thedsDNA, giving rise to 8-oxoG, since the dGuo and dAdo nucleotidesin contact with the electrode surface were easily oxidized. Amechanism for oxidized quercetin-induced damage to DNA-electrochemical biosensors, prepared after dsDNA immobilizationonto the GCE surface, was proposed and the formation of 8-oxoGwas explained.

The antioxidant and prooxidant properties of a semi-syntheticflavonolignan 7-O-galloylsilybin (7-GSB) were described, and it hasbeen shown that the presence of a galloyl moiety significantlyenhances the antioxidant capacity of 7-GSB compared to that ofsilybin (SB) [57]. These findings were supported by electrochemistry,DPPH• (2,2-diphenyl-1-picrylhydrazyl radical) scavenging activity,total antioxidant capacity (CL-TAC) and DFT (density functionaltheory) calculations. A three-step oxidation mechanism of 7-GSBwas proposed at pH 7.4 and confirmed bymolecular orbital analysis.The Cu(II) complexation of 7-GSB was also studied and theprooxidant effects of the metal-complexes were then testedaccording to their capacity to induce DNA oxidative modificationand cleavage. The results led to the conclusion that 7-O-galloylsubstitution to SB concomitantly enhances antioxidant (reactiveoxygen species (ROS) scavenging) capacity and decreases theprooxidant effect/DNA damage after Cu complexation. Thismultidisciplinary approach provided a comprehensive mechanisticpicture of the antioxidant vs. metal-induced prooxidant effects offlavonolignans at the molecular level, under ex vivo conditions.

3.4. Pollutants

Pollutants are chemicals purposely or accidentally introducedin the environment by agricultural and industrial processes, whichcause short- or long-term damage in plants and animals. Even if bio-degradable, their degradation products may possess toxic potential.From this point of view is of outmost importance to understand theireffects on organism as well as to develop analytical methodolo-gies for their sensitive detection.

Cyanobacterial hepatotoxins microcystin-LR (MC-LR) andnodularin (NOD) are among the most commonly reported toxinsproduced by cyanobacteria [58]. Several previous studies havebrought evidence for the possibility of direct induction of dsDNAdamage in vitro and in vivo upon interaction with any of these toxinswhereas other studies suggested that MC-LR and NOD genotoxicityand carcinogenicity arise mainly from the secondary effects of these

Fig. 7. - AFM images: (A) dsDNA and (B) Ni-DNA complex. (C) DP voltammogramsin buffer of an immobilised thin-layer film on GCE of: (Ë Ë Ë) dsDNA from controlsolution after 24 h preparation, and Ni-DNA complex from a solution incubated with2mMNi2+ during ( ) 12 h and ( ) 24 h. (D) Cross-section profile through whiteline in the image B. [From Ref. [50] with permission].

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toxins chemical degradation products rather than direct toxin-DNA interaction. The interaction between DNA andMC-LR and NODwas investigated using dsDNA-electrochemical biosensors and inincubated solutions and it was confirmed the decrease of the dsDNAoxidation peaks with time. It was shown that MC-LR, NOD and theirchemical degradation products, interacted with the dsDNA causingthe aggregation of dsDNA strands [59]. The analysis of dsDNAinteraction withMC-LR or NOD in incubated solutions, where dsDNAstrands are allowed to move freely and adopt the better conforma-tions for and after the interaction, enabled the detection of adeninefree residues (Fig. 8). The interaction between DNA and MC-LR andNOD, besides dsDNA aggregation, caused dsDNA abasic sites, whichif left unrepaired can lead to mutations during the replicationprocess.

A DNA-electrochemical biosensor based on graphene-ionic liquid-Nafion modified pyrolytic graphite electrode (PGE) was developedby layer-by-layer assembly of DNA and horseradish peroxidase (HRP)for the detection of acrylamide (AA) and its product [60]. The PGE/graphene-ionic liquid-Nafion and the construction of the (HRP/DNA)n film were characterized by electrochemical impedancespectroscopy (EIS). Using the G signal as indicator, the DNA damagewas detected by DP voltammetry after incubation in AA or AA +H2O2

solutions at 37°C. The results indicated that, in the presence of H2O2,HRP was activated and catalysed the transformation of AA toglycidamide, whichmay form DNA adducts and inducemore seriousDNA damage than the AA. The DNA damage induced by AA and itsproducts in solution were also investigated by UV–vis spectropho-tometry, and similar results were obtained.

Hydroquinone is a widely used chemical compound that can bemetabolized to benzoquinone which presented haematotoxic,genotoxic and carcinogenic potential. Detection of hydroquinonewasperformed at a DNA-electrochemical biosensor constructed usingchitosan (CTS) and polyaniline (PANI) [61]. The electrochemicalbehaviour of hydroquinone on the biosensor and its DNA-damagingmechanisms were investigated. The results showed that the DNAredox peak current was remarkably increased after GCEmodificationby PANI/CTS. The dsDNA damage by hydroquinonewas concentrationdependent and the G oxidation peak current of dsDNA decreased.The UV–vis spectrophotometry confirmed that applying dsDNA/PANI/CTS/GCE to monitor hydroquinone was accurate and reliable,and that hydroquinone intercalated in dsDNA.

Polycyclic aromatic hydrocarbons (PAHs) represent a large groupof organic contaminants widely diffused in different ecosystems.

Because of their hydrophobic nature, PAHs can easily cross cellmem-branes and tend to bioaccumulate in lipid tissues. The mutagenic/genotoxic effects of different PAHs have been proved, being classifiedas potentially carcinogenic by the International Agency for ResearchonCancer (IARC). Thebest studied among thePAHs is benzo[a]pyrene(BaP) and IARC concluded in 1987 that it is a potential humancarcinogen. The reactivity of photodegradation products of BaPversus DNA were assessed using genomic and ODN based DNA-electrochemical biosensors [62]. The kinetic of a photooxidationreaction of BaP carried out in controlled conditions using a 6WUVlamp peaked at 365 nm has been studied using HPLC withfluorimetric detection. The degradation of BaP by both UV and UV/H2O2 exhibited pseudo-first-order reaction kinetics with half-livesranging from 3.0 to 9.8 h depending on the pH and on the amountof H2O2. The oxidation products of BaP obtained in differentconditions were tested with the DNA-electrochemical biosensorsprepared after immobilization on graphite or gold screen-printedelectrodes. The G residues oxidation peak obtained usingchronopotentiometry was used to detect the interaction of the BaPproducts with DNA. The dose-response curve obtained with BaPincubatedwas different from that of the parent compound indicatinga different type of interaction with DNA. The formation of stableadducts between the G residues and the BaP oxidation productswasdescribed. HPLCwithmass spectrometry detection of the oxidationproducts confirmed the presence of chemical species potentiallyforming adducts with DNA. The data reported demonstrated thatDNA-electrochemical biosensors have the potential to be used tomonitor remediation processes and to assess the potential toxicityvs. DNA of chemicals forming stable DNA adducts.

Another strategy for the detection of BaP involved thelayer-by-layer assembling of horseradish peroxidase (HRP) anddouble-stranded DNA at nafion-solubilized single-wall carbonnanotubes-ionic liquid (SWCNTs-NA-IL) composite film [63]. Thebiosensor was characterized by cyclic and DP voltammetry, EIS,scanning electron microscopy and computational methods. UV-visspectrophotometrywas also used to investigate DNA damage inducedby BaP and its products in solution. The DNA-electrochemicalbiosensor was investigated separately in BaP, H2O2, and in theirmixture. The analysis demonstrated the unwinding of DNA helix andexposure of the bases.

3.5. Free radicals

ROS such as superoxide (O2-•), peroxyl (ROO•), and hydroxyl (•OH)radicals are generated inside cells as products of metabolism, byleakage from mitochondrial respiration, and also under theinfluence of exogenous agents such as ionizing radiation, quinones,and peroxides. Excess ROS are responsible for causing DNA oxidativemodifications and mutations, which can initiate carcinogenesis andmay play a role in the development of several age-correlateddegenerative diseases.

Under aerobic conditions, transition metal ions caused DNAdamage through production of ROS, frequently via Fenton-typereactions. Formation of strand brakes (sb) in covalently closedsupercoiled (scDNA) were detected using an electrochemical bio-sensor based on a scDNA-modified mercury electrode. [64] scDNAanchored at mercury electrode was cleaved by catalytic amountsof Fe/EDTA ions in the absence of chemical reductants whenappropriate electrode potential was applied, the process requiringoxygen or hydrogen peroxide. The extent of DNA damageincreasedwith the shift of the electrode potential to negative values,displaying a sharp inflection point matching the potential ofFe(EDTA)]2−/[Fe(EDTA)]1− redox pair. In the absence of transitionmetal ions, significant DNA damage was observed at potentialssufficiently negative for reduction of dioxygen at the mercury

Fig. 8. - DP voltammograms of 50 μg ml−1 dsDNA solution in pH = 4.5 0.1 M acetatebuffer ( ) before and after incubation with 30 μM MC-LR during (Ë Ë Ë) 0, ( ) 6and ( ) 24 h. [Adapted from Ref. [59] with permission].

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electrode, suggesting cleavage of the surface-attached scDNA byradical intermediates of oxygen reduction.

Damage to DNA immobilized at the surface of GCEmodified withsilver nanoparticles and covalently attached neutral red conductivepolymer was studied in amodel system based on the Fenton reagent.[65] The oxidation process resulted in synchronous increase ofelectron transfer resistance and capacitance measured by EIS. Thecontribution of each sensor component on the signal was specifiedand sensitivity estimated against similar surface coatings. The shiftof EIS parameters was found to be higher than that of similarbiosensors. The DNA-electrochemical biosensor was tested for theevaluation of antioxidant capacity of green tea infusions.

Among the electrochemical transducers, the boron-dopeddiamond electrode (BDDE) presents unique properties. The BDDEbehaviour is strongly related to the controlled in situ electrochemi-cal generation (by water discharge) of hydroxyl radicals and theirsubsequent reactions [66]. The effect of BDDE surface termination,immediately after cathodic and anodic electrochemical pre-treatment, and the influence of the pre-treatment, in differentsupporting electrolytes, on the electrochemical oxidation potentialsof dsDNA, DNA bases, nucleotides, homopolynucleotides andbiomarker 8-oxoG, in aqueous media at different pHs, wereinvestigated [67]. The results demonstrated that the BDDE electro-chemical propertiesweredependent on the surface functional groups.The interaction and adsorption of DNA and its components on theBDDE surface pre-treated cathodicallywas facilitated due to a BDDEhigher conductivity. On the other hand, after anodic pre-treatmenta wider potential window of BDDE was obtained enabling thedetection of the pyrimidine bases. However, the hydroxyl radicalsproducedonBDDEduring anodic pre-treatmentwerehighly reactive,and consequently the BDDE surface was not completely inert.

The in situ interaction and oxidative damage caused by hydroxylradicals to dsDNAwas also investigated using a thickmultilayer DNA-electrochemical biosensor prepared onto the oxidized BDDE surface[68]. The BDDE allowed the generation of •OH at approximately +3.00 V (vs. Ag/AgCl in pH = 4.5 0.1M acetate buffer) [69] in agreementwith the reaction:

BDD H O BDD OH H e+ → + +( )• + −2

The DNA-electrochemical biosensor on the BDDE enabledpreconcentration of the •OH electrogenerated at the BDDE surface.Controlling the applied potential, different concentrations of •OHwere electrochemically generated in situ on the BDDE surface. Aftermonitoring the modification of the oxidation peak currents of thepurine deoxynucleoside residues, it was found that •OH oxidativelydamaged the immobilized dsDNA on the BDDE surface, leading tomodifications in the dsDNA structure, exposingmore purine residuesto the electrode surface and facilitating their oxidation (Fig. 9). ThedsDNA structural modifications were confirmed by electrophoresisand the DP voltammetric results demonstrated the occurrence ofthe 8-oxoG oxidation peak, due to the occurrence of DNA oxidativedamage.

3.6. Radiation

The interaction of electromagnetic radiation with living organ-isms has been studied for a long time but is still an actual topic ofresearch. The irradiation outcomes on living cells or tissues is difficultto analyse, since many biochemical processes are taking place atthe same time, competing with the radiation effects [70]. Ionizingradiation can change the structure of biomolecules, creatingpotentially harmful effects. Although radiation has the potential todamage a multitude of biomolecules inside a cell, the structure ofmost concern is DNA. Radiation can be absorbed directly by DNA,leading to ionization of both the bases and sugar in a mechanism

described as the direct effect of generating single and tandem DNAdamage. Contrary, approximately 65% of the DNA damage is causedby the indirect effect of free radicals such as hydroxyl radicals thatare formed from the radiolysis of surrounding water molecules andthat successively attack DNA [71]. The interaction of γ radiation withpoly(G), poly(A), poly(T), poly(C), ssDNA and dsDNAwas first studiedby DP voltammetry at a GCE in aqueous solutions.

A DNA-electrochemical biosensor was used for the detection ofDNA damage by UV-C radiation and reactive oxygen species producedby the Fenton type reaction model as well as mineral water sampleswith additives [72]. Among different detection strategies, squarewavevoltammetry of DNA bases was used to characterize the time changeson the dsDNA structure. It has been shown that the G residuesintrinsic response presented a method for the detection of dsDNAhelix changes and single-strand breaks that depended on theincubation time in the cleavage medium.

A similar approach has been used in the presence of CdTequantum dots (QDs) [73]. In this report, the sensor was a GCEmodified with a layer of dsDNA and another layer of CdTe QDs. Ithas been demonstrated that the size of the QDs exerted a significanteffect on the rate of the degradation of dsDNA by UV-C light, andeven by visible light. Time-dependent structural changes of DNAincluded unwinding of the double helix indicated by the increaseof the redox response of the Gmoiety, due to easy electron exchangewith the electrode surface when compared to the original doublehelix. The effects of QDs were verified for salmon sperm DNA andcalf thymus DNA, and corroborated by experiments in which DNAsolutions were irradiated in the presence of QDs.

4. Conclusion

The development of DNA-electrochemical biosensors, that usedirect electrochemistry of DNA as a detection platform, designedfrom engineered DNA structures that self-assembled in unusual butbiologically relevant structures, such as G-quadruplexes and i-motifs,were revisited. The influence of DNA sequence composition on DNAself-assembling properties and the understanding of the key factors

Fig. 9. - DP voltammograms in pH = 4.5 0.1 M acetate buffer with a thick multi-layer dsDNA-BDDE biosensor: ( ) control and ( ) first and ( ) 4th scans afterapplying + 3.0 V during 2 h to the BDDE surface causing electrogeneration of hy-droxyl radicals. [Adapted from Ref. [68] with permission].

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in controlling the distribution, size and shape of highly ordered two-and three-dimensional DNA nanostructures on the electrode surfacewere referred.

Combining the characteristics of DNA probes with the capacityof direct and label-free electrochemical detection has allowedapplications in many different fields, from the investigation andevaluation of DNA oxidative damage and interaction mechanismswith pharmaceutical compounds, such as anticancer kinaseinhibitors, anthracyclines, anti-cancer drugs, antipsychotic andantifungal drugs, to rapid monitoring metals, pollutant agents, andfree radicals from homogenous redox reactions.

The electrochemical and AFM investigation has been of greatrelevance to explain many biological mechanisms, and for nano- andbiosensor technology applications of the DNA-electrochemicalbiosensors.

Acknowledgements

Financial support from Fundação para a Ciência e a Tecnologia(FCT), grant SFRH/BPD/92726/2013 (A.-M. Chiorcea-Paquim), projectsPTDC/SAU-BMA/118531/2010, PTDC/QEQ-MED/0586/2012, PTDC/DTP-FTO/0191/2012, and CEMUC-R (Research Unit 285),(co-financed by the European Community Fund FEDER), and FEDERfunds through the program COMPETE – Programa OperacionalFactores de Competitividade, is gratefully acknowledged.

References

[1] E. Katz, B. Willner, I. Willner, Amplified electrochemical andphotoelectrochemical analysis of DNA, in: E. Palecek, F. Scheller, J. Wang(Editors), Perspectives in Bioanalysis, vol. 1, Electrochemistry of nucleic acidsand proteins – Towards electrochemical sensors for genomicss and proteomics,Elsevier, Amsterdam, 2005, pp. 195–246. and references therein.

[2] L. Soleymani, Z. Fang, E.H. Sargent, S.O. Kelley, Programming the detection limitsof biosensors through controlled nanostructuring, Nat. Nanotechnol. 4 (2009)844–848.

[3] L. Soleymani, Z. Fang, X. Sun, H. Yang, B.J. Taft, E.H. Sargent, et al.,Nanostructuring of patterned microelectrodes to enhance the sensitivity ofelectrochemical nucleic acids detection, Angew. Chem. 121 (2009) 8609–8612.

[4] A.M. Chiorcea Paquim, P.V. Santos, A.M. Oliveira Brett, Atomic force microscopyand voltammetric characterisation of synthetic homo-oligodeoxynucleotides,Electrochim. Acta 110 (2013) 599–607.

[5] E. Palecek, M. Bartošík, Electrochemistry of nucleic acids, Chem. Rev. 112 (2012)3427–3481 and references therein.

[6] E. Palecek, Past, present and future of nucleic acids electrochemistry, Talanta56 (2002) 809–819 and references therein.

[7] A.M. Oliveira Brett, F.-M. Matysik, Voltammetric and sonovoltammetric studieson the oxidation of thymine and cytosine at a glassy carbon electrode,J. Electroanal. Chem. 429 (1997) 95–99.

[8] A.M. Oliveira-Brett, J.A.P. Piedade, L.A. Silva, V.C. Diculescu, Voltammetricdetermination of all DNA nucleotides, Anal. Biochem. 332 (2004) 321–329.

[9] A.M. Oliveira Brett, S.H.P. Serrano, J.A.P. Piedade, Comprehensive ChemicalKinetics, Applications of Kinetic Modelling, R.G. Compton (Editor), vol. 37,Elsevier, Oxford, UK, 1999, p. 91. (Chapter 3), and references therein.

[10] A.M. Oliveira Brett, V.C. Diculescu, A.M. Chiorcea Paquim, S.H.P. Serrano,DNA-electrochemical biosensors for investigating DNA damage, in: S. Alegret,A. Merkoçi (Editors), Comprehensive Analytical Chemistry, ElectrochemicalSensor Analysis, Elsevier, The Netherlands, 2007, pp. 413–437. (Chapter 20),and references therein.

[11] A.M. Oliveira-Brett, Electrochemistry for probing DNA damage, in: C. Grimes(Editor), Encyclopedia of Sensors, vol. 3, American Scientific Publishers, USA,2006, pp. 301–314, and references therein.

[12] A.M. Oliveira, A.M. Brett, Chiorcea, Atomic forcemicroscopy of DNA immobilizedonto a highly oriented pyrolytic graphite electrode surface, Langmuir 19 (2003)3830–3839.

[13] A.M. Oliveira, A.M. Brett, Chiorcea, DNA imaged on a HOPG electrode surfaceby AFM with controlled potential, Bioelectrochemistry 66 (2005) 117–124.

[14] V. Brabec, E. Palecek, Interactions of nucleic acids with electrically chargedsurfaces: Part IV. Local changes in the structure of DNA adsorbed on mercuryelectrode in the vicinity of zero charge, J. Electroanal. Chem. 88 (1978) 373–385.

[15] A.M. Oliveira, A.M. Brett, Chiorcea, Effect of pH and applied potential on theadsorption of DNA on highly oriented pyrolytic graphite electrodes. Atomic forcemicroscopy surface characterisation, Electrochem. Commun. 5 (2003) 178–183.

[16] A.M. Chiorcea, A.M. Oliveira Brett, Atomic force microscopy characterisationof an electrochemical DNA-biosensor, Bioelectrochemistry 63 (2004) 229–232.

[17] A.M. Chiorcea Paquim, V. Diculescu, A.T.S. Oretskaya, A.M. Oliveira Brett, AFMand electroanalytical studies of synthetic oligonucleotide hybridization, Biosens.Bioelectron. 20 (2004) 933–944.

[18] A.M. Oliveira Brett, A.M. Chiorcea Paquim, V. Diculescu, T.S. Oretskaya, Syntheticoligonucleotides: AFM characterisation and electroanalytical studies,Bioelectrochemistry 67 (2005) 181–190.

[19] V.C. Diculescu, A.M. Chiorcea Paquim, A.M. Oliveira Brett, Electrochemical DNAsensors for detection of DNA damage, Sensors (Basel) 5 (2005) 377–393.

[20] A.M. Chiorcea Paquim, T.S. Oretskaya, A.M. Oliveira Brett, Adsorption of synthetichomo– and hetero–oligodeoxynucleotides onto highly oriented pyrolyticgraphite: atomic force microscopy characterization, Biophys. Chem. 121 (2006)131–141.

[21] A.M. Chiorcea Paquim, T.S. Oretskaya, A.M. Oliveira Brett, Atomic forcemicroscopy characterization of synthetic pyrimidinic oligodeoxynucleotidesadsorbed onto an HOPG electrode under applied potential, Electrochim. Acta51 (2006) 5037–5045.

[22] A.M. Oliveira Brett, A.M. Chiorcea Paquim, V.C. Diculescu, J.A.P. Piedade,Electrochemistry of nanoscale DNA surface films on carbon, Med. Eng. Phys.28 (2006) 963–970.

[23] A.M. Chiorcea Paquim, J.A.P. Piedade, R. Wombacher, A. Jäschke, A.M.Oliveira Brett, Atomic forcemicroscopy and anodic voltammetry characterizationof a 49-mer Diels-Alderase ribozyme, Anal. Chem. 78 (2006) 8256–8264.

[24] V.C. Diculescu, A.M. Chiorcea Paquim, R. Eritja, A.M. Oliveira Brett, ThrombineBinding Aptamer Quadruplex Formation: AFM and VoltammetricCharacterization, Journal Nucleic Acids (2010) doi:10.4061/2010/841932 ArticleID 841932.

[25] V.C. Diculescu, A.M. Chiorcea Paquim, R. Eritja, A.M. Oliveira Brett, Evaluationof the structure-activity relationship of thrombin with thrombin bindingaptamers by voltammetry and atomic force microscopy, J. Electroanal. Chem.656 (2011) 159–166.

[26] A.M. Chiorcea Paquim, P. Santos, V.C. Diculescu, R. Eritja, A.M. Oliveira Brett,Electrochemical characterization of guanine quadruplexes, in: G.P. Spada(Editor), Guanine Quartets-Structure and Application, RCS Publishing, 2013,pp. 100–109.

[27] A.M. Chiorcea Paquim, A.M. Oliveira Brett, Redox behavior of G-quadruplexes,Electrochim. Acta 126 (2014) 162–170.

[28] A.M. Chiorcea Paquim, P.V. Santos, R. Eritja, A.M. Oliveira Brett, Self-assembledG-quadruplex nanostructures: AFM and voltammetric characterization, Phys.Chem. Chem. Phys. 15 (2013) 9117–9124.

[29] A.D.R. Pontinha, A.M. Chiorcea Paquim, R. Eritja, A.M. Oliveira Brett, Quadruplexnanostructures of d(TGGGGT): influence of sodium and potassium ions, Anal.Chem. 86 (2014) 5851–5857.

[30] A.M. Chiorcea Paquim, A.D.R. Pontinha, A.M. Oliveira Brett, Time-dependentpolyguanylic acid structural modifications, Electrochem. Commun. 45 (2014)71–74.

[31] L.I.N. Tomé, N.V. Marques, V.C. Diculescu, A.M. Oliveira-Brett, In situ dsDNA-bevacizumab anticancer monoclonal antibody interaction electrochemicalevaluation, Anal. Chim. Acta (2015) doi:10.1016/j.aca.2015.09.049.

[32] I.B. Santarino, S.C.B. Oliveira, A.M. Oliveira-Brett, In Situ Evaluation of theAnticancer Antibody Rituximab-dsDNA Interaction Using a DNA-ElectrochemicalBiosensor, Electroanalysis 26 (2014) 1304–1311.

[33] V.C. Diculescu, M. Vivan, A.M. Oliveira Brett, Voltammetric Behavior ofAntileukemia Drug Glivec. Part III – In Situ DNA Oxidative Damage by the GlivecElectrochemical Metabolite, Electroanalysis 18 (2006) 1963–1970.

[34] V.C. Diculescu, A.M. Oliveira Brett, In situ electrochemical evaluation of dsDNAinteraction with anticancer drug danusertib nitrenium radical metabolite usingthe DNA-electrochemical biosensor, Bioelectrochemistry (2015) doi:10.1016/j.bioelechem.2015.10.004.

[35] B. Dogan-Topal, B. Bozal-Palabiyik, S.A. Ozkan, B. Uslu, Investigation of anticancerdrug lapatinib and its interaction with dsDNA by electrochemical andspectroscopic techniques, Sensor. Actuat. B-Chem. 194 (2014) 185–194.

[36] A.D.R. Pontinha, S.M.A. Jorge, A.-M. Chiorcea Paquim, V.C. Diculescu, A.M.Oliveira-Brett, In situ evaluation of anticancer drug methotrexate-DNAinteraction using a DNA-electrochemical biosensor and AFM characterization,Phys. Chem. Chem. Phys. 13 (2011) 5227–5234.

[37] A.D.R. Pontinha, S. Sparapani, S. Neidle, A.M. Oliveira-Brett, Triazole-acridineconjugates: Redox mechanisms and in situ electrochemical evaluation ofinteraction with double-stranded DNA, Bioelectrochemistry 89 (2013) 50–56.

[38] A.M. Chiorcea Paquim, A.D.R. Pontinha, R. Eritja, G. Lucarelli, S. Sparapani, S.Neidle, et al., Atomic force microscopy and voltammetric investigation ofquadruplex formation between a triazole-acridine conjugate and guanine-containing repeat DNA sequences, Anal. Chem. 87 (2015) 6141–6149.

[39] V.C. Diculescu, A.M. Oliveira-Brett, DNA-electrochemical Biosensors andOxidative Damage to DNA: Application to Cancer, in: V.R. Preedy, V.B. Patel(Editors), Biosensors and Cancer, CRC Press, London, 2012, pp. 187–210.

[40] A.M. Oliveira-Brett, M. Vivan, I.R. Fernandes, J.A.P. Piedade, Electrochemicaldetection of in situ adriamycin oxidative damage to DNA, Talanta 56 (2002)959–970.

[41] J.A. Piedade, I.R. Fernandes, A.M. Oliveira Brett, Electrochemical sensing ofDNA-adriamycin interactions, Bioelectrochemistry 56 (2002) 81–83.

[42] A.M. Oliveira-Brett, J.A.P. Piedade, A.-M. Chiorcea, Anodic voltammetry and AFMimaging of picomoles of adriamycin adsorbed onto carbon surfaces,J. Electroanal. Chem. 538–539 (2002) 267–276.

[43] H.E.S. Kara, Redox mechanism of anticancer drug idarubicin and in-situevaluation of interaction with DNA using an electrochemical biosensor,Bioelectrochemistry 99 (2014) 17–23.

35V.C. Diculescu et al. / Trends in Analytical Chemistry 79 (2016) 23–36

[44] S.C.B. Oliveira, A.M. Chiorcea Paquim, S.M. Ribeiro, A.T.P. Melo, M. Vivan, A.M.Oliveira Brett, In situ electrochemical and AFM study of thalidomide-DNAinteraction, Bioelectrochemistry 76 (2009) 201–207.

[45] S. Kurbanoglu, B. Dogan-Topal, L. Hlavat, J. Labuda, S.A. Ozkan, B. Uslu,Electrochemical investigation of an interaction of the antidepressant drugaripiprazole with original and damaged calf thymus dsDNA, Electrochim. Acta169 (2015) 233–240.

[46] G. Aydogdu, G. Gunendi, D.K. Zeybek, B. Zeybek, S. Pekyardimci, A novelelectrochemical DNA biosensor based on poly-(5-amino-2-mercapto-1,3,4-thiadiazole) modified glassy carbon electrode for the determination ofnitrofurantoin, Sensor. Actuat. B-Chem. 197 (2014) 211–219.

[47] M.C. de Vasconcellos, C. de Oliveira Costa, E.G. da Silva Terto, M.A.F.B. de Moura,C.C. de Vasconcelos, T.L. Gomez de Lemos, et al., Electrochemical, spectroscopicand pharmacological approaches toward the understanding of biflorin DNAdamage effects, J. Electroanal. Chem. (2015) doi:10.1016/j.jelechem.2015.09.040.

[48] S.C.B. Oliveira, A.M. Oliveira-Brett, In situ evaluation of chromium-DNA damageusing a DNA-electrochemical biosensor, Anal. Bioanal. Chem. 398 (2010)1633–1641.

[49] J. Vacek, T. Mozga, K. Cahova, H. Pivonkova, M. Fojta, Electrochemical sensingof chromium-induced DNA damage: DNA strand breakage by intermediates ofChromium(VI) electrochemical reduction, Electroanalysis 19 (2007) 2093–2102.

[50] A.-M. Chiorcea-Paquim, O. Corduneanu, S.C.B. Oliveira, V.C. Diculescu, A.M.Oliveira-Brett, Electrochemical and AFM evaluation of hazard compounds-DNAinteraction, Electrochim. Acta 54 (2009) 1978–1985.

[51] S.C.B. Oliveira, O. Corduneanu, A.M. Oliveira-Brett, In situ evaluation ofheavy metal-DNA interactions using an electrochemical DNA biosensor,Bioelectrochemistry 72 (2008) 53–58.

[52] O. Corduneanu, A.-M. Chiorcea-Paquim, M. Garnett, A.M. Oliveira-Brett, Lipoicacid-palladium complex interaction with DNA, voltammetric and AFMcharacterization, Talanta 77 (2009) 1843–1853.

[53] O. Corduneanu, A.-M. Chiorcea-Paquim, V. Diculescu, S.M. Fiuza, M.P.M. Marques,A.M. Oliveira-Brett, DNA Interaction with Palladium Chelates of BiogenicPolyamines Using Atomic Force Microscopy and Voltammetric Characterization,Anal. Chem. 82 (2010) 1245–1252.

[54] P. Babula, J. Vanco, L. Krejcova, D. Hynek, J. Sochor, V. Adam, et al., VoltammetricCharacterization of Lawsone-Copper(II) Ternary Complexes and TheirInteractions with dsDNA, Int. J. Electrochem. Sc. 7 (2012) 7349–7366.

[55] A.M. Oliveira-Brett, V.C. Diculescu, Electrochemical study of quercetin-DNAinteractions. Part I – Analysis in incubated solutions, Bioelectrochemistry 64(2004) 133–141.

[56] A.M. Oliveira-Brett, V.C. Diculescu, Electrochemical study of quercetin-DNAinteractions. Part II – In situ sensing with DNA-biosensors, Bioelectrochemistry64 (2004) 143–150.

[57] J. Vacek, M. Zatloukalova, T. Desmier, V. Nezhodova, J. Hrbac, M. Kubala, et al.,Antioxidant, metal-binding and DNA-damaging properties of flavonolignans:A joint experimental and computational highlight based on 7-O-galloylsilybin,Chem. Biol. Interact. 205 (2013) 173–180.

[58] I.C. Lopes, P.V.F. Santos, V.C. Diculescu, F.M.P. Peixoto, M.C.U. Araújo, A.A. Tanaka,et al., Microcystin-LR and chemically degraded microcystin-LR electrochemicaloxidation, Analyst 137 (2012) 1904–1912.

[59] P.V.F. Santos, I.C. Lopes, V.C. Diculescu, A.M. Oliveira-Brett, DNA-cyanobacterialhepatotoxins microcystin-LR and nodularin interaction electrochemicalevaluation, Electroanalysis 24 (2012) 547–553.

[60] Y. Qiu, X. Qu, J. Dong, S. Ai, R. Han, Electrochemical detection of DNA damageinduced by acrylamide and its metabolite at the graphene-ionic liquid-Nafionmodified pyrolytic graphite electrode, J. Hazard. Mater. 190 (2011) 480–485.

[61] W. Tanga, M. Zhang, W. Li, X. Zeng, An electrochemical sensor based onpolyaniline for monitoring hydroquinone and its damage on DNA, Talanta 127(2014) 262–268.

[62] M. Del Carlo, M. Di Marcello, M. Giuliani, M. Sergi, A. Pepe, D. Compagnone,Detection of benzo(a)pyrene photodegradation products using DNAelectrochemical sensors, Biosens. Bioelectron. 31 (2012) 270–276.

[63] A.R. Jalalvand, M.-B. Gholivand, H.C. Goicoechea, T. Skov, K. Mansouri, Mimickingenzymatic effects of cytochrome P450 by an efficient biosensor for in vitrodetection of DNA damage, Int. J. Biol. Macromol. 79 (2015) 1004–1010.

[64] M. Fojta, T. Kubicarova, E. Palecek, Electrode potential-modulated cleavage ofsurface-confined DNA by hydroxyl radicals detected by an electrochemicalbiosensor, Biosens. Bioelectron. 15 (2000) 107–115.

[65] Y. Kuzin, A. Porfireva, V. Stepanova, V. Evtugyn, I. Stoikov, G. Evtugyn, et al.,Impedimetric Detection of DNA Damage with the Sensor Based on SilverNanoparticles and Neutral Red, Electroanalysis 27 (2015) doi:10.1002/elan.201500312.

[66] S.C.B. Oliveira, A.M. Oliveira-Brett, Voltammetric and electrochemical impedancespectroscopy characterization of a cathodic and anodic pre-treated boron dopeddiamond electrode, Electrochim. Acta 55 (2010) 4599–4605.

[67] S.C.B. Oliveira, A.M. Oliveira Brett, Boron doped diamond electrode pre-treatments effect on the electrochemical oxidation of dsDNA, DNA bases,nucleotides, homopolynucleotides and biomarker 8-oxoguanine, J. Electroanal.Chem. 648 (2010) 60–66.

[68] S.C.B. Oliveira, A.M. Oliveira-Brett, In situ DNA oxidative damage byelectrochemically generated hydroxyl free radicals on a boron-doped diamondelectrode surface, Langmuir 28 (2012) 4896–4901.

[69] T.A. Enache, A.-M. Chiorcea-Paquim, O. Fatibello-Filho, A.M. Oliveira-Brett,Hydroxyl radicals electrochemically generated in situ on a boron-dopeddiamond electrode, Electrochem. Commun. 11 (2009) 1342–1345.

[70] A. Deppman, J.O. Echeimberg, A.N. Gouveia, J.D.T. Arruda-Neto, F.M. Milian, N.Added, et al., Radiation interaction with DNA, Braz. J. Phys. 34 (2004) 958–961.

[71] J.A.P. Piedade, P.S.C. Oliveira, M.C. Lopes, A.M. Oliveira-Brett, Voltammetricdetermination of γ radiation-induced DNA damage, Anal. Biochem. 355 (2006)39–49.

[72] L. Hlavata, K. Benikova, V. Vyskocil, J. Labuda, Evaluation of damage to DNAinduced by UV-C radiation and chemical agents using electrochemical biosensorbased on low molecular weight DNA and screen-printed carbon electrode,Electrochim. Acta 71 (2012) 134–139.

[73] L. Hlavata, I. Striesova, T. Ignat, J. Blaskovisova, B. Ruttkay-Nedecky, P. Kopel,et al., An electrochemical DNA-based biosensor to study the effects of CdTequantum dots on UV-induced damage of DNA, Microchim. Acta 182 (2015)1715–1722.

36 V.C. Diculescu et al. / Trends in Analytical Chemistry 79 (2016) 23–36