past, present and future challenges of biosensors and ... anal chem-2… · 1. introduction 287 2....

CHAPTER NINE

Applications of DNA-Electrochemical Biosensors inCancer ResearchAna-Maria Chiorcea-Paquim1, Severino Carlos B. Oliveira1,2,Victor C. Diculescu1,3 and Ana Maria Oliveira-Brett1,*1University of Coimbra, Coimbra, Portugal2Universidade Federal Rural de Pernambuco, Recife, Brazil3National Institute of Material Physics, Magurele, Romania*Corresponding author: E-mail: [email protected]

Contents

1. Introduction 2872. DNA-Electrochemical Biosensor Development 2893. DNA-Electrochemical Biosensor in Evaluation of Cancer Treatment Drugs 302

3.1 Monoclonal antibodies 3023.2 Nucleoside analogues 3033.3 Kinase inhibitors 3063.4 Antimetabolites 3083.5 G-quadruplex ligands 3103.6 Antibiotics 3123.7 Thalidomide 3123.8 Metal complexes 3133.9 Natural compounds 315

3.10 Free radicals 3173.11 Ionizing radiation 319

4. DNA-Electrochemical Biosensor in Detection of Cancer Biomarker Proteins 3205. DNA-Electrochemical Biosensor in Risk Assessment of Hazardous Compounds 323

5.1 Metal ions 3245.2 Pollutants 325

6. Conclusions 328Acknowledgements 329References 329

1. INTRODUCTION

Despite the unremitting efforts in prevention, early detection, andtreatment, cancer remains one of the four leading threats to human health

Comprehensive Analytical Chemistry, Volume 77ISSN 0166-526Xhttp://dx.doi.org/10.1016/bs.coac.2017.06.003

© 2017 Elsevier B.V.All rights reserved. 287 j

http://dx.doi.org/10.1016/bs.coac.2017.06.003

and development. According to the International Agency for Research on Cancer(IARC), in 2012 there were 14.1 million new cancer cases and 8.2 millioncancer deaths worldwide, and it is estimated that there will be 21.7 millionnew cases of cancer each year by 2030.

Nanotechnology and biosensor technology have the potential to impactcancer treatment, detection, and prevention, through the development ofbiotechnological devices, for screening new therapeutic agents that interactwith specific targets, detection of cancer biomarkers, and analysis of newenvironmental hazard compounds. Electrochemical biosensors receivedparticular attention, due to their robustness, easy miniaturization, excellentdetection limits, use of small analyte volumes, and ability to be used in turbidbiofluids, which make them exceptional tools for rapid and simple on-fielddetection.

The DNA molecule can be seen from different viewpoints in molecularpharmacology. DNA represents the primary element of the genetic machin-ery, it is involved in complex interactions with different proteins and proteincomplexes, and it is the primary target for many drugs, including antibioticsand antineoplastic drugs. DNA, its components, and derivatives can act asdrugs by themselves (e.g., azidothymidine used in AIDS and cancertreatment), and DNA is the basis for the development of DNA moleculardiagnostics (e.g. polymerase chain reaction assay) for numerous diseases[1]. Due to its important chemical and biophysical characteristics, and itshigh specificity for recognition and binding to other molecules, nanotech-nology and biosensor technology consider DNA for the construction ofDNA-based sensor devices.

The DNA-electrochemical biosensor represents a good model forsimulating nucleic acid interactions with cell membranes, specific DNAsequences, proteins, drugs, or harmful carcinogenic compounds [2e8], andpresent nowadays well-established applications in medical, environmental,and food control areas.

ADNA-electrochemical biosensor is a sensing device composed of an elec-trode, the electrochemical transducer, interfaced with a DNA immobilizedlayer on the electrode surface that acts as a biological recognition element,to detect target analytes that interact with DNA at nanoscale, inducingmorphological, structural, and electrochemical changes in the DNA layer,which are further translated into an electrochemical signal, Scheme 1[2,3,5e7,9e11]. Several electrochemical strategies are used in DNA-electrochemical biosensors applications: (i) the direct label-free detection ofDNA bases’ electrochemical signals, (ii) the detection of redox reactions of

288 Ana-Maria Chiorcea-Paquim et al.

reporter label molecules, or (iii) the detection of charge transport reactionsmediated by the p-p interaction between DNA stacked bases.

In this chapter, the advances on the DNA-electrochemical biosensorsdesign and its applications in detection of cancer treatment drugs, proteincancer biomarkers, and carcinogens will be briefly discussed. Special atten-tion is given to the label-free DNA-electrochemical biosensor devices thatdirectly monitor the changes in the oxidation and/or reduction peaks ofthe DNA after the interaction with the analyte [2e8]. This type of DNA-electrochemical biosensor is highly sensitive, up to femtomoles of analyte,and allows the use of different electrode substrates, which makes it suitablefor inexpensive miniaturization for clinical diagnosis and on-site environ-mental monitoring.

2. DNA-ELECTROCHEMICAL BIOSENSORDEVELOPMENT

The development and miniaturization of DNA-electrochemical bio-sensors requires both (i) the correct assessment of the DNA immobilizationon the electrode surface, Scheme 2A-middle and (ii) a good understandingof the electrochemical response of the immobilized DNA, Scheme 2A-right. The DNA adsorption and stability on the surface of the electrode isa complicated process and requires special attention, since it influences theDNA characteristics, the chemical compounds accessibility to the DNA,the sensor response and its performance. Several internal (DNA basesequence, length, concentration) and external parameters (the electrode

ANALYTE

DNA RECOGNITION

LAYER

ELECTRODE

DNA - ANALYTE INTERACTION

ELECTROCHEMICAL DETECTION

Scheme 1 DNA-electrochemical biosensor: the analyte interaction with the DNArecognition layer immobilized at the electrode surface causes DNA damage that iselectrochemically detected.

Applications of DNA-Electrochemical Biosensors in Cancer Research 289

0.4 0.6 0.8 1.0 1.2 1.4

ArGr

E / V vs. Ag/AgCl

I / n

A

G

8-oxoG/2,8-DHA

REDOX BEHAVIOUR

MODIFICATIONS

dsDNA recognition layer

DNA REDOX BEHAVIOURBottom-up

immobilisation

followINTERACTION

(A) - DEVELOPMENT

8-oxoG/2,8-DHA

- APPLICATIONS

0.4 0.6 0.8 1.0 1.2 1.4

ArGr

E / V vs. Ag/AgCl

I / n

A

A

A

dsDNA-analyteMORPHOLOGICAL MODIFICATIONS

A

B

500nm

500nm

(B)

Scheme 2 DNA-electrochemical biosensor and DNA oxidative damage detection, showing DNA immobilization, and surface and voltam-metric characterization. (A) Development and (B) applications.

290Ana-M

ariaChiorcea-Paquim

etal.

characteristics and electrode pretreatment conditions, DNA adsorption pro-cedure, pH, ionic strength, applied potential, ion concentration, time)directly influence the immobilized DNA structural conformation and bio-physical properties.

DNA is composed of nucleotides, each containing a phosphate group, asugar group, and a nitrogen base, the purines adenine (A) and guanine (G)and the pyrimidines thymine (T) and cytosine (C), Scheme 3A. The mainstructural conformation for natural DNA is the double-stranded DNA(dsDNA), Scheme 3D-left, in Watson-Crick base-pairs, Scheme 3B, thecellular DNA being almost exclusively in this form [1]. However, DNAcan be found in a variety of other conformations, such as double helixeswith different types of loops (bulge, internal, hairpin, junction, knottedloops, etc.), single helixes, triplex helixes, or four-stranded secondary struc-tures, such as i-motifs formed by DNA sequences rich in C or G-quadruplex(GQ) structures, formed by DNA sequences rich in G that occur in the telo-meric regions at the ends of the chromosomes, Scheme 3C and D [1,8,12].Many electrochemical studies were devoted to the investigation of the DNAand DNA constituents’ redox behaviour, and to understand, dependent onDNA conformation, the nature of the DNA-electrode surface interactions[4,11,13e20].

The free A and C bases can be reduced at mercury electrodes, in aqueousmedia at acidic pH, in a process involving the transfer of four electrons for Aand three electrons for C [21,22]. The reduction behaviour of A did notchange when A is incorporated into nucleosides or nucleotides [21,22]. Atnegative potentials, close to the electrolyte discharge, G free base is reducedto an unstable, oxidizable product [21,22]. In single-stranded DNA(ssDNA), the A and C residues are also reducible at mercury electrodes,and G residues are reduced at very negative potentials of w�1.80 V inneutral pH, the G residues reduction product, with a lower reductionpotential, being more easily studied [21,22]. The T residues reduction inDNA has not been observed [21,22].

All free DNA bases present pH-dependent oxidation at carbonelectrodes, displaying anodic peaks at wþ0.70 V for G, þ0.96 V for A,þ1.16 V for T, and þ1.31 V for C, in neutral pH w7.0, Fig. 1A [23,24].The G oxidation product is 8-oxoguanine (7,8-dihydro-8-oxoguanine or8-oxoG) [25], and the A oxidation product is 2,8-oxoadenine (8-oxoA or2,8-DHA) [26], which are oxidized at a lower potential, of w0.35 V atglassy carbon electrode (GCE), Fig. 1B [25,26]. The DNA nucleosidesand nucleotides oxidation at GCE occurs at potentials w0.20 V more


N

NN

N

O

NH

H

H

H

N

N

N

O

H H

H

HH

N

N

O

O

CH3

HH

H

N

NN

N

N

H

H H

HH

G-G-G-G (G-quartet)

Cytosine (C)Thymine (T)

Adenine (A)

N

NHN

N

O

N

N

N NH

N

O

N

NN

NHN

O

N

NN

HN N

O

NH

H

H

H

HH

HH

H

HH

H

AH+-AH+

+HN

N NH

N

HNH

NH+

NHN

N

NHH

N+

N

N

O

H H

H

HH

N

N

N

O

HH

H

HH

H

C-CH+

Guanine (G)

K+

NH

N

N

O

NH2N

O

H

HHHHOH

OPOOH

O

NUCLEOSIDEdeoxyguanosine

BASEguanine

NUCLEOTIDEdeoxyguanosine monophosphate

A-T G-C

N NH

O

O

H3C

HN

NNH

N

O

NH

H

H

H

NNH

HN

O

H

N

NNH

N

HNH

H

Watson-Crick base-pairing

(A)

(B)

(C)

(D)

Double-strand G-quadruplex i-motif

Scheme 3 (A) Chemical structure of DNA nucleotides, nucleosides, and bases; (B) Watson-Crick base-pairing; (C) Homo-ODNs base-pairing and (D) Schematic representation ofthe DNA double strand, G-quadruplex, and i-motif configurations. Adapted from V.C.Diculescu, A.-M. Chiorcea-Paquim, A.M. Oliveira-Brett, Applications of a DNA-electrochemical


positive than the corresponding free bases [24], due to steric effects imposedby the glycosidic bond on the p-system of purine and pyrimidine rings thatare now further away from the GCE surface, and more difficult to beoxidized.

The dsDNA oxidation in solution, at GCE, is pH dependent, showingtwo small anodic peaks, corresponding to the G residues oxidation (Gr),and A residues oxidation (Ar) in the double-helix DNA, Fig. 1D[3,5,8,9,27]. The T and C residues oxidation is more difficult to detect, sinceit occurs with a very low current, at very high positive potentials, near to thepotential of oxygen evolution. Differential pulse (DP) voltammograms ofssDNA in solution, at GCE, also show the G and A residues oxidation peaks,Fig. 1D, but the ssDNA oxidation peak currents are much higher than thecurrents of dsDNA, due to easier transfer of electrons from the inside of thesingle helix that presents the base residues in closer proximity to the elec-trode surface.

The advances in the electrochemical and atomic force microscopy(AFM) characterization of short- and long-chain DNA sequences havedetermined in detail the key factors controlling the distribution, size, andshape of highly ordered two- and three-dimensional DNA nanostructureson the electrode surface [4,11,13e20].

DNA adsorption was initially investigated on mercury [3,22,28], and lateron carbon electrodes [3,5,9]. At both electrodes, dsDNA presents smalleradsorption than ssDNA, since it is less flexible, and has the hydrophobicbase residues more protected inside the double helix. Electrochemicalcharacterization of the DNA adsorption process onto electrode surfaces wascombined with other analytical methods, such as ellipsometry and surfaceenhanced Raman spectroscopy [3], and now with AFM [4,11,13e20].

Long-chain dsDNA and ssDNA adsorb spontaneously at the surface ofcarbon electrodes, forming thin two-dimensional network films, whosecharacteristics are directly dependent on the DNA concentration and pH,Fig. 2A, B, E, and F. Applying a positive potential of wþ0.30 V,versus AgQRE to the carbon electrode surface, which is not sufficientlyhigh to oxidize the G and A residues on the dsDNA and ssDNA, morerobust and stable DNA films are formed, Fig. 2C, D, G, and H.

=-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------biosensor, TrAC Trends Anal. Chem. 79 (2016) 23e36, http://dx.doi.org/10.1016/j.trac.2016.01.019; A.-M. Chiorcea-Paquim, P.V. Santos, A.M. Oliveira-Brett, Atomic force microscopyand voltammetric characterisation of synthetic homo-oligodeoxynucleotides, Electrochim.Acta 110 (2013) 599e607, http://dx.doi.org/10.1016/j.electacta.2013.03.103 with permission.


http://dx.doi.org/10.1016/j.trac.2016.01.019


http://dx.doi.org/10.1016/j.electacta.2013.03.103

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

20 nA

8-oxoG

2,8-DHA

2 nA

pU

(B)

(C)

(D)

pCpTpApG

U

(A)

CT

GA

100 nA

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

ssDNA

dsDNA

10 nA

E / V vs. Ag/AgCl

Figure 1 DP voltammograms baseline corrected at GCE, in pH 7.0 solutions:(A, ▬) 20 mM equimolar mixture of guanine (G), adenine (A), thymine (T) and cytosine(C), and (A, nnn) 20 mM uracil (U), (B) 0.5 mM 8-oxoG and 2.8-DHA, (C) 40 mg/mLpoly(dG) and poly(dA), 100 mg/mL poly(dT) and poly(dC), 250 mg/mL poly(dU), and(D) 60 mg/mL ( ) dsDNA and ( ) ssDNA. Adapted from A.M. Oliveira-Brett, J.A.P.Piedade, L.A. Silva, V.C. Diculescu, Voltammetric determination of all DNA nucleotides,Anal. Biochem. 332 (2004) 321e329, http://dx.doi.org/10.1016/j.ab.2004.06.021; S.C.B.Oliveira, A.M. Oliveira-Brett, DNA-electrochemical biosensors: AFM surface characteri-sation and application to detection of in situ oxidative damage to DNA, Comb. Chem.High Throughput Screen. 13 (2010) 628e640 with permission.


http://dx.doi.org/10.1016/j.ab.2004.06.021

(1)

(2)

dsDNA

ssDNA

(B)(A)

(E) (F) (G) (H)

(C) (D)

Figure 2 AFM images of an HOPG electrode modified by: (1) (AeD) 60 mg/mL dsDNA (A and B) free adsorption in (A) pH 5.3 and (B) pH 7.0,(C and D) at applied potential ofþ0.30 V (vs. AgQRE) in (C) pH 5.3 and (D) pH 7.0; (2) (EeF) 5 mg/mL ssDNA (E and F) free adsorption in (E) pH 5.3and (F) pH 7.0, (G and H) at applied potential ofþ0.30 V (vs. AgQRE) in (G) pH 5.3 and (H) pH 7.0. Adapted from A.M. Oliveira Brett, A.-M. Chiorcea,Effect of pH and applied potential on the adsorption of DNA on highly oriented pyrolytic graphite electrodes. Atomic force microscopy surfacecharacterisation, Electrochem. Commun. 5 (2003) 178e183 with permission.

Applications

ofDNA-Electrochem

icalBiosensorsin

Cancer

Research295

Thin dsDNA adsorbed films that form network structures with holesexposing the electrode surface define different active surface areas on theDNA-electrochemical biosensor, Fig. 3A. The uncovered electrode surfacemay act as an array of microelectrodes with nanometer or micrometerdimensions [29]. In a thin network film of DNA-electrochemical biosensor,the incompletely covered electrode surface will allow the diffusion andnonspecific adsorption of the analyte molecules from the bulk solution tothe electrode surface, Fig. 3A-right, blue arrows. This can lead to twocontributions to the electrochemical signal, one from the simple adsorbedanalyte, and other from the damage caused by the analyte to the immobi-lized dsDNA, being difficult to distinguish between the two signals.

The DNA-electrochemical biosensors built with a thick multilayerimmobilized DNA film have the advantage, due to a complete surfacecoverage, that the undesired nonspecific adsorption of the analyte moleculesto the electrode surface is impossible, Fig. 3B [29], the sensor response will beonly determined by the interaction of the compound with the immobilized

THIN FILMdsDNA-ELECTROCHEMICAL BIOSENSOR

THICK FILMdsDNA-ELECTROCHEMICAL BIOSENSOR

(A)

(B)

Figure 3 (A and B) AFM three-dimensional images of a (A) thin film and a (B) thick filmdsDNA-electrochemical biosensor in pH 4.5, and schematic representation of theirinteraction with the analyte. Adapted from A.-M. Chiorcea, A.M. Oliveira Brett, Atomicforce microscopy characterization of an electrochemical DNA-biosensor, Bioelectrochemistry63 (2004) 229e932, http://dx.doi.org/10.1016/j.bioelechem.2003.09.029 with permission.


http://dx.doi.org/10.1016/j.bioelechem.2003.09.029

dsDNA on the electrode surface, without any electrochemical contributionfrom the analyte.

Short-chain oligodeoxyribonucleotides (ODNs) are used as recognitionelements in DNA-electrochemical biosensor devices. Adaptation of immo-bilization techniques to miniaturization allows the manufacture of DNAmicroarrays with several hundred thousand sequences per square centimetre.The sensor response is particularly sensitive to ODN’s small structural vari-ations, since base residues can easily associate in a variety of arrangements,Scheme 3C, different from the Watson-Crick base-pairs, Scheme 3B.

The adsorption morphology and the redox behaviour of the shorthomo-ODNs containing only one type of base, d(A)10, d(C)10, andd(T)10, were studied using AFM at highly oriented pyrolytic graphite(HOPG) and DP voltammetry at GCE, concerning their ability to formcomplex secondary structures and higher-order nanostructures, dependenton the incubation time, solution pH, and ODN concentration [12]. Theshort homo-ODNs, d(A)10, d(C)10, and d(T)10, are single stranded underphysiological pH, and the flexible single helixes aggregate and coil,adsorbing as network films with knobby appearance. In mild acid pHsolutions, d(A)10 double-helical conformations were observed by AFMas network films with lower surface coverage, and detected by DPvoltammetry by the occurrence of the A residues oxidation peak currentdecrease, whereas d(C)10 forms i-motifs, and were observed by AFM asspherical aggregates [12].

G-rich ODNs have great medical and nanotechnological potential,because they can self-assemble into GQs, Scheme 3D-middle, andhigher-order nanostructures. They are also considered important cancer-specific molecular targets for anticancer drugs, since the GQ stabilizationby small organic molecules can lead to telomerase inhibition and telomeredysfunction in cancer cells.

The electrochemical behaviour of DNA sequences to self-assembleinto GQ configurations has been studied [30e33]. The first report onthe electrochemical oxidation of GQs concerned the investigation oftwo different length thrombin-binding aptamer (TBA) sequences,d(G2T2G2TGTG2T2G2) and d(G3T2G3TGT3T2G3) [30]. The differentadsorption patterns and degree of surface coverage observed by AFMwere correlated with the sequence base composition, presence/absence ofKþ, and voltammetric behaviour observed by DP voltammetry. In theabsence of Kþ, in Naþ containing solutions, the formation of GQs isslow, and the oxidation of both TBA sequences showed one anodic peak,corresponding to the G residues oxidation in the TBA single strands. In


the presence of Kþ, both TBA sequences fold into GQs, and only a fewsingle-stranded sequences are observed on the HOPG surface, since noadsorption of the stable and rigid GQs occurs. DP voltammetry showedthe decrease of the G residues oxidation peak and the occurrence of anew GQ peak at a higher potential, corresponding to the G residuesoxidation in the GQs. The GQ higher oxidation potential is due to thegreater difficulty of electron transfer from the inside of the GQ structureto the electrode surface than the electron transfer from the G residues inthe more flexible single strands.

The redox behaviour and adsorption of the d(G)10, d(TG9), andd(TG8T) ODN sequences that fold into parallel tetramolecular GQstructures, Scheme 3D-middle, were studied by AFM and DP voltammetryat carbon electrodes [32e34]. The results demonstrated that GQ formationis directly influenced by the ODN sequence and concentration, pH, andpresence of monovalent cations (Naþ vs. Kþ). DP voltammetry allowedthe detection of the ODN single strands folding into GQs and G-basednanostructures, in freshly prepared solutions, at concentrations 10 timeslower than usually detected using other techniques currently employed tostudy the formation of GQs. Single-stranded ODNs, in Naþ containingsolutions and for short incubation times, were detected using AFM asnetwork films and polymeric structures and using DP voltammetry by theoccurrence of only the G residues oxidation peak (Gr), Fig. 4. GQ structures,in Naþ containing solutions and for long incubation times or in Kþ contain-ing solutions, were detected using AFM as spherical aggregates and using DPvoltammetry by the decrease of the G residues oxidation peak and the GQoxidation peak occurrence, increase, and shift to more positive potentials, ina time-dependent manner, Fig. 4 [12,34]. Concerning the self-assemblinginto higher-order nanostructures, the homo-ODN sequence d(G)10 wasthe only sequence forming G-nanowires observed using AFM, Fig. 4C,d(TG9) forms short G-based super structures that adsorbed as rod-shapedaggregates, and d(TG8T) forms no nanostructures, due to the presence ofT residues at both 50 and 30 ends [34].

Thed(TG4T) telomeric repeat sequence, of the free-living ciliate protozoaTetrahymena, forms tetramolecular GQ structures that are considered simplermodels of biologically relevant human quadruplexes, being used to obtainhigh resolution data on drug-DNA interactions [35]. The well-knownconformation of the d(TG4T) GQs, and their extraordinary stiffness, enabledthe d(TG4T) molecules to be considered good building block candidatesfor the development of novel devices, with medical and nanotechnology


applications. The transformation of the d(TG4T) sequence from single strandinto GQ configuration, influenced by the Naþ and Kþ ions concentration,was successfully detected using AFM on HOPG and DP voltammetry atGCE [35]. The d(TG4T) GQs self-assembled very fast in Kþ ions solutions,and slowly in Naþ ions solutions, revealing a time and a Kþ ions concentra-tion-dependent adsorption process and redox behaviour. The optimum Kþ

ions concentration for the formation of d(TG4T) GQs was similar to the

0.8 0.9 1.0 1.1

GQ

Gr5 nA

E / V (vs. Ag/AgCl)0.7 0.8 0.9 1.0 1.1

GQ

Gr

5 nA

E / V (vs. Ag/AgCl)0.8 0.9 1.0 1.1

GQ

Gr

5 nA

E / V (vs. Ag/AgCl)

50nm 50nm 50nm 50nm50nmd(G)10, without K+ d(G)10, 100 mM K+ d(G)10, 200 mM K+

N

NHN

NO

N

N

N NH

NO

N

NN

NHN

O

N

NN

HN N

O

NH H

H

H

HH

HHH

HH

HN

NHN

NO

N HH

H

(A)

(C)

(B) K+

ions concentration dependenceIncubation time dependence

0 h 14 days 24 h 24 h

Figure 4 (A and B) DP voltammograms baseline corrected in 3.0 mM d(G)10, in pH 7.0:(A) Incubation time dependence e in the absence of Kþ ions (nCn) 0, (···) 24,(nnn) 48 h and (▬) 14 days incubation, and in the presence of 1 mM Kþ ions ( ) 0and ( ) 24 h incubation; (B) Kþ ions concentration dependence, 0 h incubation(•••) in the absence and in the presence of (left, ▬) 100 mM, (left, nnn) 5 mM, ( ),100 mM, (right, nCn) 200 mM, (right, ···) 500 mM and (right, ▬) 1 M Kþ ions;(C) AFM images of 0.3 mM d(G)10, onto HOPG in the absence and in the presenceof 100 mM and 200 mM Kþ ions, for different incubation times. Adapted from A.-M.Chiorcea-Paquim, P.V. Santos, R. Eritja, A.M. Oliveira-Brett, Self-assembled G-quadruplexnanostructures: AFM and voltammetric characterization, Phys. Chem. Chem. Phys. 15(2013) 9117e9124, http://dx.doi.org/10.1039/c3cp50866h with permission.


http://dx.doi.org/10.1039/c3cp50866h

healthy cells’ intracellular Kþ ions concentration. The d(TG4T) higher-ordernanostructures self-assembled slowly in Naþ ion solutions and were detectedby AFM as short nanowires and nanostructured films. The absence of higher-order nanostructures in Kþ ion solutions showed that the rapid formation ofstable GQ structures induced by the Kþ ions is relevant for the good functionof cells.

Long-chain polynucleotides poly(dG) and poly(G) are widely prevalentin the human and other genomes at both DNA and RNA levels and are usedin DNA-electrochemical biosensor, as models to determine the preferentialinteraction of drugs with G-rich segments of DNA. AFM at HOPG and DPvoltammetric studies at GCE, showed that the poly(G) single strands self-assemble into short GQ regions for short incubation times, while largepoly(G)-GQ aggregates with low adsorption are formed after long incuba-tion times, in the presence of monovalent Naþ or Kþ ions, Fig. 5AeC [36].DP voltammograms in freshly prepared poly(G) solutions showed only theG residues oxidation peak (Gr), Fig. 5D, due to the G residues oxidation inthe poly(G) single strand. Increasing the incubation time, the G residuesoxidation peak decreases and disappears, and a GQ oxidation peak in thepoly(G)-GQ morphology appears, at a higher oxidation potential,dependent on the incubation time, presenting a maximum after 10 days in-cubation, and reaching a steady value after w17 days incubation, Fig. 5D.

The bottom-up immobilization of DNA sequences that self-assemble ineither double- and single-stranded form, or in unusual but biologicallyrelevant configurations, such as GQs and i-motifs, and large nanostructuresallows different applications. Long chains dsDNA, ssDNA, and thepurine homo-polynucleotide poly(A) and poly(G), as well as short-chainODNs in single-stranded, double-stranded and GQ configurations, weresuccessfully used for screening cancer therapeutic agents, cancer biomarkers,and hazard compounds.

In DNA-electrochemical biosensors applications, the target analyteinteracts with the DNA recognition layer immobilized at the electrodesurface, Scheme 2B-left, inducing morphological changes in the dsDNAhelical structure, Scheme 2B-middle. The sensing strategy of label-freeDNA-electrochemical biosensors consists in the electrochemical detectionof the difference between the intact dsDNA, Scheme 2A-right, and theDNA damaged structure, after the interaction with the analyte, Scheme2B-right.

When the DNA interaction with the chemical compound in the cellinduce oxidation of G and A residues in the dsDNA, the formation of


8-oxoG or 2,8-DHA causes important transversion mutagenic lesions, andcan be the starting point for cellular dysfunction, which in turn could leadto a state of illness [25,26]. Therefore, the DP voltammetric detectionof 8-oxoG and 2,8-DHA biomarkers of DNA bases oxidative stressusing DNA-electrochemical biosensor, Scheme 2B-right, allows the directdetection of DNA oxidative damage after interaction with the analyte.

0.7 0.8 0.9 1.0 1.1

10nA

GQGr

10 days

21 days24 h

0 h

E / V (vs. Ag/AgCl)

d(G)10

50nm

24 h

50nm

0 h(A) (B)

50nm

21 days(C) (D)

(E) (F) (G)

Figure 5 (AeC) AFM images of onto HOPG, of 5 mg/mL poly(G), in the presence of Kþ

ions after: (A) 0 h, (B) 24 h, and (C) 21 days incubation; (D) DP voltammogramsbaseline corrected in 100 mg/mL poly(G), in pH 7.0, in the presence of Kþ ions after:( ) 0 h, (▬) 24 h, (nnn) 10 days and ( ) 21 days; and (nCn) 3 mM d(G)10 in thepresence of Kþ ions after 24 h incubation; (EeG) Schematic representation ofthe poly(G) adsorption process: (E) poly(G) single strand, (F) poly(G) single strandwith short GQ regions and (G) poly(G) single strand with larger GQ regions.Reproduced from A.-M. Chiorcea-Paquim, A.D.R. Pontinha, A.M. Oliveira-Brett, Time-dependent polyguanylic acid structural modifications, Electrochem. Commun. 45 (2014)71e74, http://dx.doi.org/10.1016/j.elecom.2014.05.019 with permission.


http://dx.doi.org/10.1016/j.elecom.2014.05.019

3. DNA-ELECTROCHEMICAL BIOSENSOR INEVALUATION OF CANCER TREATMENT DRUGS

A variety of new biopharmaceutical drugs have been developed in thelast years, to bind and interact strongly with dsDNA, providing newtherapeutic opportunities for cancer and many inherited diseases. The under-standing of the mechanisms involved in the drug-dsDNA specific interactionis essential to comprehend the toxicity, as well as chemotherapeutic effects,of those compounds [37,38]. DNA-electrochemical biosensors are importanttools for the detection and evaluation of pharmaceutical compounds’ specificbinding to nucleic acids, due to their high sensitivity for the detection ofsmall perturbations of DNA morphology. The DNA-electrochemicalbiosensor use in the analysis of drugs, medicines, and other analytes of interestin the pharmaceutical area have received increased attention.

3.1 Monoclonal antibodiesImmunotherapy is a treatment that uses the patient body’s own immunesystem to help fight cancer, being considered one of the most promisingstrategies for the treatment of specific types of cancer. Monoclonal antibodies(mAb) recognize and attach to specific proteins produced by cells. Severaldifferent mAbs are successfully used in clinical treatments, and some newertypes are in clinical trials. In the light of the mAbs’ advances, the study of theinteraction between mAbs and dsDNA is of utmost importance to predict itsaction mechanism and to understand its toxicity.

Rituximab (RTX) is a human/murine chimeric mAb that specifi-cally targets the transmembrane protein CD20 of B-cells. The oxidationmechanism of native and denatured RTXwas investigated by DP voltammetryat GCE. Native RTX presented only one oxidation peak of tyrosine andtryptophan residues, whereas in denatured RTX were detected three peakscorresponding to the oxidation of tyrosine, tryptophan, and histidine residues[39]. The dsDNA-RTX interaction was investigated by DP voltammetry inincubated samples and using a multilayer dsDNA-electrochemical biosensor,and by gel electrophoresis [40]. The voltammetric study showed a strongcondensation of the DNA double-helical structure promoted by thedsDNA-RTX interaction, detected by the decrease and disappearance of theA residues oxidation peak current, the decrease of the G residues oxidationpeak current, and the occurrence of free G and A bases oxidation peaks, butno DNA base oxidative damage was detected.


Bevacizumab (BEVA) is a mAb used in clinical oncology to treat severaltypes of cancer, such as colon, lung, kidney, ovarian, and brain cancers. Theelectrochemical oxidation of native and denatured BEVA was investigatedin solution over a wide pH range and using BEVA-thin film voltammetryon a GCE [41]. For native BEVA, only one pH-dependent oxidation peak,corresponding to tyrosine and tryptophan amino acid residues oxidation,was observed. The unfolding of BEVA-thin film on GCE surface afterdenaturation with chemical agents enabled the exposure of more electroac-tive amino acid residues to the electrode surface, and additional peaks, dueto cysteine and histidine amino acid residues oxidation were observed [41].The interaction of BEVA with dsDNA was investigated by DP voltamme-try and gel electrophoresis in incubated solutions and using the dsDNA-electrochemical biosensor [42]. The voltammetric results showed thedecrease/disappearance of the G and A residues oxidation peaks after incu-bation, Fig. 6, indicating that the BEVA-dsDNA interaction leads to theformation of a complex adduct that prevents the interaction of DNA purinebases with the electrode surface. For long incubation times the occurrenceof the free G base oxidation peak was observed, but no oxidative damageDNA was electrochemically detected [42].

3.2 Nucleoside analoguesNucleoside analogues represent a major class of chemotherapeutic agents forthe treatment of cancer, especially leukaemia. Because of their structuralsimilarity with the natural nucleosides, these analogues are taken up by cells,turned into their triphosphate species, and incorporated into DNA or RNA.

The redox behaviour of the adenosine analogues clofarabine (CLF) [43]and cladribine (CLD) [44] was investigated at GCE using cyclic, DP, andsquare wave (SW) voltammetry, in different pH supporting electrolytes.The CLF and CLD oxidation are irreversible and pH-dependent processes,which occur with the transfer of two protons and two electrons, followingdiffusion-controlled mechanisms. The in situ evaluation of the DNA inter-action with CLF, Fig. 7A [43], and CLD [44], using a dsDNA-electrochemicalbiosensor, was investigated. The interaction with both purine nucleosideanalogues caused dsDNA structural modifications in a time-dependentmanner, but no DNA oxidative damage was observed. The results wereconfirmed using poly(G)- and poly(A)-electrochemical biosensors.

The electrochemical behaviour of the cytosine nucleosideanalogue gemcitabine (GEM) was investigated and no electrochemical pro-cess was observed [45]. The interaction of GEMwith DNA was investigated


(A)

(B)

(C)

Figure 6 DP voltammograms baseline corrected at GCE, in 100 mg/mL dsDNA, in pH7.0: ( ) before and (A) after incubation with 100 mg/mL BEVA during (▬) 0 and (···)48 h, and (B and C) after incubation with (···) 10 mg/mL and (▬) 500 mg/mL BEVAduring (B) 0 and (C) 48 h. Reproduced from L.I.N. Tomé, N.V. Marques, V.C. Diculescu,A.M. Oliveira-Brett, In situ dsDNA-bevacizumab anticancer monoclonal antibody interac-tion electrochemical evaluation, Anal. Chim. Acta. 898 (2015) 28e33, http://dx.doi.org/10.1016/j.aca.2015.09.049 with permission.


http://dx.doi.org/10.1016/j.aca.2015.09.049


0.3 0.5 0.7 0.9 1.1 1.3 1.5

CLF10 nA

E / V (vs. Ag/AgCl)

0.4 0.6 0.8 1.0 1.2 1.4

E / V (vs. Ag/AgCl)

10 nA

(A)

(B)

Gr

Gr

Ar

Ar

G

Figure 7 DP voltammograms baseline corrected, at a dsDNA-electrochemicalbiosensor, in pH 4.5 (···) before and after incubation with: (A) 100 mM CLF, during(▬) 5 and ( ) 15 min, and (B) 10 mM GEM, during (▬) 15 min and ( ) 4 h. Adaptedfrom H.E. Satana, A.D.R. Pontinha, V.C. Diculescu, A.M. Oliveira-Brett, Nucleoside analogueelectrochemical behaviour and in situ evaluation of DNAeclofarabine interaction,Bioelectrochemistry 87 (2012) 3e8,http://dx.doi.org/10.1016/j.bioelechem.2011.07.004;R.M. Buoro, I.C. Lopes, V.C. Diculescu, S.H.P. Serrano, L. Lemos, A.M. Oliveira-Brett, In situevaluation of gemcitabineeDNA interaction using a DNA-electrochemical biosensor,Bioelectrochemistry 99 (2014) 40e45, http://dx.doi.org/10.1016/j.bioelechem.2014.05.005 with permission.





in incubated solutions and at a DNA-electrochemical biosensor, and showedmodifications of the DNA morphological structure, but no oxidative dam-age to DNA was observed, Fig. 7B. The interaction mechanism occurred intwo consecutive steps. The initial process was independent of the DNAsequence and led to the condensation/aggregation of dsDNA. The forma-tion of a GEM-DNA rigid structure promoted a second step, favouringthe interaction between the G residues and the fluorine atoms in theGEM ribose moiety and provoking the release of G bases detected at theelectrode surface.

3.3 Kinase inhibitorsSmall molecules able to target and inhibit protein kinases, enzymesresponsible for phosphorylation processes, have been recognized to beefficient in anticancer therapy. In vitro studies demonstrated that kinaseinhibitors and/or their metabolites can increase the amount of DNAdamage, and showed that they act through intercalation into DNA orformation of alkali-labile sites and/or DNA strand breaks [46]. The inter-action between different kinases inhibitors and dsDNA, using label-freeelectrochemical biosensors, has been investigated [46e48].

Imatinib is a relatively small molecule with activity against the proteintyrosine kinase, a protein expressed by all patients with chronic myelogenousleukaemia, which is currently undergoing extensive evaluation of its activityagainst other types of tumour [49,50]. The interaction of DNA withimatinib in bulk solution and at a dsDNA-electrochemical biosensor wasinvestigated [46]. The imatinib-dsDNA interaction led to modifications inthe dsDNA structure, detected by changes of the G and A residues oxidationpeaks, Fig. 8A, and the imatinib redox product, the electrochemicalgenerated metabolite, caused DNA oxidative damage. Using poly(A)- andpoly(G)-electrochemical biosensors, it was proved that the interactionbetween imatinib and DNA takes place preferentially at the A-richsegments, and an imatinib-dsDNA interaction mechanism was proposed.The electrochemical generation of imatinib oxidation product inside theDNA double helix leads to A residues oxidation and the electrochemicaldetection of 2,8-DHA, marker of dsDNA oxidative damage [46].

Danusertib is an Aurora kinases inhibitor, which also binds and inhibitswith high affinity other tyrosine kinases, which makes danusertib aneffective drug for the treatment of multiple tumours. Danusertib interactionwith dsDNA was studied in solution and using a dsDNA-electrochemical


(A)

(B)

Figure 8 DP voltammograms baseline corrected, in pH 4.5, at a dsDNA-electrochemicalbiosensor, after incubation with: (A) 5 mM imatinib, during 2 min, (▬) without and ( )with þ0.90 V conditioning potential during 2 min, and (B) 10 mM danusertib, during60 min, (▬) without and ( ) with þ0.85 V conditioning potential during 30 min.Adapted from V.C. Diculescu, M. Vivan, A.M.O. Brett, Voltammetric behavior of anti-leukemia drug glivec. Part III: in situ DNA oxidative damage by the glivec electrochemicalmetabolite, Electroanalysis 18 (2006) 1963e1970, http://dx.doi.org/10.1002/elan.200603602; V.C. Diculescu, A.M. Oliveira-Brett, In situ electrochemical evaluation ofdsDNA interaction with the anticancer drug danusertib nitrenium radical product using theDNA-electrochemical biosensor, Bioelectrochemistry 107 (2016) 50e57, http://dx.doi.org/10.1016/j.bioelechem.2015.10.004 with permission.


http://dx.doi.org/10.1002/elan.200603602




biosensor, Fig. 8B [47]. The dsDNA-danusertib interaction occurs in twosequential steps: first, the danusertib’s positively charged piperazine moietyis bound electrostatically to the dsDNA phosphate backbone; then, in asecond step, a more stable danusertib-DNA complex is formed, that involvesthe danusertib pyrrolo-pyrazole moiety, which causes small morphologicalmodifications in the DNA double helix, detected by DP voltammetry bychanges of the G and A residues oxidation peaks and confirmed by electro-phoretic and spectrophotometric measurements [47].

Controlling the applied potential to the dsDNA-electrochemical biosensorsurface, the danusertib amino group oxidation enabled the in situ electrochem-ical generation of nitrenium cation radical, and the study of its interaction withdsDNA,Fig. 8B [47]. The decrease of G residues oxidation peak was in agree-ment with the covalent attachment of the danusertib nitrenium cation radicalredox metabolite, to the C8 position of the G residues, protecting them andpreventing their oxidation. Using homo-polynucleotides of known sequenceit was confirmed that the danusertib-dsDNA interaction takes place preferen-tially at G-rich segments, an interaction mechanism was proposed, and theformation of danusertib redox metabolite-G adduct formation was explained[47].

Lapatinib (LPT) is a dual kinase inhibitor used for breast cancer and othersolid tumours. The electrochemical oxidation mechanism of LPT was studiedin detail by cyclic, DP, and SW voltammetry, in the pH range 0.30e10.00[48]. LPT exhibits three anodic peaks, corresponding to the transfer of twoelectrons for each peak. The oxidation mechanism of the first anodic peakwas associated with the (2-methylsulfonylethylamino)methyl oxidation toalcohol, and the second anodic peak with the 4-methoxyaniline oxidationto quinone imine. The dsDNA-LPT interaction was studied using a DNA-electrochemical biosensor, and by spectroscopic techniques [48]. Based onelectrochemical and spectroscopic methods, it was confirmed that LPT inter-calates into the DNA double helix.

3.4 AntimetabolitesAntimetabolites are a group of drugs that became important for cancerchemotherapy, being highly effective in targeting and inhibiting theenzymes involved in malignant cells lines. Methotrexate (MTX) is an anti-metabolite of folic acid that targets the enzyme dehydrofolate reductase,which plays a supporting but essential role for the synthesis of thyminenucleotide. MTX is used in the treatment of many neoplasms, such as acuteleukaemia, head and neck cancer, and micrometastases of osteosarcoma,


being also used as alternative treatment for psoriasis and rheumatoid arthritisresistant to conventional therapies. The MTX-dsDNA interaction was stud-ied by DP voltammetry at GCE and AFM at HOPG, Fig. 9 [51], and themechanism of interaction in incubated solutions was explained taking intoconsideration the correlation between the redox behaviour and morpholog-ical modifications. AFM images showed reorganization of the immobilizedDNA on the surface of HOPG after incubation with MTX, and theformation of a more densely packed and thicker MTX-DNA lattice. Inagreement with the AFM, the voltammetric data showed that structuraldsDNA modifications occur in a time-dependent manner. The decreaseof dsDNA oxidation peaks observed at short incubation times was consistentwith a mechanism that involves condensation and formation of more rigidstructures due to the intercalation of MTX into DNA strands. ThedsDNA-electrochemical biosensor enabled in situ monitoring of both

0.60.8

1.01.2

1.4

MTX

Gr

t / hours

72

240

10 nA

E / V vs. A

g/AgCl

Ar

(A) (B)

50nm 50nm

(C)

Figure 9 (A and B) AFM images at HOPG: (A) 10 mg/mL dsDNA and (B) 10 mg/mL dsDNAincubated with 0.5 mM MTX, during 24 h (C) DP voltammograms, baseline corrected atGCE in solutions in pH 4.5 of ( ) 100 mg/mL dsDNA and (▬) 100 mg/mL dsDNAincubated with 5 mM MTX, during 0, 24 and 72 h. Adapted from A.D.R. Pontinha,S.M.A. Jorge, A.-M. Chiorcea Paquim, V.C. Diculescu, A.M. Oliveira-Brett, P.T.R. Rajagopalan,et al., In situ evaluation of anticancer drug methotrexateeDNA interaction using a DNA-electrochemical biosensor and AFM characterization, Phys. Chem. Chem. Phys. 13 (2011)5227, http://dx.doi.org/10.1039/c0cp02377a with permission.


http://dx.doi.org/10.1039/c0cp02377a

DNA and MTX oxidation peaks, showing that the dsDNA bending processimposes kinking of the strands, which facilitates the intercalation of MTX,causing unwinding of the dsDNA structure and the purinic bases’ exposureto the GCE surface. The preferred affinity of MTX to A-rich segments,explained by the weakness of the H-bonds between AeT, was confirmedusing single-stranded poly(A)- and poly(G)-electrochemical biosensors [51].

3.5 G-quadruplex ligandsAnother class of anticancer drugs is represented by small ligand moleculesthat are specifically designed to bind to G-rich DNA sequences, at theends of chromosomes. Their ability to induce and stabilize the DNA foldinginto four-stranded GQ configurations at the level of telomeres prevent thetelomeric DNA from unwinding and opening to telomerase, thus indirectlytargeting the telomerase and inhibiting its catalytic activity, which furtherleads to the senescence and apoptosis of tumour cells. Remarkable progresshas been made in the development of selective GQ ligands; some entered inclinical trials for cancer therapy, presenting significant telomerase inhibitionor suppression of the transcription activity of oncogenes. The trisubstitutedacridine compound BRACO-19 has been developed as a ligand forstabilizing GQ structures that produce short- and long-term growth arrestin cancer cell lines. However, BRACO-19 revealed to be relatively nonGQ-selective, having also significant binding affinity for duplex DNA.Therefore, a series of new triazole-linked acridine ligands, for example,GL15 and GL7, with enhanced selectivity for human telomeric GQ bindingversus duplex DNA binding has been designed, synthesized, and evaluated.BRACO-19, GL15, and GL7 present a complex, pH-dependent, andadsorption-controlled irreversible oxidation mechanism, at a GCE [52,53].The interaction between DNA and the acridine ligands GL15 and GL7was investigated in incubated solutions and using dsDNA-, poly(G)-, andpoly(A)-electrochemical biosensors [53]. Both GL15 and GL7 interactwith dsDNA in a time-dependent manner, causing condensation of thedsDNA morphological structure, with preferential affinity for The G-richsegments, but do not cause DNA oxidative damage.

The interactions of the GQ-targeting triazole-linked acridine ligand GL15with the short-chain length Tetrahymena telomeric DNA repeat sequenced(TG4T) and with the long poly(G) sequence have been reported at the sin-gle-molecule level [54]. In the presence of GL15, GQ formation was detectedby AFM via the adsorption of GL15-d(TG4T)-GQ and GL15-poly(G)-GQsmall spherical aggregates, and large GL15-poly(G)-GQ assemblies, and by


DP voltammetry via GL15 and Gr oxidation peak current decrease and disap-pearance, and the occurrence of a GQ oxidation peak, Fig. 10. The AFM andvoltammetric results showed that the GL15 molecule interacts with bothsequences in a time-dependent manner. An excellent correlation wasobserved between the d(TG4T) and poly(G) structural changes and redoxbehaviour, before and after interaction with GL15, and was directly influ-enced by the presence of monovalent Naþ or Kþ ions in solution. Theseresults are consistent with the interaction of triazole-linked acridine deriva-tives with terminal G-quartets in an individual GQ, Fig. 10A-right. The

(A)

(B)

0.6 0.7 0.8 0.9 1.0 1.1

0day28days35days42days

GL15

GQ

Gr

E / V (vs. Ag/AgCl)

0.6 0.7 0.8 0.9 1.0 1.1

0day3days28days42days

GL15

GQGr

E / V (vs. Ag/AgCl)

In the presence of Na+ ions

In the presence of K+ ions

Figure 10 DP voltammograms baseline corrected at GCE, in solutions of 3.0 mMd(TG4T)incubated with 4.0 mM GL15, in pH 7.0, for a range of incubation times, in the presence:(A) Naþ and (B) Kþ ions. Adapted from A.-M. Chiorcea-Paquim, A.D.R. Pontinha, R. Eritja, G.Lucarelli, S. Sparapani, S. Neidle, et al., Atomic force microscopy and voltammetricinvestigation of quadruplex formation between a triazole-acridine conjugate and guanine-containing repeat DNA sequences, Anal. Chem. 87 (2015) 6141e6149. http://dx.doi.org/10.1021/acs.analchem.5b00743 with permission.


http://dx.doi.org/10.1021/acs.analchem.5b00743


binding of GL15 to d(TG4T) and poly(G) both strongly stabilized the GQsand accelerated GQ formation, in both Naþ and Kþ ions solutions, althoughonly the Kþ-containing solution promoted the formation of perfectly alignedtetramolecular GQs [54].

3.6 AntibioticsAnthracycline antibiotics are a class of anticancer drugs, originally derivedfrom Streptomyces bacterium, which are active against a wide variety of solidtumours and haematological malignancies [55]. However, their use isconsiderably limited due to tumour resistance and their toxicity to healthytissue, especially cardiotoxicity [56]. Daunorubicin was the first anthracy-cline compound to be characterized. Doxorubicin, also known by the tradename Adriamycin, is a hydroxyl derivative of daunorubicin, being one of themost widely used chemotherapeutic agents, generally prescribed in combi-nation with other drugs. It is approved for a long list of cancer types, beingone of the most effective drugs for solid tumour treatment (breast cancer,small cell lung and ovarian cancer) [56e58]. Doxorubicin intercalationand in situ interaction with dsDNA were investigated using a DNA-electro-chemical biosensor [56,57]. Interaction of doxorubicin with DNA is depen-dent on the applied potential. The reduction of DNA intercalateddoxorubicin leads to the formation of a doxorubicin radical that oxidativelydamages DNA and the formation of biomarker 8-oxoG. A mechanism fordoxorubicin reduction and oxidation in situ, when intercalated into thedouble helix, was presented and the formation of the mutagenic 8-oxoGexplained [56,57].

Idarubicin (IDA) is a synthetic analogue of daunorubicin, presenting animproved activity for different types of leukaemia. IDA oxidation showedan irreversible, pH-dependent process that occurs with the transfer of oneelectron and one proton, by cyclic and DP voltammetry, at GCE [59]. Themechanism of interaction of IDA with dsDNA was determined in incu-bated solutions and using multilayer dsDNA and poly(A) and poly(G)-electrochemical biosensors. Intercalation of IDA in the DNA double helixwas detected by the decrease of the G and A residues oxidation peak cur-rents with increasing incubation time. However, no oxidation peaks of thepurine base oxidation products, 8-oxoG and 2,8-DHA, were observed.

3.7 ThalidomideThalidomide (TD) is an immunomodulatory, antineoplastic, and leprostaticagent. Initially used as a sedative and an anti-emetic drug to combat morning


sickness, it was banned from clinical use in the 1960s due to its severeteratogenic effects. TD came back to be used initially to treat erythemanodosum leprosum, and then various cancers, particularly multiplemyeloma. Voltammetric, AFM, UV-vis and electrophoresis studies showedthat TD interacts specifically with the dsDNA by intercalation, inducingdsDNA structural changes in a time-dependent manner [60]. TD has affinityfor both G and A residues, and the interaction between TD and dsDNAinduced major changes in the oxidation peaks of both TD, and G and Aresidues, directly dependent on the dsDNA and TD concentrations andincubation time. Oxidative damage caused to DNA by TD was detectedelectrochemically by the occurrence of the 8-oxoGua and/or 2,8-DHAoxidation peak. Multiple time- and concentration-dependent effects onthe dsDNA structure are induced by TD. Initially, the aggregation anddisappearance of the G and A residues oxidation peaks is related to theTD induced DNA condensation. Afterwards, the formation of thinnerTD-DNA films, and the increase of the G and A residues oxidation peaks,with increasing the incubation time, is related to the TD intercalation intothe DNA, followed by the double helix unwinding.

3.8 Metal complexesThe discovery of the Pt(II) complex cisplatin was considered a significantachievement in cancer therapy [61], cisplatin being widely used to treat avariety of tumours including ovarian, cervical, head and neck, and testicularcancers. However, its clinical use decreased over time, due to its toxicity andacquired resistance, which triggered the interest in the development of moreefficient Pt(II)- and other metal-containing complexes [61].

As alternatives to first-generation agents such as cisplatin, the developmentof new chemotherapeutic agents led to the synthesis of polynuclear metalcomplexes, a new class of third-generation anticancer agents with specificchemical and biological properties. In particular, Pd(II) complexes aimedto achieve lower toxicity, enhanced solubility, and tumour selectivity,when compared with the Pt(II) ones. The interaction with dsDNA oftwo polynuclear Pd(II) chelates with the biogenic polyamines spermidine(Spd) and spermine (Spm) was studied at room temperature, using AFM,DP voltammetry, and gel electrophoresis [62]. After the interaction withPd(II)-Spd and Pd(II)-Spm, Fig. 11, a reorganization of the DNA self-assembled network on the surface of the HOPG and a decrease in G andA residues oxidation peaks at GCE were observed, consistent with themodel mechanism of interaction of Pd(II) and Pt(II) complexes with dsDNA


that leads to the formation of Pd(II)-DNA adducts and/or aggregates. ThePd(II)-Spd complex presents a stronger interaction with dsDNA, owing toits molecular structure comprising three Pd(II) centres and two Spd mole-cules, with six possible coordination sites to dsDNA, as compared toPd(II)-Spm that contains only two Pd(II) ions and one Spm ligand, yieldingonly four possible coordination sites. Furthermore, the voltammetric resultsshowed that the interaction with either of the Pd(II) polyamine complexescaused no oxidative damage to dsDNA. Due to their polycationic chains,the polyamines Spd and Spm interact specifically with the negativelycharged phosphate groups of the DNA double helix by electrostatic forces,thus stabilizing its structure. The voltammetric results showed that the inter-action is observed even at polyamines’ low concentration, but causing nooxidative damage to DNA [62].

50nm100nm

0.2 0.4 0.6 0.8 1.0 1.2 1.4

5 nA

E / V vs. Ag/AgCl

Gr

Ar

G

(A)

(C)

(B)

Figure 11 (A and B) AFM images at HOPG modified by: (A) 10 mg/mL dsDNA, and(B) 10 mg/mL dsDNA incubated with 50 mM Pd(II)-Spm, during 24 h (C) DPvoltammograms baseline corrected at GCE modified by: (▬) 10 mg/mL dsDNA and10 mg/mL dsDNA incubated with 100 mM Pd(II)-Spm, in pH 4.5, during (nnn) 10and (···) 30 min. Adapted from O. Corduneanu, A.-M. Chiorcea-Paquim, V. Diculescu,S.M. Fiuza, M.P.M. Marques, A.M. Oliveira-Brett, DNA interaction with palladium chelatesof biogenic polyamines using atomic force microscopy and voltammetric characteriza-tion, Anal. Chem. 82 (2010) 1245e1252, http://dx.doi.org/10.1021/ac902127d withpermission.


http://dx.doi.org/10.1021/ac902127d

The lipoic acid-palladium (LAPd) complex was originally designed as anon-toxic chemotherapeutic agent, in a prescription version called DNAReductase, and consists of a palladium bonded to both end-groups of alipoic acid (LA). The interaction of dsDNA with LAPd and Poly-MVA, adietary supplement containing LAPd polymer that exists as trimers ofLAPd complex joined to thiamine was studied at room temperature, usingAFM and DP voltammetry [63]. The DP voltammetric results demonstratedthe interaction of both LAPd and Poly-MVAwith dsDNA, but no oxidativedamage caused to dsDNA was detected. The interaction of dsDNA withlow concentrations of LAPd complex leads to less knotted and bended,and more extended, dsDNA molecules, when compared with controldsDNA adsorbed on the HOPG surface. The LAPd molecules interactand adsorb strongly on HOPG, in comparison with LA, probably due tothe incorporation of palladium into the ligand structure. The stability ofboth the LAPd complex and the LAPd-containing Poly-MVA solution,after applying high negative or high positive potentials, was tested. TheDP voltammetry and AFM showed that, while in the case of LAPd complexpalladium removal is still possible, in the Poly-MVA solution the same wasnot achieved [63].

3.9 Natural compoundsNaphthoquinones are secondary metabolites of some fungi, plants, and bac-teria, with antibiotic, antiviral, antifungal, antiparasitic, anti-inflammatory,antiproliferative, and cytotoxic activity. Biflorin is an ortho-naphthoquinoneisolated from Capraria biflora, a perennial shrub distributed in countries oftropical America, that is used as cytotoxic and antioxidant agent. Biflorinshowed a cytotoxic activity against different tumour cell lines, demon-strating a good antitumour therapeutic potential, especially for skin, breast,and colon cancer cells. The pharmacokinetics of biflorin was evaluated byelectrochemistry and spectrophotometry [64], showing that biflorin interactswith dsDNA by intercalation, and also with ssDNA, which may lead toDNA damage. The effects of biflorin-dsDNA interaction were addressedthrough a molecular cytogenetic approach, using the comet assay and thechromosome aberration induction evaluation. Biflorin, compared to thenegative control, presented approximately four- and six-fold increase inDNA damage. Also, like other naphthoquinones such as b-lapachone andlapachol, biflorin presents antitumour and cytotoxic activities, but lacksmutagenic potential. The absence of either clastogenic or aneuploidogenicactivity of the compound reinforced its safety.


Naphthoquinone-based complexes with metal ions may present highercytotoxic properties in comparison with naphthoquinone itself or withmetal ions. Lawsone is a derivate of 1,4-napthoquinone with significantcytotoxic properties, demonstrated on different cell lines, due to its abilityto induce production of reactive oxygen species (ROS). Therefore, ternarylawsone-copper(II) complexes are a very interesting group of compoundsin cancer research [65]. The voltammetric behaviour of seven ternary cop-per(II) complexes of lawsone with additional O-donor (water) and N-donorligands (pyridine, 2-, 3-, and 4-aminopyridine, 3-hydroxypyridine, and3,5-dimethylpyrazole) was studied by cyclic and DP voltammetry [65].Their interaction with DNA was investigated using a DNA-electrochemicalbiosensor prepared on carbon paste electrodes. All lawsone complexesshowed ability to interact with dsDNA. The most simple complex isCu(lawsone)2(H2O)2$0.5H2O, with significant prooxidant properties,which may contribute to its cytotoxicity.

Flavonoids are naturally occurring polyphenolic compounds withantioxidant activity that, depending on the concentration, can also act asprooxidants, to produce free radicals and cause DNA damage andmutagenesis. The prooxidant activity is generally catalyzed by metals, suchas Fe and Cu, present in biological systems. Knowledge about the malignanttissue-specific anticancer effects of flavonoids can be applied in chemopre-vention, as well as in cancer treatment [66].

Quercetin exhibits several anticancer properties, such as cell signalling,pro-apoptotic, antiproliferative, and antioxidant effects, and growth suppres-sion. On the other hand, synergistic effects of chemotherapeutic agents whencombined with quercetin were described [67], quercetin acting both aschemosensitization and chemoprotective agent in anticancer treatment.Quercetin interaction with dsDNA was investigated electrochemically usingtwo types of DNA-electrochemical biosensors at GCE in order to evaluatethe occurrence of DNA damage caused by oxidized quercetin [68]. Theresults showed that quercetin binds to dsDNA where it can undergooxidation; the radicals formed during quercetin oxidation cause breaks ofthe hydrogen bonds in the dsDNA finally giving rise to 8-oxoG, biomarkerof DNA oxidative damage. A mechanism for oxidized quercetin-induceddamage to dsDNA immobilized onto a GCE surface was proposed. Inbulk solution, a very weak interaction between quercetin and DNA wasfound to take place [69]. In addition, extensive quercetin-induced DNAdamage via reaction with Cu(II) has been reported. An electrochemical studyof the DNA-Cu(II)-quercetin system in solution showed a time-dependent


damage on the dsDNA structure, recognized via the G and A residues oxida-tion peaks increase, and confirmed spectrophotometrically via the 260 nmadsorption band increase [69].

The antioxidant and prooxidant properties of the semi-synthetic flavo-nolignan 7-O-galloylsilybin (7-GSB) have been studied [70]. The presenceof a galloyl moiety enhances the antioxidant capacity of 7-GSB, whencompared to that of silybin (SB). These results were supported by electro-chemistry, DPPH$(2,2-diphenyl-1-picrylhydrazyl radical) scavenging activ-ity, total antioxidant capacity (CL-TAC), and DFT (density functionaltheory) calculations. A three-step oxidation mechanism of 7-GSB wasproposed at pH 7.4 and confirmed by molecular orbital analysis. The com-plex formed by 7-GSB with Cu(II) was also studied and the prooxidanteffects of the metal complexes were tested according to their capacity toinduce DNA oxidative modification and cleavage. The results showedthat 7-O-galloyl substitution to SB concomitantly enhances the antioxidantcapacity and decreases the prooxidant effect/DNA damage after coppercomplexation.

3.10 Free radicalsROS species, such as superoxide radical, hydrogen peroxide, singlet oxygen,and the highly reactive hydroxyl radical, are very reactive radicals, ions, ormolecules that have a single unpaired electron in their outermost shell ofelectrons [71]. ROS species are known to be cytotoxic and have been asso-ciated with the occurrence of several diseases, including cancer, heart disease,and aging [72]. Most ROS are generated as by-products during mitochon-drial electron transport; also, various carcinogens may act by generatingROS species during their metabolism. Oxidative damage to DNA byROS has been established as cause of cancer since the 80’s [72,73], andthe majority of mutations involve modification of G residues, causingG/ T transversions, and accumulation of 8-oxoG [72]. Compared withnormal cells, cancer cells present higher levels of endogenous oxidative stressin culture and in vivo, resulting in very high levels of ROS. Therefore,studies suggested that these excessive levels of ROS in cancer cells can beexploited for selectively eliminating the tumour cells by further pharmaco-logical ROS attacks [74].

Oxygen radicals can be electrochemically generated in situ, from redoxreactions, at the electrode surface. Immobilizing DNA at the electrodesurface allows the potential control of the oxygen radicals’ formation (theDNA-damaging species) and subsequent electrochemical detection of the


damage they cause to the surface-attached DNA. The DNA damage causingstrand brakes formation was detected using an electrochemical biosensor,based on a covalently closed supercoiled DNA-modified mercury electrode[75]. The hydroxyl radicals generated in electrochemically controlled iron/EDTA-mediated Fenton reaction, as well as radical intermediates of oxygenelectroreduction, cleaved the DNA anchored at the mercury electrodesurface. The extent of DNA damage increased with the shift of the electrodepotential to negative values, displaying a sharp inflection point matching thepotential of [Fe(EDTA)]2�/[Fe(EDTA)]1� redox pair [75].

A DNA-electrochemical biosensor, based on native DNA adsorbed onhybrid coating consisting of nanoparticles of silver decorated with a macro-cyclic receptor with catechol groups and covalently attached to a neutral redconductive polymer, was used for the estimation of DNA damage, in amodel system based on the Fenton reagent [76]. Oxidative damage ofDNA caused by the Fenton reagent resulted in synchronous increase ofthe electron transfer resistance and capacitance measured by EIS. TheDNA-electrochemical biosensor was tested for the evaluation of antioxidantcapacity of green tea infusions [76].

The hydroxyl radicals electrochemically generated in situ on a boron-doped diamond electrode (BDDE) have been investigated, in differentelectrolyte media, over a wide pH range. An oxidation peak at high positivepotentials, corresponding to the transfer of one electron and one proton inpH < 9 electrolytes, associated with the electrochemical generation ofhydroxyl radicals from the water discharge process, was observed [77].The effect of BDDE surface termination (causing oxygen or hydrogenterminations), immediately after cathodic or anodic electrochemical pre-treatment, and the influence of the pretreatment on the electrochemicaloxidation of dsDNA, poly(G) and poly(A) homopolynucleotides, GMPand AMP nucleotides, DNA bases, and biomarker 8-oxoG, were investigated[78,79]. The interaction and adsorption of DNA and its components on theBDDE surface pretreated cathodically was facilitated due to the BDDEhigher conductivity. On the other hand, after anodic pretreatment a widerpotential window of BDDE was obtained enabling the detection of thepyrimidine bases. The hydroxyl radicals produced on BDDE surface athigh positive potentials were highly reactive, and consequently the BDDEsurface was not completely inert.

The dsDNA-BDDE-electrochemical biosensor, prepared byimmobilization of dsDNA on an oxidized BDDE surface, was successfullyused to detect the dsDNAoxidative damage caused by in situ electrogenerated


hydroxyl radicals [80]. Controlling the time of BDDE applied potential,different hydroxyl radical concentrations were electrochemically generatedin situ, enabling the hydroxyl radicals pre-concentration onto the thickmultilayer immobilized dsDNA. Monitoring the G and A residues oxidationpeak currents modification, Fig. 12, it was concluded that the hydroxylradicals oxidatively damage the immobilized dsDNA, leading to the dsDNAstructure modifications, demonstrated by the occurrence of the 8-oxoGoxidation peak, a biomarker of DNA oxidative damage, and confirmedby electrophoresis [80].

3.11 Ionizing radiationThe application of ionizing radiation in cancer treatment was recognizedsoon after the discovery of X-rays and has been used for several decadesin curative and palliative treatments of cancerous solid tumours [81,82].Ionizing radiation can change the structure of biomolecules, creatingpotentially harmful effects. Although radiation has the potential to damagea multitude of biomolecules, inside a cell, the structure of most concern isDNA, to which different types of damage can be induced: single- and

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

G

8-oxoG

Ar

A

Gr0.5

E / V (vs. Ag/AgCl)

µA

Figure 12 DP voltammograms of a thick multilayer dsDNA-BDDE biosensor, in pH 4.5:( ) control and (▬) first and ( ) forth scans after applying þ3.0 V during 2 h tothe BDDE surface causing electrogeneration of hydroxyl radicals. Adapted from S.C.B.Oliveira, A.M. Oliveira-Brett, In situ DNA oxidative damage by electrochemically generatedhydroxyl free radicals on a boron-doped diamond electrode, Langmuir. 28 (2012) 4896e4901, http://dx.doi.org/10.1021/la300070x with permission.


http://dx.doi.org/10.1021/la300070x

double-strand breaks, modifications of the base residues, destruction of thesugar, cross-links, and formation of dimers.

In particular, g-radiation has been used in radiotherapy for severaldecades, and it is known that exposure gives rise to genomic instability lead-ing to mutagenesis, carcinogenesis, and cell death. The g-radiation can beabsorbed directly by DNA, leading to ionization of both the bases and sugar,in a mechanism described as the direct effect of generating single and tandemDNA damage.

However, approximately 65% of the DNA damage is not caused directlyby radiation, but is an indirect effect of free radicals, such as hydroxyl radi-cals, that are formed from the radiolysis of surrounding water molecules thatattack DNA [82]. The characterization of different kinds of DNA damagecaused by g-radiation exposure was studied by DP voltammetry at GCE,using dsDNA, ssDNA, poly(dG), poly(dA), poly(dT), and poly(dC) aqueoussolutions previously exposed to g-radiation doses between 2 and 35 Gy [82].The generation of 8-oxoG, 2,8-DHA, 5-formyluracil, base-free sites, andsingle- and double-stranded breaks, in the g-irradiated DNA samples, wasdetected electrochemically, the damage depending on the g-irradiationtime. It was found that the peak currents for 8-oxoG increase linearlywith the g-radiation dose applied to the different nucleic acid samples,and values between 8 and 446 8-oxoG molecules per 106 G moleculesper applied Gy were obtained. The electrochemical observations wereconfirmed by the electrophoretic migration profile, obtained for the samessDNA and dsDNA g-irradiated samples, by non-denaturing agarose gelelectrophoresis [82].

4. DNA-ELECTROCHEMICAL BIOSENSOR INDETECTION OF CANCER BIOMARKER PROTEINS

The NCI defines a biomarker as a biological molecule found in blood,body fluids (e.g., stool, urine, sputum, or nipple discharge), or tissues, whosepresence represents a sign of an abnormal process, condition or disease [83].Cancer biomarkers include proteins, nucleic acids, antibodies, peptides, etc.,and can be used to non-invasively differentiate a healthy person from apatient with a specific cancer disease [84]. Among cancer biomarkers, pro-teins are particularly important, since more than 1261 proteins are believedto be differentially expressed in human cancer [85]. Cancer biomarkerprotein candidates include proteins involved in a variety of processes, suchas oncogenesis, angiogenesis, development, differentiation, proliferation,


apoptosis, hematopoiesis, immune and hormonal responses, cell signalling,nucleotide function, hydrolysis, cellular homing, cell cycle and structure,the acute phase response, and hormonal control [85].

The identification and quantification of proteins is essential in manymedical and biomedical applications related to cancer early detection andtreatment. Protein detection has been investigated by different techniques,such as AFM, molecular affinity scanning force microscopy, ellipsometry,fluorescence spectroscopy, acoustic plate mode sensors, surface plasmonresonance, etc.

DNA-electrochemical biosensors have started to be used in the directdetection of different proteins-DNA interaction. DNA-electrochemicalbiosensors, designed to specifically detect emerging cancer biomarker pro-teins, can open the perspective of many potential applications in oncology,including risk assessment, differential diagnosis, determination of prognosis,prediction of response to treatment, and monitoring of disease progression.

Cancer risk can be determined by various genetic, metabolic, and life-style factors that alter the metabolism. Cytochrome P450 1A2 (CYP1A2)is responsible for the metabolism of oestrogens, and many exogenous com-pounds, including caffeine, and its activity can be correlated with hormones,blood lipids, and lifestyle factors associated with breast cancer [86]. Thedirect, label-free detection of the human cytochrome CYP1A2 interactionwith dsDNA was investigated by DP voltammetry, in incubated solutionsand at the surface of a dsDNA-electrochemical biosensor, and by UV-Visspectrophotometry. CYP1A2 interacted with dsDNA causing its condensa-tion and DNA oxidative damage, Fig. 13. A preferential interaction betweenthe dsDNA G residues and CYP1A2 was found, as free G and 8-oxoGoxidation peaks were detected. This was confirmed using poly(G)- andpoly(A)-electrochemical biosensors [87].

Pur proteins are sequence-specific single-stranded nucleic acid-bindingproteins, which in humans are critical for myeloid cell, muscle and braindevelopment. Pura and Purbmembers have been implicated in several typesof diseases, as diverse as cancer (e.g., acute myelogenous leukaemia), prema-ture aging, and fragile-X mental retardation syndrome [88].

The interaction of the protein Purb of mouse (mPurb) with ssDNA, andof Escherichia coli protein MutH with dsDNA, was studied at a DNA-electro-chemical biosensor [89]. Impedance spectroscopy and DP voltammetrydistinguished their interaction specificity and recognition, the mPurb forsingle strands and the MutH for double strands. DP voltammogramsrecorded for the DNA before and after the interaction with proteins,


confirmed the DNA residues oxidation peak modifications after binding tothe proteins. The DNA-electrochemical response depended on the proteinsconcentration, and the DNA-proteins complexes’ formation constants wereestimated based on the DP voltammetric results [89].

Aptamers are small single-stranded nucleic acids molecules thatpresent specific three-dimensional structure, synthesized by chemical orenzymatic procedures, to specifically bind to target molecules and inhibittheir biological functions [90,91]. Due to their high affinity for a seriesof proteins, they are largely used in biosensor development applied toprotein quantification assays. Aptamers can fold into different three-dimensional configurations, such as GQ, stem-and-loop, hairpin, andthree-stem helix junctions [92].

Particular attention received the short aptamers that adopt GQconfigurations, which can bind to a wide variety of molecular targets, mainlyproteins (such as thrombin, nucleolin, signal transducer and activator oftranscription STAT3, human RNase H1, protein tyrosine phosphataseShp2, VEGF, HIV-1 integrase, HIV-1 reverse transcriptase, HIV-1 reversetranscriptase, HIV-1 nucleocapsid protein, M. tuberculosis polyphosphatekinase 2, sclerostin, insulin, etc.), but also to some other targets(hematoporphyrin IX, hemin, ochratoxin, potassium ions, ATP) [93e96].

54

32

10 0.2

0.40.6

0.81.01.2

Ar

Gr

Gr8-oxoG/2,8-DHA

2 nA

E / V (vs. A

g/AgCl)Time / h

ArCYP1A2 + G

Figure 13 DP voltammograms baseline corrected, in pH 7.0: ( ) control dsDNA-electrochemical biosensor, and (▬) dsDNA-electrochemical biosensor incubatedwith 0.5 mg/mL CYP1A2, during 10 min, 1, 3, and 5 h. Adapted from I.C. Lopes, A.M.Oliveira-Bret, Human cytochrome P450 (CYP1A2)-dsDNA interaction in situ evalua-tion using a dsDNA-electrochemical biosensor, Electroanalysis (2017) (in press) withpermission.


Therefore, the development of electrochemical biosensors using aptamersas recognition units in cancer diagnosis early stages become a promisingstrategy [97].

Thrombin is a serine protease and a coagulation protein in the bloodstream that controls the coagulation mechanism. Cancer patients are atincreased risk of deep vein thrombosis and pulmonary embolism, thereforethe need for monitoring the thrombin generation during cancer treatments[98]. In addition, thrombin induces invasion and metastasis in various can-cers, although the mechanisms by which it promotes tumorigenesis arenot well understood [99]. For this reason, the detection of thrombin usingTBAs has been extensively studied. In fact, the TBA-thrombin model hasbeen used for the development of electrochemical aptasensors with differentarchitectures [33,100].

A systematic study of the interaction between the serine proteasethrombin and two different TBA sequences, d(G2T2G2TGTG2T2G2) andd(G3T2G3TGT3T2G3), was carried out on two different types of carbonelectrode, GCE using DP voltammetry and HOPG using AFM [31,34].The thrombin interaction with the TBA’s primary and secondary structures,were detected through changes in G and A residues oxidation peaks in theTBA sequences. The complex between thrombin and single-stranded TBAsequences involved coiling of the single strands around thrombin, leading tothe formation of a robust TBA-thrombin complex that maintains thesymmetry and conformation of thrombin. The thrombin oxidation peaksoccurred at more positive potentials after single-stranded TBA-thrombincomplex formation. In the presence of Kþ ions, the aptamers folded intoGQ structures that facilitate the interaction with thrombin. The TBA-thrombin complex adsorbs with the aptamer GQ always in preferentialcontact with the electrode surface and the thrombin molecules on top ofthe aptamer GQ structure.

5. DNA-ELECTROCHEMICAL BIOSENSOR IN RISKASSESSMENT OF HAZARDOUS COMPOUNDS

Identification of unknown carcinogenic compounds and screeningthe presence of these hazardous substances in the environment are crucialstrategies for cancer risk assessment and prevention.

Carcinogens are hazardous compounds that increase the risk of cancer, byincreasing the opportunities for DNA changes to occur. NCI’s list of possiblecancer-causing substances include aflatoxins, aristolochic acids, arsenic,asbestos, aspartame, benzene, cadmium, ethylene oxide, formaldehyde,


hexavalent chromium compounds, nickel compounds, strong inorganic acidmists containing sulphuric acid, radon, vinyl chloride, and several compoundsfound in cosmetics, antiperspirants, or hair days beauty salon treatments[101]. However, new harmful compounds are evaluated and identifiedevery day. Furthermore, the toxicity of many well-known carcinogens(e.g., the previous described metal complexes, naphthoquinones, cyanotox-ins, free radicals, and ionizing radiation) is now exploited as anticancer drugs,for selectively eliminating the tumour cells. In this context, DNA-electro-chemical biosensors can be effectively used from a cancer preventionperspective, for rapid preliminary screening of chemical mutagens andcarcinogens in environmental and food samples.

5.1 Metal ionsEpidemiological studies have shown that occupational and environmentalexposure to specific metals is associated with an increased risk of differentcancers and adverse health effects. Transition and post-transition metalions can interact specifically with DNA inducing changes of the DNAdouble helix. In particular, arsenic and arsenic compounds, beryllium andberyllium compounds, cadmium and cadmium compounds, hexavalentchromium compounds, lead, cobalt, and nickel compounds are provencarcinogens in laboratory animals [102].

DP voltammetry was used to study the in situ dsDNA interaction mech-anism with different chromium species, Cr(III), Cr(IV), Cr(V), and Cr(VI)[103]. The Cr(III) reactive intermediates oxidation by the O2 dissolved insolution cause DNA oxidative damage, and the Cr(VI) reactive intermedi-ates oxidation leads to conformational modifications of the dsDNA doublehelix. The Cr(IV) and Cr(V) reactive intermediates are produced, in situ, inCr(III) oxidation by O2 dissolved in solution, and, in vivo, in Cr(VI) reduc-tion by ascorbate and glutathione. The DNA-electrochemical biosensorenabled the separate investigation of the interaction and toxicity of eachchromium species with DNA and a better understanding of the molecularmechanisms involved in chromium species-induced neoplasia. Using poly-nucleotides of known sequences, it was confirmed that chromium reactiveintermediates preferentially interact with dsDNA at G-rich segments, lead-ing to oxidative damage and formation of 8-oxoG [103].

DNA damage by Cr(V) and/or Cr(IV) intermediates of Cr(VI)electrochemical reduction was also evaluated using a supercoiled DNA-modified mercury electrode [104]. DNA at the mercury electrode surfaceis cleaved in the presence of the chromium species in an electrode


potential-dependent manner. The AC voltammetric signal sensitive to theformation of DNA strand breaks increased after incubation of the DNA-modified electrode in solutions of Cr(VI), at potentials sufficiently negativefor the Cr(VI) reduction. These processes lead to DNA damage, and mayinvolve reactive intermediates of Cr(VI) reduction and/or ROS [104].

Heavy metal ions, lead, cadmium and nickel, are well-known carcinogenswith different natural origins [105,106]. A dsDNA-electrochemical biosensorwas used for the in situ evaluation of Pb(II), Cd(II), and Ni(II) interactionwith dsDNA [105], showing that the interaction leads to differentmodifications in the dsDNA structure, electrochemically recognized aschanges in the G and A residues oxidation peaks. Using poly(G)- andpoly(A)-electrochemical biosensors, it has been confirmed that the interactionbetween Pb(II) and DNA causes oxidative damage and takes placepreferentially at A-rich segments, with the formation of 2,8-DHA, the Aresidues oxidation product, biomarker of DNA oxidative damage. ThePb(II) bound to dsDNA can still undergo oxidation. The interaction ofCd(II) and Ni(II) causes conformational changes, destabilizing the doublehelix, which can facilitate the action of other oxidative agents on DNA [105].

AFM and DP voltammetry at spontaneously adsorbed DNAemetalcomplexes film also showed that Pb(II), Ni(II), Cd(II), and Pd(II) interactspecifically with the dsDNA, due to a high affinity to form covalent bondswith nitrogenous bases, inducing conformational modifications in theB-DNA structure [106]. An increase of the electrode surface coverage bythe PbeDNA, CdeDNA and NieDNA complexes, when comparedwith control dsDNA, was observed. The local denaturation of the doublehelix, due to the metaleDNA interaction, facilitates the hydrophobicinteractions between the DNA bases and the hydrophobic carbon electrodesurface. The Pd(II) cations induced the greatest morphological changes inthe DNA adsorption pattern, leading to the formation of PdeDNAcomplex thicker aggregates, caused by DNA condensation. The compactPdeDNA structures observed by AFM and the DP voltammetry suggestthat the Pd(II)eDNA interaction is very strong and stable [106].

5.2 PollutantsPollutants are chemical substances present in greater concentration thannormal, and introduced in the environment (air, water, soil, and biota), asa result of agricultural and industrial activities, and presenting detrimentaleffect on human health [107]. Many pollutants have been found to cause


cancer in animal species or humans. DNA-electrochemical biosensors can beused to further predict and understand the pollutant effects on organisms, aswell as to develop analytical methodologies for their sensitive detection.

Acrylamide (AA) is an a,b-unsaturated carbonyl compound, used as abuilding block in making polyacrylamide and acrylamide copolymers,widely used in industrial processes, such as the production of paper, dyes,and plastics, and in the treatment of drinking water and wastewater.Acrylamide can also be found in certain foods when heated above 120�C.IARC considers acrylamide to be a probable human carcinogen, based onits neurotoxicity, genotoxicity, and reproductive toxicity found in rodentmodels [108]. A DNA-electrochemical biosensor based on graphene-ionicliquid-Nafion/(horseradish peroxidase/DNA)3 modified pyrolytic graphiteelectrode was prepared by the layer-by-layer method, which provided aneffective strategy for mimicking and detecting DNA damage induced byAA in vivo [108]. The results indicated that under the coexistence of horse-radish peroxidase (HRP), H2O2, and AA, HRP was activated by H2O2 andcatalyzed the transformation of AA into glycidamide. The formation ofDNA adducts with AA and glycidamide disturbed the double helix ofDNA, which exposed the G residues inside the dsDNA. Based on thechanges of the G residues oxidation peak, DNA damage was directlydetected. In addition, glycidamide induced more serious DNA damagethan AA, which provided further evidence for the mainly carcinogenicactivity of glycidamide.

Hydroquinone is a chemical compound used for decades as a skin light-ening agent, banned initially from cosmetics due to mid-term effects such asleucoderma/vitiligo or exogenous ochronosis. However, more studiessuggest that carcinogenesis may be expected as well, due to the hydroqui-none metabolites formed in the liver, such as hydroquinone’s conjugatesp-benzoquinone and glutathione [109]. The electrochemical behaviour ofhydroquinone and its DNA-damaging mechanisms were investigated usinga DNA-electrochemical biosensor constructed with chitosan (CTS) andpolyaniline (PANI) [110]. The results showed that the redox peak currentwas remarkably increased after GCEmodification by PANI/CTS. The elec-trochemical oxidation of hydroquinone on the dsDNA/PANI/CTS/GCEelectrode was an adsorption-controlled irreversible and a two-electrontwo-proton transfer process. The dsDNA damage by hydroquinone wasconcentration dependent, increasing along with the hydroquinone oxida-tion peak current increase, and the dsDNA G residues oxidation peakcurrent decrease [110].


Polycyclic aromatic hydrocarbons (PAHs) are a group of different chem-icals, formed during the incomplete burning of coal, oil and gas, garbage, orother organic substances like tobacco or charbroiled meat, being currentlyclassified as human carcinogens [111]. Benzo(a)pyrene (BaP) is one of themost studied PAHs, classified by IARC as probable human carcinogen.BaP and its metabolite were detected at a DNA-electrochemical biosensor,built by layer-by-layer assembling of HRP and dsDNA at nafion-solubilizedsingle-wall carbon nanotubes-ionic liquid (SWCNTs-NA-IL) compositefilm [112]. The biosensor was characterized by cyclic and DP voltammetry,EIS, SEM, and computational methods. UV-vis spectrophotometry was alsoused to investigate DNA damage induced by BaP and its products in solu-tion. The DNA-electrochemical biosensor was investigated separately inBaP, H2O2, and in their mixture. The results showed that the interactionwith BaP caused unwinding of DNA double helix and exposure of the bases.

The cyanobacterial blooms in aquatic environments are of increasedconcern in many lakes, rivers, and brackish waters worldwide and morethan 40 types of cyanobacteria are known to produce a variety of harmfulcompounds as secondary metabolites called cyanotoxins. Microcystin-LR(MC-LR) and nodularin (NOD) are among the most commonly reportedtoxins produced by cyanobacteria, with strong hepatotoxic, genotoxic,and carcinogenic potential, which have been associated with the inductionof DNA damage in vitro and in vivo [113,114]. Apart from the environ-mental, hazardous, tumour promoting activity of MC-LR and NOD,especially liver cancer, based on the strong MC-LR cytotoxicity in vitro,it was proposed that MC-LR could be held as structural basis for thedevelopment of novel targeted compounds against pancreatic cancer[115]. From the pharmacological viewpoint, cyanotoxins represent a richsource of natural cytotoxic compounds with a potential to target specificcancers [116].

The MC-LR electrochemical behaviour at GCE was investigated bycyclic, DP, and SW voltammetry [113], showing that MC-LR oxidationis a diffusion-controlled irreversible and pH-independent process that occurswith the transfer of one electron and does not involve the formation of anyelectroactive oxidation product. Upon incubation during 24 h, in differentpH electrolytes, homogeneous chemical degradation of MC-LR in solutionoccurred. The chemically degradedMC-LRwas electrochemically detectedby the occurrence of a new oxidation peak, at a lower oxidation potential,corresponding to an irreversible, pH-dependent process, and involving theformation of two new redox products that undergo reversible oxidation.


The chemical degradation of MC-LR in solution was confirmed by high-performance liquid chromatography with UV detection at roomtemperature.

The interaction of MC-LR and NOD with dsDNA was investigated inincubated solutions and at DNA-electrochemical biosensors [114]. It wasshown that MC-LR, NOD, and their chemical degradation products interactwith dsDNA, causing the aggregation of DNA strands, detected by the time-dependent decrease of the G and A residues oxidation peaks, Fig. 14 [114].In incubated solutions, where dsDNA strands are allowed to move freely,the interaction leads to the release of free A bases. The interaction betweenDNA and MC-LR or NOD also causes a basic damage, upon cleavage ofthe bond between the DNA phosphate sugar backbone that, if leftunrepaired, can lead to mutations during the replication process.

6. CONCLUSIONS

This chapter presents advances on the design and applications oflabel-free DNA-electrochemical biosensors, in a cancer research perspective.

0.2 0.4 0.6 0.8 1.0 1.2 1.4

A

Gr

cdMC-LR

Ar

Gr + MC-LR

E / V vs. Ag/AgCl

10 nA

Figure 14 DP voltammograms baseline corrected of 50 mg/mL dsDNA solution, in pH4.5: ( ) before and after incubation with 30 mM MC-LR, during (nnn) 0, (▬) 6 and ( )24 h. Adapted from P.V.F. Santos, I.C. Lopes, V.C. Diculescu, A.M. Oliveira-Brett, DNA -cyanobacterial hepatotoxins microcystin-LR and nodularin interaction: electrochemicalevaluation, Electroanalysis. 24 (2012) 547e553, http://dx.doi.org/10.1002/elan.201100516 withpermission.



Screening new antineoplastic drugs with increased specificity, early diagnosisof cancer biomarkers, identification of unknown carcinogenic compounds,and detection of the presence of hazardous substances in the environment arecrucial strategies for evaluation of new effective cancer treatment drugs, riskassessment of hazardous compounds, and prevention.

The development of DNA-electrochemical biosensors, built by bottom-upimmobilization of DNA sequences that self-assemble in either double- andsingle-stranded form, or in unusual but biologically relevant configu-rations, such as GQs and i-motifs, is revised. The influence of the DNAimmobilization parameters and the analytical characterization, using com-bined electrochemical and surface characterization techniques is crucial andemerges as a necessary step for the development of new, more sensitiveDNA-electrochemical biosensors. The applications of DNA-electrochemicalbiosensors, for label-free detection of anticancer pharmaceutical compounds,protein cancer biomarkers, and carcinogens, can now provide new informa-tion very useful for planning therapeutic intervention, in a wide variety ofhuman cancers.

ACKNOWLEDGEMENTSFinancial support from Fundaç~ao para a Ciência e Tecnologia (FCT), Grant SFRH/BPD/92726/2013 (A.-M. Chiorcea-Paquim), project UID/EMS/00285/2013 (co-financedby the European Community Fund FEDER), FEDER funds through the programCOMPETE e Programa Operacional Factores de Competitividade, Innovec’EAU(SOE1/P1/F0173), and projects (APQ/MCTI/CNPq/Universal/456725/2014-8),(FACEPE-APQ-0535-1.06/14), and FACEPE-CNPq-BRAZIL (S.C.B. Oliveira), aregratefully acknowledged.

REFERENCES[1] J.H. Shibata, Nucleic acids: structures, properties, and functions (Bloomfield, Victor

A.; Crothers, Donald M.; Tinoco, Ignacio, Jr.; contributions from Hearst, John E.;Wemmer, David E.; Kollman, Peter A.; Turner, Douglas H.), J. Chem. Educ. 78(2001) 314, http://dx.doi.org/10.1021/ed078p314.2.

[2] A.M. Oliveira Brett, Biosensors and Modern Biospecific Analytical Techniques,Elsevier, 2005, http://dx.doi.org/10.1016/S0166-526X(05)44004-0.

[3] A.M.O. Brett, Electrochemistry for probing DNA damage, in: M.V. Pishko,C.A. Grimes, E.C. Dickey (Eds.), Encycl. Sensors, American Scientific Publishers,2006, p. 301. https://estudogeral.sib.uc.pt/jspui/handle/10316/13388.

[4] A.M. Oliveira-Brett, A.M.C. Paquim, V.C. Diculescu, J.A.P. Piedade, Electrochem-istry of nanoscale DNA surface films on carbon, Med. Eng. Phys. 28 (2006) 963e970,http://dx.doi.org/10.1016/j.medengphy.2006.05.009.

[5] A.M. Oliveira Brett, V.C. Diculescu, A.-M. Chiorcea Paquim, S. Serrano, Chapter 20DNA-electrochemical biosensors for investigating DNA damage, Compr. Anal.Chem. 49 (2007) 413e437, http://dx.doi.org/10.1016/S0166-526X(06)49020-6.


http://dx.doi.org/10.1021/ed078p314.2

http://dx.doi.org/10.1016/S0166-526X(05)44004-0

https://estudogeral.sib.uc.pt/jspui/handle/10316/13388

http://dx.doi.org/10.1016/j.medengphy.2006.05.009

http://dx.doi.org/10.1016/S0166-526X(06)49020-6

[6] A.M. Oliveira Brett, Electrochemical DNA assays, in: P.N. Bartlett (Ed.), Bio-electrochemistry Fundam. Exp. Tech. Appl., John Wiley & Sons, Ltd, Chichester,UK, 2008, p. 411, http://dx.doi.org/10.1002/9780470753842.

[7] A.-M. Oliveira-Brett, Nanobioelectrochemistry, in: P. Schmuki, S. Virtanen (Eds.),Electrochem. Nanoscale, Nanostrutures Sci. Technol., Springer New York, NewYork, NY, 2009, p. 407, http://dx.doi.org/10.1007/978-0-387-73582-5.

[8] V.C. Diculescu, A.-M. Chiorcea-Paquim, A.M. Oliveira-Brett, Applications of aDNA-electrochemical biosensor, TrAC Trends Anal. Chem. 79 (2016) 23e36,http://dx.doi.org/10.1016/j.trac.2016.01.019.

[9] A.M. Oliveira Brett, S.H.P. Serrano, A.J.P. Piedade, Applications of KineticModelling, Elsevier, 1999, http://dx.doi.org/10.1016/S0069-8040(99)80008-9.

[10] A.M. Oliveira Brett, V.C. Diculescu, A.M. Chiorcea-Paquim, S.H.P. Serrano, Elec-trochemical Sensor Analysis, Elsevier, 2007, http://dx.doi.org/10.1016/S0166-526X(06)49020-6.

[11] V.C. Diculescu, A.-M. Chiorcea Paquim, A.M. Oliveira Brett, Electrochemical DNAsensors for detection of DNA damage, Sensors 5 (2005) 377e393.

[12] A.-M. Chiorcea-Paquim, P.V. Santos, A.M. Oliveira-Brett, Atomic force microscopyand voltammetric characterisation of synthetic homo-oligodeoxynucleotides, Electro-chim. Acta 110 (2013) 599e607, http://dx.doi.org/10.1016/j.electacta.2013.03.103.

[13] A.M. Oliveira Brett, A.-M. Chiorcea, Effect of pH and applied potential on theadsorption of DNA on highly oriented pyrolytic graphite electrodes. Atomic force mi-croscopy surface characterisation, Electrochem. Commun. 5 (2003) 178e183.

[14] A.M. Oliveira Brett, A.-M. Chiorcea, Atomic force microscopy of DNA immobilizedonto a highly oriented pyrolytic graphite electrode surface, Langmuir 19 (2003) 3830e3839.

[15] A.-M. Chiorcea Paquim, V.C. Diculescu, T.S. Oretskaya, A.M. Oliveira Brett, AFMand electroanalytical studies of synthetic oligonucleotide hybridization, Biosens. Bio-electron. 20 (2004) 933e944, http://dx.doi.org/10.1016/j.bios.2004.06.018.

[16] A.M. Oliveira Brett, A.-M. Chiorcea Paquim, DNA imaged on a HOPG electrodesurface by AFM with controlled potential, Bioelectrochemistry 66 (2005) 117e124,http://dx.doi.org/10.1016/j.bioelechem.2004.05.009.

[17] A.M. Oliveira Brett, A.-M. Chiorcea Paquim, V. Diculescu, T.S. Oretskaya, Syntheticoligonucleotides: AFM characterisation and electroanalytical studies, Bio-electrochemistry 67 (2005) 181e190, http://dx.doi.org/10.1016/j.bioelechem.2004.06.008.

[18] A.-M. Chiorcea Paquim, T.S. Oretskaya, A.M. Oliveira Brett, Adsorption of synthetichomo- and hetero-oligodeoxynucleotides onto highly oriented pyrolytic graphite:atomic force microscopy characterization, Biophys. Chem. 121 (2006) 131e141.

[19] A.-M. Chiorcea Paquim, T.S. Oretskaya, A.M. Oliveira Brett, Atomic force micro-scopy characterization of synthetic pyrimidinic oligodeoxynucleotides adsorbed ontoan HOPG electrode under applied potential, Electrochim. Acta 51 (2006) 5037e5045, http://dx.doi.org/10.1016/j.electacta.2006.03.041.

[20] A.-M. Chiorcea Paquim, J.A.P. Piedade, R. Wombacher, A. J€aschke, A.M. OliveiraBrett, Atomic force microscopy and anodic voltammetry characterization of a 49-mer diels-alderase ribozyme, Anal. Chem. 78 (2006) 8256e8264.

[21] E. Palecek, Past, present and future of nucleic acids electrochemistry, Talanta 56 (2002)809e819. http://www.ncbi.nlm.nih.gov/pubmed/18968559.

[22] E. Pale�cek, M. Barto�sík, Electrochemistry of nucleic acids, Chem. Rev. 112 (2012)3427e3481, http://dx.doi.org/10.1021/cr200303p.

[23] A.M. Oliveira Brett, F.-M. Matysik, Voltammetric and sonovoltammetric studies onthe oxidation of thymine and cytosine at a glassy carbon electrode, J. Electroanal.Chem. 429 (1997) 95e99, http://dx.doi.org/10.1016/S0022-0728(96)05018-8.


http://dx.doi.org/10.1002/9780470753842

http://dx.doi.org/10.1007/978-0-387-73582-5


http://dx.doi.org/10.1016/S0069-8040(99)80008-9

http://dx.doi.org/10.1016/S0166-526X(06)49020-6

http://dx.doi.org/10.1016/S0166-526X(06)49020-6

http://refhub.elsevier.com/S0166-526X(17)30038-7/sref11







http://refhub.elsevier.com/S0166-526X(17)30038-7/bib14




http://dx.doi.org/10.1016/j.bios.2004.06.018














http://www.ncbi.nlm.nih.gov/pubmed/18968559

http://dx.doi.org/10.1021/cr200303p

http://dx.doi.org/10.1016/S0022-0728(96)05018-8

[24] A.M. Oliveira-Brett, J.A.P. Piedade, L.A. Silva, V.C. Diculescu, Voltammetric deter-mination of all DNA nucleotides, Anal. Biochem. 332 (2004) 321e329, http://dx.doi.org/10.1016/j.ab.2004.06.021.

[25] A.M.O. Brett, J.A.P. Piedade, S.H.P. Serrano, Electrochemical oxidation of8-oxoguanine, Electroanalysis 12 (2000) 969e973, http://dx.doi.org/10.1002/1521-4109(200008)12:12<969::AID-ELAN969>3.0.CO;2-O.

[26] A.M. Oliveira-Brett, V. Diculescu, J.A.P. Piedade, Electrochemical oxidation mecha-nism of guanine and adenine using a glassy carbon microelectrode, Bioelectrochemistry55 (2002) 61e62. http://www.ncbi.nlm.nih.gov/pubmed/11786341.

[27] S.C.B. Oliveira, A.M. Oliveira-Brett, DNA-electrochemical biosensors: AFM surfacecharacterisation and application to detection of in situ oxidative damage to DNA,Comb. Chem. High Throughput Screen 13 (2010) 628e640. http://www.ncbi.nlm.nih.gov/pubmed/20402643.

[28] V. Brabec, E. Pale�cek, Interactions of nucleic acids with electrically charged surfaces, J.Electroanal. Chem. Interfacial Electrochem. 88 (1978) 373e385, http://dx.doi.org/10.1016/S0022-0728(78)80125-9.

[29] A.-M. Chiorcea, A.M. Oliveira Brett, Atomic force microscopy characterization of anelectrochemical DNA-biosensor, Bioelectrochemistry 63 (2004) 229e232, http://dx.doi.org/10.1016/j.bioelechem.2003.09.029.

[30] V.C. Diculescu, A.M. Chiorcea-Paquim, Thrombin-binding aptamer quadruplex for-mation: AFM and voltammetric characterization, J. Nucleic Acids (2010). http://www.hindawi.com/journals/jna/2010/841932/abs/.

[31] V.C. Diculescu, A.-M. Chiorcea-Paquim, R. Eritja, A.M. Oliveira-Brett, Evaluationof the structureeactivity relationship of thrombin with thrombin binding aptamers byvoltammetry and atomic force microscopy, J. Electroanal. Chem. 656 (2011) 159e166, http://dx.doi.org/10.1016/j.jelechem.2010.11.037.

[32] A.-M. ChiorceaePaquim, P. Santos, V.C. Diculescu, R. Eritja, A.M. Oliveira-Brett,A.-M. ChiorceaePaquim, et al., Electrochemical characterization of guanine quadru-plexes, in: Guanine Quartets, Royal Society of Chemistry, Cambridge, 2012,pp. 100e109, http://dx.doi.org/10.1039/9781849736954-00100.

[33] A.-M. Chiorcea-Paquim, A.M. Oliveira-Brett, Redox behaviour of G-quadruplexes,Electrochim. Acta 126 (2014) 162e170, http://dx.doi.org/10.1016/j.electacta.2013.07.150.

[34] 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) 9117e9124, http://dx.doi.org/10.1039/c3cp50866h.

[35] A.D. Rodrigues Pontinha, A.-M. Chiorcea-Paquim, R. Eritja, A.M. Oliveira-Brett,Quadruplex nanostructures of d(TGGGGT): influence of sodium and potassiumions, Anal. Chem. 86 (2014) 5851e5857, http://dx.doi.org/10.1021/ac500624z.

[36] A.-M. Chiorcea-Paquim, A.D.R. Pontinha, A.M. Oliveira-Brett, Time-dependentpolyguanylic acid structural modifications, Electrochem. Commun. 45 (2014) 71e74, http://dx.doi.org/10.1016/j.elecom.2014.05.019.

[37] S.Wu-Pong, Y. Rojanasakul (Eds.), Biopharmaceutical Drug Design and Development,Humana Press, Totowa, NJ, 2008, http://dx.doi.org/10.1007/978-1-59745-532-9.

[38] M.C. Perry, D.C. Doll, C.E. Freter, Perry’s the Chemotherapy Source Book, WoltersKluwer Health/Lippincott Williams & Wilkins, 2012.

[39] S.C.B. Oliveira, I.B. Santarino, A.M. Oliveira-Brett, Direct electrochemistry of nativeand denatured anticancer antibody rituximab at a glassy carbon electrode, Electroanal-ysis 25 (2013) 1029e1034, http://dx.doi.org/10.1002/elan.201200552.

[40] I.B. Santarino, S.C.B. Oliveira, A.M. Oliveira-Brett, In situ evaluation of the anti-cancer antibody rituximab-dsDNA interaction using a DNA-electrochemicalbiosensor, Electroanalysis 26 (2014) 1304e1311, http://dx.doi.org/10.1002/elan.201300488.




http://dx.doi.org/10.1002/1521-4109(200008)12:12<969::AID-ELAN969>3.0.CO;2-O







http://dx.doi.org/10.1016/S0022-0728(78)80125-9

http://dx.doi.org/10.1016/S0022-0728(78)80125-9



http://www.hindawi.com/journals/jna/2010/841932/abs/

http://www.hindawi.com/journals/jna/2010/841932/abs/

http://dx.doi.org/10.1016/j.jelechem.2010.11.037

http://dx.doi.org/10.1039/9781849736954-00100



http://dx.doi.org/10.1039/c3cp50866h

http://dx.doi.org/10.1021/ac500624z


http://dx.doi.org/10.1007/978-1-59745-532-9






[41] F.Z. Issaad, L.I.N. Tomé, N.V. Marques, C. Mouats, V.C. Diculescu, A.M. Oliveira-Brett, Bevacizumab anticancer monoclonal antibody: native and denatured redoxbehaviour, Electrochim. Acta 206 (2016) 246e253, http://dx.doi.org/10.1016/j.electacta.2016.04.097.

[42] 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 898 (2015) 28e33, http://dx.doi.org/10.1016/j.aca.2015.09.049.

[43] H.E. Satana, A.D.R. Pontinha, V.C. Diculescu, A.M. Oliveira-Brett, Nucleosideanalogue electrochemical behaviour and in situ evaluation of DNAeclofarabineinteraction, Bioelectrochemistry 87 (2012) 3e8, http://dx.doi.org/10.1016/j.bioelechem.2011.07.004.

[44] A.D.R. Pontinha, H.E. Satana, V.C. Diculescu, A.M. Oliveira-Brett, Anodic oxida-tion of cladribine and in situ evaluation of DNA-cladribine interaction, Electroanalysis23 (2011) 2651e2657, http://dx.doi.org/10.1002/elan.201100320.

[45] R.M. Buoro, I.C. Lopes, V.C. Diculescu, S.H.P. Serrano, L. Lemos, A.M. Oliveira-Brett, In situ evaluation of gemcitabineeDNA interaction using a DNA-electrochem-ical biosensor, Bioelectrochemistry 99 (2014) 40e45, http://dx.doi.org/10.1016/j.bioelechem.2014.05.005.

[46] V.C. Diculescu, M. Vivan, A.M.O. Brett, Voltammetric behavior of antileukemiadrug glivec. Part III: in situ DNA oxidative damage by the glivec electrochemicalmetabolite, Electroanalysis 18 (2006) 1963e1970, http://dx.doi.org/10.1002/elan.200603602.

[47] V.C. Diculescu, A.M. Oliveira-Brett, In situ electrochemical evaluation of dsDNAinteraction with the anticancer drug danusertib nitrenium radical product using theDNA-electrochemical biosensor, Bioelectrochemistry 107 (2016) 50e57, http://dx.doi.org/10.1016/j.bioelechem.2015.10.004.

[48] B. Dogan-Topal, B. Bozal-Palabiyik, S.A. Ozkan, B. Uslu, Investigation of anticancerdrug lapatinib and its interaction with dsDNA by electrochemical and spectroscopictechniques, Sens. Actuators B Chem. 194 (2014) 185e194, http://dx.doi.org/10.1016/j.snb.2013.12.088.

[49] V.C. Diculescu, M. Vivan, A.M.O. Brett, Voltammetric behavior of antileukemiadrug glivec. Part I e electrochemical study of glivec, Electroanalysis 18 (2006)1800e1807, http://dx.doi.org/10.1002/elan.200603591.

[50] V.C. Diculescu, M. Vivan, A.M.O. Brett, Voltammetric behavior of antileukemiadrug glivec. Part II e redox processes of glivec electrochemical metabolite, Electro-analysis 18 (2006) 1808e1814, http://dx.doi.org/10.1002/elan.200603592.

[51] A.D.R. Pontinha, S.M.A. Jorge, A.-M. Chiorcea Paquim, V.C. Diculescu,A.M. Oliveira-Brett, P.T.R. Rajagopalan, et al., In situ evaluation of anticancerdrug methotrexateeDNA interaction using a DNA-electrochemical biosensor andAFM characterization, Phys. Chem. Chem. Phys. 13 (2011) 5227, http://dx.doi.org/10.1039/c0cp02377a.

[52] A.-M. Chiorcea-Paquim, A.D. Rodrigues Pontinha, A.M. Oliveira-Brett, Quadru-plex-targeting anticancer drug BRACO-19 voltammetric and AFMcharacterization, Electrochim. Acta 174 (2015) 155e163, http://dx.doi.org/10.1016/j.electacta.2015.05.146.

[53] A.D.R. Pontinha, S. Sparapani, S. Neidle, A.M. Oliveira-Brett, Triazole-acridine con-jugates: redox mechanisms and in situ electrochemical evaluation of interaction withdouble-stranded DNA, Bioelectrochemistry 89 (2013) 50e56, http://dx.doi.org/10.1016/j.bioelechem.2012.08.005.















http://dx.doi.org/10.1016/j.snb.2013.12.088

http://dx.doi.org/10.1016/j.snb.2013.12.088









[54] A.-M. Chiorcea-Paquim, A.D.R. Pontinha, R. Eritja, G. Lucarelli, S. Sparapani,S. Neidle, et al., Atomic force microscopy and voltammetric investigation of quadru-plex formation between a triazole-acridine conjugate and guanine-containing repeatDNA sequences, Anal. Chem. 87 (2015) 6141e6149, http://dx.doi.org/10.1021/acs.analchem.5b00743.

[55] G.N. Hortob�agyi, Anthracyclines in the treatment of cancer. An overview, Drugs 54(Suppl. 4) (1997) 1e7. http://www.ncbi.nlm.nih.gov/pubmed/9361955.

[56] 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) 959e970, http://dx.doi.org/10.1016/S0039-9140(01)00656-7.

[57] J.A.P. Piedade, I.R. Fernandes, A.M. Oliveira-Brett, Electrochemical sensing ofDNAeadriamycin interactions, Bioelectrochemistry 56 (2002) 81e83, http://dx.doi.org/10.1016/S1567-5394(02)00013-0.

[58] 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. 538e539 (2002) 267e276.

[59] H. Eda Satana Kara, Redox mechanism of anticancer drug idarubicin and in-situ evalu-ation of interaction with DNA using an electrochemical biosensor, Bioelectrochemistry99 (2014) 17e23, http://dx.doi.org/10.1016/j.bioelechem.2014.06.002.

[60] 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 thalidomideeDNAinteraction, Bioelectrochemistry 76 (2009) 201e207, http://dx.doi.org/10.1016/j.bioelechem.2009.03.003.

[61] M. Frezza, S. Hindo, D. Chen, A. Davenport, S. Schmitt, D. Tomco, et al., Novelmetals and metal complexes as platforms for cancer therapy, Curr. Pharm. Des. 16(2010) 1813e1825. http://www.ncbi.nlm.nih.gov/pubmed/20337575.

[62] O. Corduneanu, A.-M. Chiorcea-Paquim, V. Diculescu, S.M. Fiuza,M.P.M. Marques, A.M. Oliveira-Brett, DNA interaction with palladium chelates ofbiogenic polyamines using atomic force microscopy and voltammetriccharacterization, Anal. Chem. 82 (2010) 1245e1252, http://dx.doi.org/10.1021/ac902127d.

[63] O. Corduneanu, A.-M. Chiorcea-Paquim, M. Garnett, A.M. Oliveira-Brett, Lipoicacid-palladium complex interaction with DNA, voltammetric and AFMcharacterization, Talanta 77 (2009) 1843e1853, http://dx.doi.org/10.1016/j.talanta.2008.10.046.

[64] M.C. de Vasconcellos, C. de Oliveira Costa, E.G. da Silva Terto, M.A.F.B. de Moura,C.C. de Vasconcelos, F.C. de Abreu, et al., Electrochemical, spectroscopic andpharmacological approaches toward the understanding of biflorin DNA damageeffects, J. Electroanal. Chem. 765 (2016) 168e178, http://dx.doi.org/10.1016/j.jelechem.2015.09.040.

[65] P. Babula, J. Vanco, L. Krejcova, Voltammetric Characterization ofLawsone-Copper(II) Ternary Complexes and Their Interactions with dsDNA, 2012.

[66] K. Sak, Cytotoxicity of dietary flavonoids on different human cancer types, Pharma-cogn. Rev. 8 (2014) 122e146, http://dx.doi.org/10.4103/0973-7847.134247.

[67] A.F. Brito, M. Ribeiro, A.M. Abrantes, A.S. Pires, R.J. Teixo, J.G. Tralh~ao, et al.,Quercetin in cancer treatment, alone or in combination with conventionaltherapeutics? Curr. Med. Chem. 22 (2015) 3025e3039. http://www.ncbi.nlm.nih.gov/pubmed/26264923.

[68] A.M. Oliveira-Brett, V.C. Diculescu, Electrochemical study of quercetineDNAinteractions, Bioelectrochemistry 64 (2004) 143e150, http://dx.doi.org/10.1016/j.bioelechem.2004.05.002.





http://dx.doi.org/10.1016/S0039-9140(01)00656-7

http://dx.doi.org/10.1016/S1567-5394(02)00013-0

http://dx.doi.org/10.1016/S1567-5394(02)00013-0












http://dx.doi.org/10.1016/j.talanta.2008.10.046






http://dx.doi.org/10.4103/0973-7847.134247





[69] A.M. Oliveira-Brett, V.C. Diculescu, Electrochemical study of quercetineDNA in-teractions: Part I. Analysis in incubated solutions, Bioelectrochemistry 64 (2004)133e141, http://dx.doi.org/10.1016/j.bioelechem.2004.05.003.

[70] J. Vacek, M. Zatloukalov�a, T. Desmier, V. Nezhodov�a, J. Hrb�a�c, M. Kubala, et al.,Antioxidant, metal-binding and DNA-damaging properties of flavonolignans: a jointexperimental and computational highlight based on 7-O-galloylsilybin, Chem. Biol.Interact. 205 (2013) 173e180, http://dx.doi.org/10.1016/j.cbi.2013.07.006.

[71] G.-Y. Liou, P. Storz, Reactive oxygen species in cancer, Free Radic. Res. 44 (2010)479e496, http://dx.doi.org/10.3109/10715761003667554.

[72] G. Waris, H. Ahsan, Reactive oxygen species: role in the development of cancer andvarious chronic conditions, J. Carcinog. 5 (2006) 14, http://dx.doi.org/10.1186/1477-3163-5-14.

[73] B.N. Ames, Dietary carcinogens and anticarcinogens. Oxygen radicals and degenera-tive diseases, Science 221 (1983) 1256e1264. http://www.ncbi.nlm.nih.gov/pubmed/6351251.

[74] D. Trachootham, J. Alexandre, P. Huang, Targeting cancer cells by ROS-mediatedmechanisms: a radical therapeutic approach? Nat. Rev. Drug Discov. 8 (2009) 579e591, http://dx.doi.org/10.1038/nrd2803.

[75] M. Fojta, T. Kubic�arov�a, E. Palecek, Electrode potential-modulated cleavage of sur-face-confined DNA by hydroxyl radicals detected by an electrochemical biosensor,Biosens. Bioelectron. 15 (2000) 107e115. http://www.ncbi.nlm.nih.gov/pubmed/11286327.

[76] Y. Kuzin, A. Porfireva, V. Stepanova, V. Evtugyn, I. Stoikov, G. Evtugyn, et al.,Impedimetric detection of DNA damage with the sensor based on silver nanoparticlesand neutral red, Electroanalysis 27 (2015) 2800e2808, http://dx.doi.org/10.1002/elan.201500312.

[77] T.A. Enache, A.-M. Chiorcea-Paquim, O. Fatibello-Filho, A.M. Oliveira-Brett, Hy-droxyl radicals electrochemically generated in situ on a boron-doped diamondelectrode, Electrochem. Commun. 11 (2009) 1342e1345, http://dx.doi.org/10.1016/j.elecom.2009.04.017.

[78] S.C.B. Oliveira, A.M. Oliveira-Brett, Voltammetric and electrochemical impedancespectroscopy characterization of a cathodic and anodic pre-treated boron doped dia-mond electrode, Electrochim. Acta 55 (2010) 4599e4605, http://dx.doi.org/10.1016/j.electacta.2010.03.016.

[79] S.C.B. Oliveira, A.M. Oliveira-Brett, Boron doped diamond electrode pre-treatmentseffect on the electrochemical oxidation of dsDNA, DNA bases, nucleotides, homopo-lynucleotides and biomarker 8-oxoguanine, J. Electroanal. Chem. 648 (2010) 60e66,http://dx.doi.org/10.1016/j.jelechem.2010.06.020.

[80] S.C.B. Oliveira, A.M. Oliveira-Brett, In situ DNA oxidative damage by electrochem-ically generated hydroxyl free radicals on a boron-doped diamond electrode, Langmuir28 (2012) 4896e4901, http://dx.doi.org/10.1021/la300070x.

[81] P.P. Connell, S.J. Kron, R.R. Weichselbaum, Relevance and irrelevance of DNAdamage response to radiotherapy, DNA Repair (Amst) 3 (2004) 1245e1251,http://dx.doi.org/10.1016/j.dnarep.2004.04.004.

[82] J.A.P. Piedade, P.S.C. Oliveira, M.C. Lopes, A.M. Oliveira-Brett, Voltammetricdetermination of g radiation-induced DNA damage, Anal. Biochem. 355 (2006)39e49, http://dx.doi.org/10.1016/j.ab.2006.05.022.

[83] NCI Dictionary of Cancer Terms, (n.d.). https://www.cancer.gov/publications/dictionaries/cancer-terms.

[84] N.L. Henry, D.F. Hayes, Cancer biomarkers, Mol. Oncol. 6 (2012) 140e146, http://dx.doi.org/10.1016/j.molonc.2012.01.010.



http://dx.doi.org/10.1016/j.cbi.2013.07.006

http://dx.doi.org/10.3109/10715761003667554

http://dx.doi.org/10.1186/1477-3163-5-14

http://dx.doi.org/10.1186/1477-3163-5-14



http://dx.doi.org/10.1038/nrd2803










http://dx.doi.org/10.1021/la300070x

http://dx.doi.org/10.1016/j.dnarep.2004.04.004


https://www.cancer.gov/publications/dictionaries/cancer-terms

https://www.cancer.gov/publications/dictionaries/cancer-terms

http://dx.doi.org/10.1016/j.molonc.2012.01.010

http://dx.doi.org/10.1016/j.molonc.2012.01.010

[85] M. Polanski, N.L. Anderson, A list of candidate cancer biomarkers for targetedproteomics, Biomark. Insights 1 (2007) 1e48. http://insights.sagepub.com/a-list-of-candidate-cancer-biomarkers-for-targeted-proteomics-article-a29-abstract?article_id¼29.

[86] C.-C. Hong, B.-K. Tang, G.L. Hammond, D. Tritchler, M. Yaffe, N.F. Boyd, Cyto-chrome P450 1A2 (CYP1A2) activity and risk factors for breast cancer: a cross-sectional study, Breast Cancer Res. 6 (2004) R352eR365, http://dx.doi.org/10.1186/bcr798.

[87] I.C. Lopes, A.M. Oliveira-Bret, Human cytochrome P450 (CYP1A2)-dsDNA inter-action in situ evaluation using a dsDNA-electrochemical biosensor, Electroanalysis(2017), http://dx.doi.org/10.1002/elan.201600713.

[88] E.M. Johnson, D.C. Daniel, J. Gordon, The pur protein family: genetic and structuralfeatures in development and disease, J. Cell. Physiol. 228 (2013) 930e937, http://dx.doi.org/10.1002/jcp.24237.

[89] C. Ban, S. Chung, D.-S. Park, Y.-B. Shim, Detection of protein-DNA interactionwith a DNA probe: distinction between single-strand and double-strand DNA-proteininteraction, Nucleic Acids Res. 32 (2004) e110, http://dx.doi.org/10.1093/nar/gnh109.

[90] G. Mayer, The chemical biology of aptamers, Angew. Chem. Int. 48 (2009) 2672e2689, http://dx.doi.org/10.1002/anie.200804643.

[91] M. Mascini, I. Palchetti, S. Tombelli, Nucleic acid and peptide aptamers: fundamentalsand bioanalytical aspects, Angew. Chem. Int. 51 (2012) 1316e1332, http://dx.doi.org/10.1002/anie.201006630.

[92] E. Baldrich, Aptamers: versatile tools for reagentless aptasensing, in: Recognit. Recept.Biosens., Springer New York, New York, NY, 2010, pp. 675e722, http://dx.doi.org/10.1007/978-1-4419-0919-0_17.

[93] A.-M. ChiorceaePaquim, P. Santos, V.C. Diculescu, R. Eritja, A.M. Oliveira-Brett,Guanine quartets, in: L. Spindler, W. Fritzsche (Eds.), Guanine Quartets Struct. Appl.,Royal Society of Chemistry, Cambridge, 2012, pp. 100e109, http://dx.doi.org/10.1039/9781849736954.

[94] T. Hianik, J. Wang, Electrochemical aptasensors - recent achievements andperspectives, Electroanalysis 21 (2009) 1223e1235, http://dx.doi.org/10.1002/elan.200904566.

[95] D. Musumeci, D. Montesarchio, Polyvalent nucleic acid aptamers and modulation oftheir activity: a focus on the thrombin binding aptamer, Pharmacol. Ther. 136 (2012)202e215, http://dx.doi.org/10.1016/j.pharmthera.2012.07.011.

[96] W.O. Tucker, K.T. Shum, J.A. Tanner, G-quadruplex DNA aptamers and theirligands: structure, function and application, Curr. Pharm. Des. 18 (2012) 2014e2026. http://www.ncbi.nlm.nih.gov/pubmed/22376117.

[97] X. Wu, J. Chen, M.Wu, J.X. Zhao, Aptamers: active targeting ligands for cancer diag-nosis and therapy, Theranostics 5 (2015) 322e344, http://dx.doi.org/10.7150/thno.10257.

[98] I. Pabinger, J. Thaler, C. Ay, Biomarkers for prediction of venous thromboembolismin cancer, Blood 122 (2013).

[99] A.R. Radjabi, K. Sawada, S. Jagadeeswaran, A. Eichbichler, H.A. Kenny, A. Montag,et al., Thrombin induces tumor invasion through the induction and association ofmatrix metalloproteinase-9 and 1-integrin on the cell surface, J. Biol. Chem. 283(2008) 2822e2834, http://dx.doi.org/10.1074/jbc.M704855200.

[100] A.-M. Chiorcea-Paquim, A. Oliveira-Brett, Guanine quadruplex electrochemicalaptasensors, Chemosensors 4 (2016) 13, http://dx.doi.org/10.3390/chemosensors4030013.

[101] Cancer-Causing Substances in the Environment, (n.d.). https://www.cancer.gov/about-cancer/causes-prevention/risk/substances.


http://insights.sagepub.com/a-list-of-candidate-cancer-biomarkers-for-targeted-proteomics-article-a29-abstract?article_id=29




http://dx.doi.org/10.1186/bcr798

http://dx.doi.org/10.1186/bcr798


http://dx.doi.org/10.1002/jcp.24237

http://dx.doi.org/10.1002/jcp.24237

http://dx.doi.org/10.1093/nar/gnh109

http://dx.doi.org/10.1093/nar/gnh109

http://dx.doi.org/10.1002/anie.200804643



http://dx.doi.org/10.1007/978-1-4419-0919-0_17

http://dx.doi.org/10.1007/978-1-4419-0919-0_17

http://dx.doi.org/10.1039/9781849736954

http://dx.doi.org/10.1039/9781849736954



http://dx.doi.org/10.1016/j.pharmthera.2012.07.011


http://dx.doi.org/10.7150/thno.10257

http://dx.doi.org/10.7150/thno.10257



http://dx.doi.org/10.1074/jbc.M704855200

http://dx.doi.org/10.3390/chemosensors4030013

http://dx.doi.org/10.3390/chemosensors4030013

https://www.cancer.gov/about-cancer/causes-prevention/risk/substances

https://www.cancer.gov/about-cancer/causes-prevention/risk/substances

[102] E.J. Tokar, L. Benbrahim-Tallaa, M.P. Waalkes, Metal ions in human cancerdevelopment, Met. Ions Life Sci. 8 (2011) 375e401. http://www.ncbi.nlm.nih.gov/pubmed/21473387.

[103] S.C.B. Oliveira, A.M. Oliveira-Brett, In situ evaluation of chromiumeDNA damageusing a DNA-electrochemical biosensor, Anal. Bioanal. Chem. 398 (2010) 1633e1641, http://dx.doi.org/10.1007/s00216-010-4051-7.

[104] J. Vacek, T. Mozga, K. Cahov�a, H. Pivo�nkov�a, M. Fojta, Electrochemical sensing ofchromium-induced DNA damage: DNA strand breakage by intermediates of chro-mium(VI) electrochemical reduction, Electroanalysis 19 (2007) 2093e2102, http://dx.doi.org/10.1002/elan.200703917.

[105] S.C.B. Oliveira, O. Corduneanu, A.M. Oliveira-Brett, In situ evaluation of heavymetaleDNA interactions using an electrochemical DNA biosensor, Bio-electrochemistry 72 (2008) 53e58, http://dx.doi.org/10.1016/j.bioelechem.2007.11.004.

[106] A.-M. Chiorcea-Paquim, O. Corduneanu, S.C.B. Oliveira, V.C. Diculescu,A.M. Oliveira-Brett, Electrochemical and AFM evaluation of hazard compoundseDNA interaction, Electrochim. Acta 54 (2009) 1978e1985, http://dx.doi.org/10.1016/j.electacta.2008.07.032.

[107] K. Rajeshwar, J.G. Ibanez, K. Rajeshwar, J.G. Ibanez, Chapter one, in: Environ. Elec-trochem., 1997, pp. 1e56, http://dx.doi.org/10.1016/B978-012576260-1/50001-2.

[108] 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-Nafion modi-fied pyrolytic graphite electrode, J. Hazard. Mater. 190 (2011) 480e485, http://dx.doi.org/10.1016/j.jhazmat.2011.03.071.

[109] W. Westerhof, T.J. Kooyers, Hydroquinone and its analogues in dermatology - apotential health risk, J. Cosmet. Dermatol. 4 (2005) 55e59, http://dx.doi.org/10.1111/j.1473-2165.2005.40202.x.

[110] W. Tang, M. Zhang, W. Li, X. Zeng, An electrochemical sensor based on polyanilinefor monitoring hydroquinone and its damage on DNA, Talanta 127 (2014) 262e268,http://dx.doi.org/10.1016/j.talanta.2014.03.069.

[111] G. Mastrangelo, E. Fadda, V. Marzia, Polycyclic aromatic hydrocarbons and cancer inman, Environ. Health Perspect. 104 (1996) 1166.

[112] A.R. Jalalvand, M.-B. Gholivand, H.C. Goicoechea, T. Skov, K. Mansouri,Mimicking enzymatic effects of cytochrome P450 by an efficient biosensor for in vitrodetection of DNA damage, Int. J. Biol. Macromol. 79 (2015) 1004e1010, http://dx.doi.org/10.1016/j.ijbiomac.2015.05.047.

[113] I.C. Lopes, P.V.F. Santos, V.C. Diculescu, F.M.P. Peixoto, M.C.U. Ara�ujo,A.A. Tanaka, et al., Microcystin-LR and chemically degraded microcystin-LR electro-chemical oxidation, Analyst 137 (2012) 1904, http://dx.doi.org/10.1039/c2an16017j.

[114] P.V.F. Santos, I.C. Lopes, V.C. Diculescu, A.M. Oliveira-Brett, DNA - cyanobacterialhepatotoxins microcystin-LR and nodularin interaction: electrochemical evaluation,Electroanalysis 24 (2012) 547e553, http://dx.doi.org/10.1002/elan.201100516.

[115] V. Kounnis, G. Chondrogiannis, M.D. Mantzaris, A.G. Tzakos, D. Fokas,N.A. Papanikolaou, et al., Microcystin LR shows cytotoxic activity against pancreaticcancer cells expressing the membrane OATP1B1 and OATP1B3 transporters, Anti-cancer Res. 35 (2015) 5857e5865. http://www.ncbi.nlm.nih.gov/pubmed/26504008.

[116] I. Sainis, D. Fokas, K. Vareli, A.G. Tzakos, V. Kounnis, E. Briasoulis, Cyanobacterialcyclopeptides as lead compounds to novel targeted cancer drugs, Mar. Drugs 8 (2010)629e657, http://dx.doi.org/10.3390/md8030629.




http://dx.doi.org/10.1007/s00216-010-4051-7







http://dx.doi.org/10.1016/B978-012576260-1/50001-2

http://dx.doi.org/10.1016/j.jhazmat.2011.03.071

http://dx.doi.org/10.1016/j.jhazmat.2011.03.071

http://dx.doi.org/10.1111/j.1473-2165.2005.40202.x

http://dx.doi.org/10.1111/j.1473-2165.2005.40202.x




http://dx.doi.org/10.1016/j.ijbiomac.2015.05.047

http://dx.doi.org/10.1016/j.ijbiomac.2015.05.047

http://dx.doi.org/10.1039/c2an16017j




http://dx.doi.org/10.3390/md8030629

past, present and future challenges of biosensors and ... anal chem-2… · 1. introduction 287 2....

Documents