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Electrochemical Biosensors for DNA–Drug InteractionsSCB de Oliveira, University of Coimbra, Coimbra, Portugal; and Universidade Federal Rural de Pernambuco, Recife, PE, BrazilVC Diculescu, University of Coimbra, Coimbra, Portugal; and National Institute of Material Physics, Magurele, RomaniaAM Chiorcea Paquim and AM Oliveira-Brett, University of Coimbra, Coimbra, Portugal

© 2018 Elsevier Inc. All rights reserved.

Introduction 124DNA Electrochemical Behavior 126DNA-Electrochemical Biosensor Development 128DNA-Electrochemical Biosensors for Detection of dsDNA Damage 129Pharmaceuticals 129Nucleoside analogs 129Imatinib and danusertib 129Adriamycin 131Thalidomide 131Methotrexate 132

Proteins 133Monoclonal antibodies 133Human cytochrome P450 (CYP1A2) 133

Toxins 134Ochatoxin A 134Nodolarine and microcystine 134

Metal Ions 135Free Radicals 136Oxygen reactive species 136Nitrogen reactive species 136

Conclusion 137Acknowledgments 137References 137Further Reading 139

Introduction

The existence of a causative link between the oxidative damage caused to the double-stranded DNA (dsDNA) and cancer, as a resultof carcinogenic agent’s chemical reactions, has been demonstrated.1–3 DNA is also the pharmacological target for many antineo-plastic drugs, which were developed to bind and interact strongly with DNA in cancer treatments. These interactions cause changesin the structure of dsDNA and block the replication of tumor cells (the key process in cell growth and division) but lack selectivityand also block the growth of normal cells. The investigation of the dsDNA interaction with drugs, ions, and radicals is very impor-tant, and the causes that control the binding affinity and selectivity to the dsDNA need to be elucidated. The understanding of themechanisms involved in the dsDNA reaction sites selection is essential to comprehend the toxicity and the chemotherapeutic effectsof those chemicals and in designing new antineoplastic drugs.3–6

Antineoplastic drugs interact with DNA5–12 either by covalent interactions, which lead to chemical modification of the dsDNAconstituents, or by noncovalent interactions such as electrostatic binding and intercalation. In addition, a number of anticancerdrugs interact with dsDNA causing DNA oxidative damage, subsequently leading to a number of mutagenic alterations withinthe cell and inducing apoptosis. However, the anticancer drugs interaction mechanisms with dsDNA are not fully understood.5–12

Among the various methods used to characterize hazard compounds and anticancer drugs interaction with DNA,5,6,13 theDNA-electrochemical biosensors present the advantages of being fast, low cost, highly sensitive, and selective in detecting smallperturbations of the double-helical structure and DNA oxidative damage under different experimental conditions. TheDNA-electrochemical biosensors have been successfully used to investigate the interaction of DNA with anticancer drugs,5,6,14–19

mutagens, carcinogens, and pollutants,5,6,14–19 allowing the unraveling of detailed mechanistic interactions. Another importantadvantage in using the DNA-electrochemical biosensors is the possibility of generation of highly reactive intermediates in situ,on the modified electrode surface, followed by the electrochemical detection of their direct interaction with dsDNA.5,14–19

A very important factor for the optimal construction of a DNA-electrochemical biosensor is the immobilization of the dsDNAprobe on the electrode surface.5,14–19 Electrode surface modification has been done by different DNA immobilization procedures,such as spontaneous adsorption, adsorption under applied potential, or evaporation, with the formation of a monolayer or a multi-layer DNA film on the electrode surface.5,14–19

124

Electrochemical studies, on carbon electrodes, concerning antineoplastic drugs, hazard compounds, or carcinogen–DNAinteractions, using different DNA-electrochemical biosensors, are summarized in Table 1.20–74

This article will focus mainly on the development of label-free DNA-electrochemical biosensors on carbon transducers and theirapplications for studying the mechanisms of interaction of pharmaceutical drugs and hazard compounds with dsDNA. Theelectrochemical behavior of dsDNA and its components will be revised, and the main procedures used for the preparation of

Table 1 Selected electrochemical studies on carbon electrodes of antineoplastic drugs, hazard compounds, or carcinogen–DNA interactions

Anticancer drug/carcinogen ProposedDNA interaction mechanism Electrode Ref.

Adriamycin Intercalation;DNA oxidative damage GCE (biosensor) 20Berberine Electrostatic GCE (biosensor) 21Berenil DNA oxidative damage GCE (biosensor) 22Bevacizumab Structural alterations GCE (biosensor) 23,24Busulfan – Screen-printed carbon electrode (SPCE) 25Calcium dobesilate Intercalation;complex formation Gold nanoparticle modified GCE 26Cladribine Structural alterations GCE (biosensor) 27Clofarabine Structural alterations GCE (biosensor) 28Cytochrome P450 (CYP1A2) Structural alterations;DNA oxidative damage GCE (biosensor) 29Danusertib Electrostatic;complex formation;adduct

formationGCE (biosensor) 30,31

Diclofenac Adduct formation GCE (in solution) 32Echinomycin Intercalation GCE (biosensor) 33Fludarabine Structural alterations GCE (biosensor) 34Furazolidone – Multiwalled carbon nanotubes modified GCE

(biosensor)35

Gemcitabine Structural alterations;DNA damage GCE (biosensor) 36Hydrazines DNA damage Carbon paste electrode (CPE) (biosensor) 37Idarubicin Structural alterations GCE (biosensor) 38Imatinib Structural alterations;DNA oxidative

damageGCE (biosensor) 39-41

Lapatinib Intercalation GCE (biosensor) 42Lipoic acid–palladium complex Structural alterations GCE (in solution) 43Metal ions Structural alterations;DNA oxidative

damageGCE (in solution/biosensor) 44-48

Metallodrugs – SPCE (biosensor) 49Methotrexate Structural alterations;intercalation;

DNA damageGCE (biosensor) 50,51

Microcystin-LR Structural alterations;DNA damage GCE (biosensor) 52Mitomycin C Structural alterations Graphene oxide (biosensor) 53Mitomycin C Structural alterations PGE (biosensor) 54Nitric oxide DNA oxidative damage GCE (biosensor) 552-(2-Nitrophenyl)-benzimidazolederivatives

Electrostatic GCE (in solution) 56

4 -Nitrophenylferrocene Intercalation GCE (in solution) 57Nodularin Structural alterations;DNA damage GCE (biosensor) 52Ochratoxin A Structural alterations GCE (biosensor) 58Palladium chelates of biogenicpolyamines

Cross-linking;adducts and aggregates GCE (in solution) 59

Photodegradation products of benzo(a)pyrene

DNA oxidative damage SPCE 60

Reactive oxygen species Structural alterations;DNA oxidative damage

BDDE (biosensor);GCE (biosensor) 61,62

Rituximab Structural alterations;DNA damage

GCE (biosensor) 63,64

Spermidine Electrostatic;minor groove binding Gold electrode 65S-Triazine derivatives DNA damage GCE (biosensor) 66Taxol Intercalation PGE (biosensor) 67Temozolomide Condensation;DNA oxidative damage GCE (biosensor) 68Thalidomide Condensation;DNA oxidative damage GCE (in solution) 69,706-Thioguanine – Pencil-graphite electrode (PGE) (biosensor) 71Topotecan Intercalation PGE (biosensor) 72Triazole–acridine conjugates Condensation GCE (biosensor) 73g Radiation DNA damage;DNA oxidative damage GCE (in solution) 74

Electrochemical Biosensors for DNA–Drug Interactions 125

DNA-electrochemical biosensors, their surface morphological and electrochemical properties, and their application for detection ofdsDNA damage induced by anticancer drugs and carcinogens will be discussed.

DNA Electrochemical Behavior

The development, design, and applications of DNA-electrochemical biosensors that use dsDNA electrochemistry as the detectionplatform require a good comprehension of the dsDNA 3D structure and biological function5,75–79 and of the electrochemicalbehavior of dsDNA and its components, on the carbon electrode electrochemical transducers, in aqueous solutions.80–87

The electrochemical oxidation of dsDNA and its components was investigated at carbon electrodes, due to their extensive poten-tial window in the positive direction, preferentially using differential pulse (DP) voltammetry, which has high sensitivity and reso-lution and enables a very good separation of the oxidation peaks.82–87

The dsDNA structure comprises two backbone chains running in opposite directions formed by a repeated pattern of a phosphategroup, a sugar group and a base (the nucleotide), all the bases being electroactive.85

The oxidation of the DNA-free bases, the purines guanine (Gua) and adenine (Ade), and the pyrimidines thymine (Thy) andcytosine (Cyt), at glassy carbon electrode (GCE), in aqueous solutions, was investigated.85 DP voltammograms for a 20 mM equi-molar mixture of all DNA bases in physiological media showed four anodic peaks, corresponding to the oxidation of Guaat þ0.70 V, Ade at þ0.96 V, Thy at þ1.16 V, and Cyt at þ1.31 V (Fig. 1A). However, the oxidation of the Thy and Cyt bases85

has a low reaction rate and occurred at very high positive potentials, and consequently, their oxidation peaks are more difficultto be detected (Fig. 1A).

The purine and pyrimidine nucleosides and nucleotides are all electroxidized at GCE (Fig. 1A), but at potentials�200 mVmorepositive than the corresponding free bases.85 As the 20-deoxyribose and the orthophosphate are not electroxidized in the experi-mental conditions employed,85 the shift in the anodic peak of nucleosides and nucleotides is due to steric effects imposed bythe glycosidic bond on the p-system of purine and pyrimidine rings, making more difficult the access to the GCE surface and theiroxidation.85

The voltammetric behavior of four single-stranded polyhomoribonucleotides was also studied88 (Fig. 1B), and their oxidationpotentials are similar to those of the corresponding purine and pyrimidine nucleotides88 (Fig. 1A).

The product of oxidation, in the C8 position, of Gua give rise to 8-oxoguanine89 (7,8-dihydro-8-oxoguanine or 8-oxoG) and ofAde to 2,8-oxoadenine90 (2,8-DHA) (Fig. 2). The formation of 8-oxoG or 2,8-DHA in the dsDNA moiety causes important trans-version mutagenic lesions and can be the starting point for cellular dysfunction, which in turn could lead to a state of illness.89,90 As

Fig. 1 DP voltammograms base line corrected at GCE: (A) 20 mM equimolar mixture of guanine (Gua), adenine (Ade), thymine (Thy), and cytosine(Cyt), and 20 mM guanosine-5-monophosphate (GMP), 20 mM adenosine-5-monophosphate (AMP), 500 mM thymidine-5-monophosphate (TMP),and 500 mM cytidine-5-monophosphate (CMP) in phosphate buffer pH ¼ 7.4, and (B) 40 mg mL�1 poly[G], 100 mg mL�1 poly[A], 100 mg mL�1

poly-[C], and 250 mg mL�1 poly[U] in phosphate buffer pH ¼ 7.1. [Adapted from Oliveira-Brett, A. M.; Piedade, J. A. P.; Silva, L. A.; Diculescu, V. C.Voltammetric Determination of all DNA Nucleotides. Anal. Biochem. 2004, 332, 321–329; Chiorcea-Paquim, A. M.; Piedade, J. A. P.; Wombacher, R.;Jäschke, A.; Oliveira-Brett, A. M. Atomic Force Microscopy and Anodic Voltammetry Characterization of a 49-mer Diels-Alderase Ribozyme. Anal.Chem. 2006, 78, 8256–8264, with permission.]

126 Electrochemical Biosensors for DNA–Drug Interactions

a consequence, 8-oxoG or 2,8-DHA are very important biomarkers of dsDNA oxidative damage; they are oxidized at a low potential,�0.45 V (Fig. 2), and can be detected by DP voltammetry.89,90

The oxidation of dsDNA at GCE, in aqueous solution, gives rise to two well-separated anodic peaks,82,86 corresponding to theoxidation of deoxyguanosine (dGuo) and deoxyadenosine (dAdo) residues in the polynucleotide chain (Fig. 3).

However, a large difference in the currents for dsDNA and single-stranded (ssDNA) was detected (Fig. 3), due to the structuraldifferences between dsDNA and ssDNA.86 The difficulty in the transfer of electrons from the inside of the more rigid double helix ofdsDNA to the electrode surface is much greater, when compared with the flexible single helix of ssDNA, where the bases are in closerproximity to the GCE surface, enabling the detection of higher oxidation peak currents in ssDNA.

Fig. 2 DP voltammograms at GCE: (A) ( ) in a solution of 50 mM Gua, fifth scan, and after transferring the electrode in acetate buffer pH ¼ 4.5,(•••) first and (–) second scan; n¼5 mV s�1, and (B) ( ) in a solution of 1 mM Ade, twentieth scan, and after transferring the electrode in acetatebuffer pH ¼ 4.5, (•••) first scan; n¼5 mV s�1. [Adapted from Oliveira-Brett, A. M.; Diculescu, V. C.; Piedade, J. A. P. Electrochemical OxidationMechanism of Guanine and Adenine Using a Glassy Carbon Microelectrode. Bioelectrochemistry 2002, 55, 61–62, with permission.]

Fig. 3 DP voltammograms base line corrected at GCE, in solutions of 60 mg mL�1 ( ) dsDNA and (–) ssDNA in acetate buffer pH ¼ 4.5. [Adaptedfrom Oliveira, S. C. B.; Oliveira-Brett, A. M. DNA-Electrochemical Biosensors: AFM Surface Characterisation and Application to Detection of In SituOxidative Damage to DNA. Comb. Chem. High Throughput Screen. 2010, 13, 628–640, with permission.]


DNA-Electrochemical Biosensor Development

A DNA-electrochemical biosensor is an integrated receptor–transducer device incorporating dsDNA as biological recognitioncomponent to detect specific binding processes with dsDNA through electrochemical transduction (Fig. 4). The most importantfactor for the construction and development of efficient and sensitive DNA-electrochemical biosensors is a very good understandingand control of the dsDNA probe immobilization, at the electrochemical carbon electrode transducer surface.17,91 In order tocharacterize the dsDNA immobilized on GCE surfaces, atomic force microscopy (AFM) was employed to clarify the nature ofthe DNA-carbon electrode surface interactions and to help establishing the optimum conditions for the dsDNA bottom-up immo-bilization.91–99

However, to obtain very reproducible results, prior to dsDNA immobilization, the GCE surface requires a pretreatment, whichgenerally consisted of mechanical polish followed by electrochemical cleaning, using DP voltammetry in the supportingelectrolyte.100

Three different immobilization procedures have been used, depending on the specific applications, for the preparation of label-free DNA-electrochemical biosensors5,17,19,91 (Fig. 4):

1. Thin-layer dsDNA biosensor prepared onto GCE (d¼1.5 mm) by 3 min free adsorption from solutions of 60 mg mL�1 dsDNA, in0.1 M acetate buffer pH¼4.5 (Fig. 4B and E);

2. Multilayer dsDNA biosensor prepared onto GCE (d¼1.5 mm) by evaporation of three consecutive drops, each containing 5 mL of50 mg mL�1 dsDNA, in 0.1 M acetate buffer pH¼4.5. After placing each drop on the electrode surface, the DNA-electrochemicalbiosensor was dried under a constant flux of N2 (Fig. 4C and F);

3. Thick-layer dsDNA biosensor prepared onto GCE (d¼1.5 mm) by evaporation of one drop containing 10 mL of 37.5 mg mL�1

dsDNA, in 0.1 M acetate buffer pH¼4.5 (Fig. 4D and F). After placing the drop on the electrode surface, the DNA-electrochemical biosensor was dried overnight in a normal atmosphere.

The thin dsDNA layer does not completely cover the GCE electrode surface, and the network structure had holes exposing the elec-trode underneath (Fig. 4B and E). Both multilayer and thick-layer dsDNA-electrochemical biosensors give rise to a very stable GCEelectrode surface complete coverage, as shown by AFM images (Fig. 4C andD).17,91 The great advantage of the multilayer and thick-layer DNA-electrochemical biosensors is that the GCE surface is completely covered by dsDNA, and consequently, the undesirednonspecific binding of molecules to the GCE surface is not possible (Fig. 4F). The multilayer dsDNA-electrochemical biosensorshort preparation time is an advantage, when compared with the thick-layer dsDNA-electrochemical biosensor longer preparationtime.

The dsDNA-electrochemical biosensors are usually electrochemically characterized by DP voltammetry in acetate buffer pH¼4.5(Fig. 4G), since the dsDNA oxidation peak currents are much higher in acid media (pH range of 4.5–5.5) than in physiologicalmedia (pH �7), thus allowing lower detection limits for the identification of DNA damage. However, the incubation of the

Fig. 4 (A–D) AFM three-dimensional images of: (A) clean HOPG, (B) thin-layer dsDNA biosensor (3 min free adsorption from 60 mg mL�1 dsDNA),(C) multi-layer dsDNA biosensor (evaporation of three consecutive drops of 5 mL from 50 mg mL�1 dsDNA) and (D) thick-layer dsDNA biosensor(evaporation from 37.5 mgmL�1 dsDNA); (E, F) Schematic models of dsDNA-anticancer drugs interaction using the (E) thin- and (F) thick-layerdsDNA-electrochemical biosensor; (G) DP voltammograms base line corrected of a multi-layer dsDNA-electrochemical biosensor in acetate bufferpH ¼ 4.5. [Adapted from Oliveira Brett, A. M.; Diculescu, V. C.; Chiorcea-Paquim, A. M.; Serrano, S. H. P. In Comprehensive Analytical Chemistry;Alegret, S.; Merkoçi, A., Eds.; 49, Elsevier: Amsterdam, 2007, pp 413–437, with permission.]


DNA-electrochemical biosensor is performed in the drug or hazard compound solution in physiological media pH �7, and onlyafterward, the DNA-electrochemical biosensor is transferred for detection in acetate buffer pH¼4.5.5,17,91

The electrochemical transduction of DNA damage is monitored measuring the following:

1. The changes in the DP voltammetric response of the DNA-electrochemical biosensors after incubation during different periodsof time with the target drug or hazard compound;

2. The shift of the formal potential of the electroactive drug or hazard compound caused by the binding to DNA;3. The occurrence of new anodic peaks associated with DNA components, such as nucleotides, nucleosides, purine free bases, and

purine bases oxidation products, biomarkers 8-oxoG and 2,8-DHA.5,17,19,91,101

Furthermore, different DNA-electrochemical biosensors have been prepared from known selected sequences of the DNA compo-nents, as in polyhomonucleotides, single-stranded sequences of guanosine and adenosine, poly(G)- and poly(A)-electrochemical biosensors, and poly-heteronucleotides, permitting biological recognition of more selective interactions.5,19

DNA-Electrochemical Biosensors for Detection of dsDNA Damage

In recent years, there has been an increasing interest in the investigations of interactions of drugs with dsDNA and the developmentof rapid, sensitive, and selective methods for detection of DNA damage mainly from hazard compounds and antineoplastic drugs,to be used in drug discovery and pharmaceutical development processes. Selected electroanalytic studies are listed in Table 1.20–74

The electrochemical investigation of the redox behavior of the drugs/hazard compounds is of key relevance and always comple-ments the study of their interaction with DNA. In fact, the drugs/hazard compounds electrochemical properties are correlated totheir 3D structure and toxic/pharmacological activity, being useful to help understand the electrochemical response of the DNA-electrochemical biosensor to the target species and also for understanding in vivo biochemical mechanisms.102,103

To illustrate in more detail the DNA-electrochemical biosensors applications and to clearly show that the DNA-electrochemicalbiosensors are very suitable for investigating DNA damage and DNA oxidative damage, the interactions with dsDNA of some anti-neoplastic drugs and hazard compounds, such as nucleoside analogs clofarabine (CLF),28 cladribine (CLD)27 and gemcitabine(GEM),36 kinase inhibitors imatinib39–41 and danusertib,30,31 adriamycin (ADM),20 thalidomide (TD),69,70 methotrexate(MTX),50,51 monoclonal antibodies (mAbs) rituximab (RTX)63,64 and bevacizumab (BEVA),23,24 human hemoprotein cytochromeP450 (CYP1A2),29 toxins ochratoxin A (OTA),58 nodularin (NOD)52 andmicrocystin-LR (MC-LR),52 transitionmetal ions and theircomplexes,43–48 and free radicals,55,61 are described.

Pharmaceuticals

Nucleoside analogsNucleoside analogs are a pharmacological class of compounds with cytotoxic, immunosuppressive, and antiviral properties. Purineand pyrimidine modified nucleobases with pharmaceutical properties represent relevant derivatives, effective in cancer treatment.The purine nucleoside analogs, CLF28 and CLD,27 and pyrimidine nucleoside analog, GEM,36 were investigated.

The electrochemical behavior of both adenosine analogs CLF and CLD oxidation process was irreversible, pH-dependent, anddiffusion controlled. The interaction of DNA with CLF and CLD, using a dsDNA-electrochemical biosensor, showed changes in theDNA morphological structure (Fig. 5A), confirmed using the purinic homopolynucleotide poly(G)- and poly(A)-electrochemicalbiosensors.

The cytosine nucleoside analog GEM36 did not undergo any electrochemical reaction. The interaction between DNA and GEM,using a dsDNA-electrochemical biosensor, showed dsDNA structural conformation modifications and damage, detected throughmodifications of the dGuo and dAdo oxidation peaks and occurrence of free Gua base oxidation peak (Fig. 5B). The interactionmechanism between dsDNA and GEM involved two sequential steps. The first step was independent of the DNA sequence andled to the condensation/aggregation of the dsDNA strands, producing rigid structures. This initial process favored a secondarystep, in which the Gua hydrogen atoms, participating in the C–G base pairs, interacted with the GEM ribose moiety fluorine atoms.

Imatinib and danusertibImatinib and danusertib are kinases inhibitors with anticancer properties.30,31,39–41 Imatinib was initially developed for treatmentof chronic myeloid leukemia, but the cross reactivity of both compounds with different kinases conferred a great therapeutic poten-tial and effectiveness for treatment of multiple tumor types.39–41

The redox mechanisms of both imatinib39,40 and danusertib,30 in a wide pH range, essential to understand and explain theirDNA binding, were investigated.

The interaction between imatinib and DNA,41 using a dsDNA-electrochemical biosensor, showed modifications in the dsDNAstructure that were electrochemically recognized through changes of the dGuo and dAdo anodic oxidation peaks (Fig. 6A). Thein situ electrochemical generation of imatinib redox metabolites allowed the detection of DNA damage at base level. Using poly-nucleotides of known sequence, it has been confirmed that the interaction took place at Ade-enriched segments, and the generationof the redox metabolite led to Ade oxidation and formation of the biomarker 2,8-DHA.


The DNA–danusertib interaction,31 using a dsDNA-electrochemical biosensor, occurred also in a two-step mechanism. The dan-usertib piperazine moiety binded first to the DNA backbone through electrostatic interactions (Fig. 6B). Next, a stable danusertib–DNA complex involved the pyrrolo-pyrazole moiety and led to small morphological modifications in the dsDNA, detected by thedGuo and dAdo oxidation peaks changes and confirmed by electrophoretic and spectrophotometric measurements. Controlling theapplied potential of the dsDNA-electrochemical biosensor surface, it was possible to in situ generate danusertib redox metabolites,

Fig. 5 DP voltammograms base line corrected, recorded at a dsDNA biosensor in acetate buffer pH 4.5, (•••) before and after incubation in solu-tions of: (A) 100 mM CLF, during (–) 5 and ( ) 15 min, and (B) 10 mM GEM, during (–) 15 min and ( ) 4 h. [Adapted from Satana, H. E.; Pontinha,A. D. R.; Diculescu, V. C.; Oliveira-Bret, A. M. Nucleoside Analogue Electrochemical Behaviour and In Situ Evaluation of DNA–Clofarabine Interaction.Bioelectrochemistry 2012, 87, 3–8; Buoro, R. M.; et al. In Situ Evaluation of Gemcitabine–DNA Interaction Using a DNA-Electrochemical Biosensor.Bioelectrochemistry 2014, 99, 40–45, with permission.]

Fig. 6 (A,B) Chemical structures: (A) imatinib (I) and (B) danusertib (D). (C,D) DP voltammograms base line corrected, at a dsDNA-electrochemicalbiosensor in acetate buffer pH ¼ 4.5, after incubation in solutions: (C) 5 mM imatinib, during 2 min, (–) without and ( ) with þ 0.90 V conditioningpotential during 2 min; (D) 10 mM danusertib, during 60 min, (–) without and ( ) with þ 0.85 V conditioning potential during 30 min. [Adapted fromDiculescu, V. C.; Oliveira-Brett, A. M. In Situ Electrochemical Evaluation of dsDNA Interaction With the Anticancer Drug Danusertib Nitrenium RadicalProduct Using the DNA-Electrochemical Biosensor. Bioelectrochemistry 2016, 107, 50–57; Diculescu, V. C.; Vivan, M.; Oliveira-Bret, A. M. Voltam-metric Behavior of Antileukemia Drug Glivec. Part III: In Situ DNA Oxidative Damage by the Glivec Electrochemical Metabolite. Electroanalysis 2006,19, 1963–1970, with permission.]


identified as nitrenium cation radicals. The dGuo oxidation peak decreased, due to the covalent attachment of the redox metaboliteto the C8 position of Gua residues, protecting them and preventing their oxidation. Using polyhomonucleotides, the preferentialinteraction with the danusertib cation nitrenium radical metabolite-Gua adduct formation occurred.

AdriamycinADM is an anthracycline antibiotic with numerous applications including antineoplastic activity by causing significant death oftumor cells. Its mode of action is not yet understood; however, it has been postulated that it intercalates mainly within CG–GCbase pairs in the minor grove of dsDNA.20

The electrochemical behavior of ADM20 has shown that different ADM groups can be oxidized and reduced. The 5,12-diquinonegroup irreversible reduction occurred at �0.4 V and �0.6 V, while the 6,11-dihidroquinone-functionality reversible oxidationoccurred at þ0.5 V.

The in situ interaction between ADM andDNA, using a thick-layer dsDNA-electrochemical biosensor,20 after applying a potentialof �0.60 V during 60 s, showed DNA oxidative damage. The reduced ADM radical produced at this applied potential was respon-sible for the DNA oxidative damage (Fig. 7A) detected by the occurrence of the biomarker 8-oxoG oxidation peak. A mechanism forDNA oxidative damage caused by ADM was proposed (Fig. 7B).

ThalidomideTD is an oral drug marketed in the 1950s mainly as an antiemetic during pregnancy, but soon removed due to its teratogenic sideeffects in newborn children. Recently, TD recovered its scientific interest, due to its potential for treating a number of other diseases,such as erythema nodosum leprosum, human immunodeficiency virus replication in acquired immune deficiency syndrome, andcancer.69,70 There are clear evidences that TD interacts with DNA due to its teratogenicity,69,70 intercalating and leading to a stackedcomplex between the flat double phthalimide rings of TD and dGuo70 or associated with TD ability to cause DNA oxidative damagemediated by free radicals.70

The electrochemical behavior of TD69 showed an irreversible pH-dependent oxidation process that occurred in two consecutivesteps to produce a cation radical that reacted with water and yielded a final hydroxylated product,69 and the reduction mechanisminvolved the protonation of the nitrogen that bridge the two cyclic groups.69

The mechanism of in vitro interaction between TD and dsDNA, by DP voltammetry and AFM, was investigated.70 A reorgani-zation of the self-assembled network on the surface of highly oriented pyrolytic graphite electrode was observed after incubation of

Fig. 7 (A) DP voltammograms, at a thick-layer dsDNA-electrochemical biosensor, in acetate buffer pH ¼ 4.5, after incubation in a solution of 5 mMADM, during 3 min: (•••) without applied potential, and (–) after applying a potential of – 0.6 V during 60 s; (B) Proposed mechanism of electro-chemical in situ ADM oxidative damage to DNA. [Adapted from Piedade, J. A. P.; Fernandes, I. R.; Oliveira-Brett, A. M. Electrochemical Sensing ofDNA-Adriamycin Interactions. Bioelectrochemistry 2002, 56, 81–83, with permission.]


dsDNA with different concentrations of TD, resulting in a decrease of the TD–DNA network film thickness, decrease number, andincrease height of TD–DNA aggregates, with increasing incubation time (Fig. 8).

The interaction between the TD and dsDNA in incubated solutions, using DP voltammetry at GCE,70 caused DNA oxidativedamage, and four well-defined anodic peaks, of 8-oxoG/2,8-DHA at þ0.45 V, Gua/TD at þ0.80 V, dGuo at þ1.03 V, and dAdoat 1.30 V,70 were observed (Fig. 8G and H). The increase of all anodic peak currents was proportional to the increase in TD concen-tration and incubation time. Agarose gel electrophoresis of TD-dsDNA confirmed the DNA condensation. The toxic effects of TDthat caused DNA condensation, TD intercalation, helix unwinding, and DNA oxidative damage were clearly demonstrated byAFM and DP voltammetry, and a TD-dsDNA interaction mechanism was proposed.70

MethotrexateMTX is an antimetabolite of folic acid and an antineoplastic drug indicated in the treatment of numerous neoplasms, such as acuteleukemia, head and neck cancer, and micrometastases of osteosarcoma.50,51 Nevertheless, several side effects indicated that MTX isgenotoxic, resulting in chromosomal/chromatid breaks, proving a direct interaction between dsDNA and MTX.

The electrochemical behavior of MTX50 showed an irreversible pH-dependent oxidation process, with the formation of one elec-troactive product, the 7-hydroxymethotrexate.

The time-dependent in situ MTX–dsDNA interaction,51 at a multilayer DNA-electrochemical biosensor (Fig. 9C), showed, fora short incubation times, the dsDNA oxidation peak current decrease, correspondent to the dGuo and dAdo residue oxidationin the polynucleotide chain, followed, for longer incubation times, by a purine oxidation peak current increase, due to theunwinding of the dsDNA. The higher affinity of MTX to Ade residues was demonstrated using poly(A)-electrochemical biosensors.51

Fig. 8 AFM images onto HOPG: (A) 10 mg mL–1 dsDNA, (B) 4 mM TD, (C-F) 10 mg mL–1 dsDNA incubated with: (C) 1 mM TD, during 24 h, and(D–F) 4 mM TD, during (D) 10 min, (E) 5 h, and (F) 24 h; (G, H) DP voltammograms base line corrected at GCE, in solutions of: ( ) control100 mg mL�1 dsDNA and (–) 100 mg mL�1 dsDNA incubated with � 40 mM TD, during different time periods. [Adapted from Oliveira, S. C. B.; et al.In Situ Electrochemical and AFM Study of Thalidomide-DNA Interaction. Bioelectrochemistry 2009, 76, 201–207, with permission.]


The AFM images of MTX–dsDNA interaction (Fig. 9A and B),51 showed structural and dsDNA conformational modificationsand DNA self-assembled network reorganization, forming a densely packed and thicker MTX–dsDNA lattice after interaction,due to the intercalation of MTX onto DNA strands.

Proteins

Monoclonal antibodiesmAbs are a new class of immunotherapeutic drugs that attach to specific receptor antigen transmembrane proteins. The RTX andBEVA are genetically engineered chimeric human/murine mAbs used in clinical oncology to treat certain types of metastaticcancers.23,24,63,64

The oxidation of RTX native and denatured63 showed significant differences, due to morphological changes and unfolding of theRTX native structure. The pH-dependent native RTX oxidation showed the tyrosine, tryptophan, and methionine residues oxida-tion.63 After denaturing, the unfolded RTX63 electrochemical detection showed the tyrosine, tryptophan, and histidine residueoxidation peaks.63 The RTX–dsDNA interaction,64 at a multilayer DNA-electrochemical biosensor, caused a strong condensationof the DNA helical structure, as the dAdo oxidation peak disappeared and dGuo oxidation peak current decreased, while freeGua and Ade were released from the DNA strands (Fig. 10). However, no DNA oxidative damage was detected. Nondenaturingagarose gel electrophoresis of RTX–dsDNA confirmed the occurrence of the dsDNA structure condensation.

The electrochemical behavior on GCE of thin-film native BEVA showed only one pH-dependent tyrosine and tryptophan aminoacid residue oxidation peak. Denatured unfolded thin-film BEVA enabled also the detection of cysteine and histidine amino acidresidues.23 The interaction of BEVA with dsDNA, in incubated solutions and using a dsDNA-electrochemical biosensor,24 showedthe decrease and disappearance of the dGuo and dAdo oxidation peaks and BEVA structural modification upon binding to DNA,24

but no DNA oxidative damage occurred, confirmed by nondenaturing agarose gel electrophoresis.

Human cytochrome P450 (CYP1A2)Human hemoprotein cytochrome CYP1A2 is a member of the P450 superfamily of proteins, highly complex and with a large fieldof activity. They are also involved in the biotransformation of drugs and chemical carcinogen activation. The CYP1A2–dsDNA inter-action in situ evaluation using a DNA-electrochemical biosensor caused DNA condensation and DNA oxidative damage29 (Fig. 11).

A preferential interaction at Gua-containing sequences, leading to DNA oxidative damage and enabling the detection of the8-oxoG biomarker, was demonstrated using poly(G)- and poly(A)-electrochemical biosensors. The molecular mechanism involvedin the CYP1A2–dsDNA interaction, by which the enzyme CYP1A2 interacted with dsDNA causing oxidative damage and mutage-nicity, in a time-dependent manner, was clarified and is now better understood.

Fig. 9 (A, B) AFM images onto HOPG: (A) 10 mg mL–1 dsDNA and (B) 10 mg mL–1 dsDNA incubated with 0.5 mM MTX, during 0 h; (C) DP voltam-mograms base line corrected, recorded at a multi-layer dsDNA-electrochemical biosensor in acetate buffer pH ¼ 4.5, ( ) before and after incubationin a solution of 100 mM MTX, during (–) 5, (– – –) 10 and (•••) 20 min. [Adapted from Pontinha, A. D. R.; Jorge, S. M. A.; Chiorcea Paquim, A.-M.;Diculescu, V. C.; Oliveira-Brett, A. M. In Situ Evaluation of Anticancer Drug Methotrexate-DNA Interaction Using a DNA-Electrochemical Biosensor andAFM Characterization. Phys. Chem. Chem. Phys. 2011, 13, 5227–5234, with permission.]


Toxins

Ochatoxin AOTA is a fungal metabolite that occurs in foods, beverages, animal tissues, and human blood and presents carcinogenic, teratogenic,and nephrotoxic properties.58 The oxidation of OTA is a pH-dependent irreversible process that involved the formation of an oxida-tion product that is reversibly oxidized.58 The in situ evaluation of the OTA interaction with DNA, using a DNA-electrochemicalbiosensor, has clearly proved that OTA interacts and binds to dsDNA, but no evidence of DNA oxidative damage was found.58

Nodolarine and microcystineNOD and MC-LR are potent cyanotoxins with strong hepatotoxic, genotoxic, and carcinogenic potential, which have been associ-ated with the induction of DNA damage in vitro and in vivo.52 The MC-LR and NOD redox mechanisms and their chemical degra-dation of metabolite oxidation were investigated.52 The NOD andMC-LR interaction with dsDNA, in incubated solutions and usinga multilayer dsDNA-electrochemical biosensor, was very similar.52 The NOD–dsDNA interaction showed, for long incubationtimes, the occurrence of free Ade, at þ1.10 V (Fig. 12).

The interaction between dsDNA and NOD or MC-LR, using poly(G)- and poly(A)-electrochemical biosensors, confirmed theliberation of free Ade, leading to the formation of DNA abasic sites, which, if left unrepaired in vivo, can lead to permanent

Fig. 10 3D plot of DP voltammograms base line corrected, at a multi-layer dsDNA-electrochemical biosensor in acetate buffer pH ¼ 4.5, ( ) beforeand (–) after incubation in solutions of 2.5 mg mL�1 RTX in phosphate buffer pH ¼ 7.0, during different time periods. [Adapted from Santarino, I.B.; Oliveira, S. C. B.; Oliveira-Brett, A. M. In Situ Evaluation of the Anticancer Antibody Rituximab-dsDNA Interaction Using a DNA-ElectrochemicalBiosensor. Electroanalysis 2014, 26, 1304–1311, with permission.]

Fig. 11 DP voltammograms baseline-corrected, in 0.1 M phosphate buffer pH 7.0: ( ) control dsDNA-electrochemical biosensor, and (–) dsDNA-electrochemical biosensor after incubation with 0.5 mg mL�1 CYP1A2 during 10 min, 1 h, 3 h, and 5 h. [Adapted from Lopes, I. C.; Oliveira-Bret, A.M. Human Cytochrome P450 (CYP1A2)-dsDNA Interaction In Situ Evaluation Using a dsDNA-Electrochemical Biosensor. Electroanalysis 2016, https://doi.org/10.1002/elan.201600713, with permission.]


https://doi.org/10.1002/elan.201600713


mutations during the replication process resulting in cell transformation.52 The toxic effects caused to dsDNA by NOD and MC-LR,such as dsDNA aggregation and dsDNA abasic damage, were clearly explained following their electrochemical behavior.52

Metal Ions

Metal ion–DNA interactions are important in nature, and in specific conditions, the DNA structural and functional modificationswere observed.43,44 The interaction of DNA with heavy metals, such as Pb, Cd, Ni, and Cr, has been extensively investigated sincethey are involved in processes leading to DNA damage.44–46

The in situ evaluation of direct interaction between Pb2þ, Cd2þ and Ni2þ, and dsDNA, at a multilayer dsDNA-electrochemicalbiosensor, showed that Pb2þ, Cd2þ, and Ni2þ bind directly to dsDNA, this interaction leading to different alterations in the dsDNAstructure.44,45 It was confirmed that Pb2þ interacts with dsDNA preferentially at Ade-containing segments, leading to DNA oxidativedamage and formation of the biomarker 2,8-DHA.44,45 No oxidative damage to dsDNA caused by Cd2þ and Ni2þwas observed, butthese heavy metal ions affected the DNA double helix, causing conformational modifications, opening the way to initiation of othercarcinogens agents’ action.44,45

The interaction with dsDNA of the Cr4þ and Cr5þ, the reactive intermediates of Cr3þ oxidation by O2, caused conformationalalterations, unfolding, breaking of hydrogen bonds, and DNA oxidative damage (Fig. 13),46 preferentially taking place at Gua-rich

Fig. 12 DP voltammograms base line corrected, at GCE, in solutions: (•••) control 50 mg mL�1 dsDNA and 50 mg mL�1 dsDNA incubated with30 mM NOD, during (– – –) 6 and ( ) 24 h. [Adapted from Santos, P. V. F.; Lopes, I. C.; Diculescu, V. C.; Oliveira-Brett, A. M. DNA-CyanobacterialHepatotoxins Microcystin-LR and Nodularin Interaction Electrochemical Evaluation. Electroanalysis 2012, 24, 547–553, with permission.]

Fig. 13 DP voltammograms base line corrected, after 60 h incubation, in acetate buffer pH ¼ 4.5: (A) 100 mg mL�1 dsDNA ( ) control and (–) with� 100 mM Cr(III) and (B) 100 mg mL�1 polyG ( ) control and (–) with � 100 mM Cr(III). [Adapted from Oliveira, S. C. B.; Oliveira-Brett, A. M. In SituEvaluation of Chromium-DNA Damage Using a DNA-Electrochemical Biosensor. Anal. Bioanal. Chem. 2010, 398, 1633–1641, with permission.]


segments, and leading to the formation of 8-oxoG.46 However, the interaction of Cr6þ with dsDNA only caused conformationchanges to the double-helical structure, and no dsDNA oxidative damage occurred.46

The polyphenolic compounds well-known for antioxidant protection are changed in the presence of metal ions, as the polyphe-nolic compounds act as prooxidants.47,48 Quercetin in the presence of Cu2þ ions undergoes homogenous oxidation, generating thehighly oxidizing hydroxyl radical (�OH).47,48 The DNA–Cu(II)–quercetin interaction, after quercetin oxidation by Cu(II), at a DNA-electrochemical biosensor, detected the occurrence of DNA oxidative damage and the biomarker 8-oxoG, clearly showing the role ofO2 in the process of DNA oxidative damage.47,48

Free Radicals

Oxygen reactive speciesReactive oxygen species (ROS) are generated inside cells as products of metabolism, by leakage from mitochondrial respirationand also under the influence of exogenous agents, such as ionizing radiation, quinones, peroxides, and transition metal ions. Thebiological function of ROS in the organism so far is ambiguous. Excess ROS are responsible for causing DNA and cellulardamage, which can contribute to development of tumors.61 However, ROS can assist the immune system, mediates cellsignaling, and is essential in apoptosis. The treatment of cancer cells by ROS-inducing antineoplastic drugs exceeds the thresholdfor ROS causing the activation of multiple cell death programs.61 The ROS–dsDNA interaction mechanism has been investigated,using various methods with great sensitivity and specificity; however, for different reasons, conflicting results have beenreported.61

The dsDNA oxidative damage caused by hydroxyl radicals electrogenerated (by water discharge), in situ at a dsDNA-electrochemical biosensor, using a boron-doped diamond electrode (BDDE) surface,61 was investigated. A large increase in theoxidation peak currents of dGuo and dAdo was observed, three new oxidation peaks, corresponding to the oxidation of 8-oxoG,and free purine bases Gua and Ade, occurred (Fig. 14), and were confirmed by nondenaturing agarose gel electrophoresis.61

The dsDNA-BDDE-electrochemical biosensors enabled, for the first time, a direct and fast detection procedure to follow theinteraction of dsDNA with hydroxyl free radicals, and the BDDE surface played a dual role being the source of the electrochemicallygenerated hydroxyl radicals and also the transducer for the detection of dsDNA oxidative damage (Fig. 14).

Nitrogen reactive speciesNitric oxide (NO), the nitrogen monoxide radical, is a physiologically active molecule regulating numerous biological processesincluding vasodilatation, neurotransmission, and cell-dependent immunity, and NO large concentrations can produce genotoxiceffects and carcinogenesis.55 The DNA-electrochemical biosensor was used for the detection of DNA damage produced by reactivenitrogen species (RNS) from diethylenetriamine/nitric oxide,55 after NO preconcentration. The peroxynitrite radicals were in situelectrochemically generated and cleaved the immobilized dsDNA causing the occurrence of 8-nitroguanine, a major product ofDNA oxidative damage by RNS.55

Fig. 14 DP voltammograms, at a thick multi-layer dsDNA-BDDE-electrochemical biosensor, in acetate buffer pH ¼ 4.5: ( ) control and ( ) first and(–) second to fourth scans after applying þ 3.0 V during 2 h to the BDDE surface, causing electrogeneration of hydroxyl radicals. [Adapted from Oli-veira, S. C. B.; Oliveira-Brett, A. M. In Situ DNA Oxidative Damage by Electrochemically Generated Hydroxyl Free Radicals on a Boron-Doped DiamondElectrode Surface. Langmuir 2012, 28, 4896–4901, with permission.]


Conclusion

The dsDNA-electrochemical biosensor has great advantages to investigate the mechanisms of antineoplastic drug/hazardcompounds–dsDNA interactions, due to the ultrasensitivity, selectivity, and fast detection of small perturbations of the dsDNAstructure, DNA damage, and DNA oxidative damage, caused directly by the drug/hazard compound and/or by in situ electrochem-ical generated radical intermediates and metabolites.

The DNA-electrochemical biosensor is an excellent analytic tool giving significant information in assessing the incidence of anyphysical, chemical, enzymatic, or oxidative damage to DNA that can cause the release of free bases, strand breaks disrupting thepolymer morphology or generating the products of purine base oxidation, in an approach to the real action scenario that occursin the living cell and without using animal tests.

Acknowledgments

Financial support from Fundação para a Ciência e Tecnologia (FCT), grant SFRH/BPD/92726/2013 (A.-M. Chiorcea-Paquim), project UID/EMS/00285/2013 (cofinanced by the European Community Fund FEDER), FEDER funds through the program COMPETEdPrograma OperacionalFactores de Competitividade, and Innovec’EAU (SOE1/P1/F0173), projects from Fundação de Amparo à Ciência e Tecnologia do Estado de Per-nambuco (FACEPE) (FACEPE/CNPq/PPP-APQ-0535-1.06/14), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (APQ/MCTI/CNPq/Universal/456725/2014-8), and FACEPE-CNPq-BRAZIL (S. C. B. Oliveira), and the National Agency for Scientific Research andInnovation through project POC-P-37-689 NANOBIOSURF (V. C. Diculescu), are gratefully acknowledged.

See also: Electrochemical Immunosensors for Clinical Diagnostics; Electrochemistry of Biofilms; Integrated Electrochemical Immunosensors.

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Chem. 1997, 429, 95–99.84. Oliveira-Brett, A. M.; Matysik, F. M. Sonoelectrochemical Studies of Guanine and Guanosine. Bioelectrochem. Bioenerg. 1997, 42, 111–116.85. Oliveira-Brett, A. M.; Piedade, J. A. P.; Silva, L. A.; Diculescu, V. C. Voltammetric Determination of all DNA Nucleotides. Anal. Biochem. 2004, 332, 321–329.86. Brett, C. M. A.; Oliveira-Brett, A. M.; Serrano, S. H. P. On the Adsorption and Electrochemical Oxidation of DNA at Glassy Carbon Electrodes. J. Electroanal. Chem. 1994,

366, 225–231.87. Oliveira, S. C. B.; Oliveira Brett, A. M. Boron Doped Diamond Electrode Pre-Treatments Effect on the Electrochemical Oxidation of dsDNA, DNA Bases, Nucleotides,

Homopolynucleotides and Biomarker 8-Oxoguanine. J. Electroanal. Chem. 2010, 648, 60–66.88. Chiorcea-Paquim, A. M.; Piedade, J. A. P.; Wombacher, R.; Jäschke, A.; Oliveira-Brett, A. M. Atomic Force Microscopy and Anodic Voltammetry Characterization of

a 49-mer Diels-Alderase Ribozyme. Anal. Chem. 2006, 78, 8256–8264.89. Oliveira-Brett, A. M.; Piedade, J. A. P.; Serrano, S. H. P. Electrochemical Oxidation of 8-Oxoguanine. Electroanalysis 2000, 12, 969–973.90. Oliveira-Brett, A. M.; Diculescu, V. C.; Piedade, J. A. P. Electrochemical Oxidation Mechanism of Guanine and Adenine Using a Glassy Carbon Microelectrode.

Bioelectrochemistry 2002, 55, 61–62.91. Chiorcea, A. M.; Oliveira Brett, A. M. Atomic Force Microscopy Characterisation of an Electrochemical DNA-Biosensor. Bioelectrochemistry 2004, 63, 229–232.92. Oliveira Brett, A. M.; Chiorcea, A. M. Effect of pH and Applied Potential on the Adsorption of DNA on Highly Oriented Pyrolytic Graphite Electrodes. Atomic Force Microscopy

Surface Characterisation. Electrochem. Commun. 2003, 5, 178–183.93. Oliveira Brett, A. M.; Chiorcea, A. M. Atomic Force Microscopy of DNA Immobilized Onto a Highly Oriented Pyrolytic Graphite Electrode Surface. Langmuir 2003, 19,

3830–3839.94. Chiorcea, A. M.; Oliveira Brett, A. M. DNA Imaged on a HOPG Electrode Surface by AFM With Controlled Potential. Bioelectrochemistry 2005, 66, 117–124.95. Chiorcea Paquim, A. M.; Oretskaya, T. S.; Oliveira Brett, A. M. Adsorption of Synthetic Homo- and Hetero-oligodeoxynucleotides Onto Highly Oriented Pyrolytic Graphite:

Atomic Force Microscopy Characterization. Biophys. Chem. 2006, 121, 131–141.96. Chiorcea Paquim, A. M.; Santos, P. V.; Oliveira Brett, A. M. Atomic Force Microscopy and Voltammetric Characterisation of Synthetic Homo-Oligodeoxynucleotides.

Electrochim. Acta 2013, 110, 599–607.97. Chiorcea Paquim, A. M.; Oliveira Brett, A. M. Redox Behavior of G-Quadruplexes. Electrochim. Acta 2014, 126, 162–170.98. Chiorcea Paquim, A. M.; Santos, P. V.; Eritja, R.; Oliveira Brett, A. M. Self-Assembled G-Quadruplex Nanostructures: AFM and Voltammetric Characterization. Phys. Chem.

Chem. Phys. 2013, 15, 9117–9124.99. Chiorcea Paquim, A. M.; Pontinha, A. D. R.; Oliveira Brett, A. M. Time-Dependent Polyguanylic Acid Structural Modifications. Electrochem. Commun. 2014, 45, 71–74.

100. Cavalheiro, E. T. G.; Brett, C. M. A.; Oliveira-Brett, A. M. Fatibello-Filho O. Bioelectroanalysis of Pharmaceutical Compounds. Bioanal. Rev. 2012, 4, 31–53.101. Rhaese, H. J.; Freese, E. Chemical Analysis of DNA Alterations: I. Base Liberation and Backbone Breakage of DNA and Oligodeoxyadenylic Acid Induced by Hydrogen

Peroxide and Hydroxylamine. Biochim. Biophys. Acta (BBA) – Nucleic Acids Protein Synth. 1968, 155, 476–490.102. Oliveira, S. C. B.; et al. Electrochemical Oxidation Mechanism of Procarbazine at Glassy Carbon Electrode. J. Electroanal. Chem. 2015, 746, 51–56.103. Fernandes, I. P. G.; et al. Isatin Halogen-Derivatives Redox Behaviour. J. Electroanal. Chem. 2016, 780, 75–83.

Further Reading

Diculescu, V. C.; Chiorcea-Paquim, A. M.; Oliveira-Brett, A. M. Applications of a DNA-Electrochemical Biosensor. TrAC Trends Anal. Chem. 2016, 79, 23–36.Chiorcea-Paquim, A.-M.; Santos, P.; Diculescu, V. C.; Eritja, R.; Oliveira-Brett, A. M. In Electrochemical Characterization of Guanine Quadruplexes; Fritzsche, W., Spindler, L., Eds.,

Royal Society of Chemistry: Cambridge, 2013; pp 100–109.Diculescu, V. C.; Oliveira-Brett, A. M. DNA-Electrochemical Biosensors and Oxidative Damage to DNA: Application to Cancer. In Biosensors and Cancer. Blood, Molecules and

Cells; Preedy, V. R., Patel, V. B., Eds., Science Publishers, CRC Press: Boca Raton, FL, 2012; pp 187–210. Ch. 10. Section 2.Oliveira-Brett, A. M.; Diculescu, V.; Chiorcea-Paquim, A. M.; Serrano, S. H. P. DNA-Electrochemical Biosensors for Investigating DNA Damage. In Comprehensive Analytical

Chemistry (CAC); Alegret, S., Merkoçi, A., Eds.; Electrochemical Sensor Analysis (ECSA), vol. 49; Elsevier: Amsterdam, The Netherlands, 2007. Ch. 20, pp 413–437, Proc.28, e203–e205, Proc. 29, e207–e211.

Oliveira-Brett, A. M. Electrochemistry for Probing DNA Damage. In Encyclopedia of Sensors, 3, Grimes, C. A., Dickey, E. C., Pishko, M. V., Eds.; American Scientific Publisher: USA,2006; pp 301–314.


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