Applications of a DNA-electrochemical biosensor

Applications of a DNA-electrochemical biosensor

Accepted Manuscript Title: Applications of a DNA-electrochemical biosensor Author: Victor Constantin Diculescu, Ana-Maria Chiorcea-Paquim, Ana Maria O...

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Accepted Manuscript Title: Applications of a DNA-electrochemical biosensor Author: Victor Constantin Diculescu, Ana-Maria Chiorcea-Paquim, Ana Maria Oliveira-Brett PII: DOI: Reference:

S0165-9936(15)30156-4 http://dx.doi.org/doi: 10.1016/j.trac.2016.01.019 TRAC 14646

To appear in:

Trends in Analytical Chemistry

Please cite this article as: Victor Constantin Diculescu, Ana-Maria Chiorcea-Paquim, Ana Maria Oliveira-Brett, Applications of a DNA-electrochemical biosensor, Trends in Analytical Chemistry (2016), http://dx.doi.org/doi: 10.1016/j.trac.2016.01.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Applications of a DNA-electrochemical biosensor

Victor Constantin Diculescu, Ana-Maria Chiorcea-Paquim and Ana Maria Oliveira-Brett*

Department of Chemistry, University of Coimbra, Portugal

* To whom correspondence should be addressed Ana Maria Oliveira-Brett Department of Chemistry University of Coimbra 3004-535 Coimbra, Portugal Phone: 00351239854487 e-mail: [email protected]

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2 Contents

1

Introduction

4

2

Development of DNA electrochemical biosensors

5

3

Applications of DNA electrochemical biosensors

10

4

3.1

Proteins

10

3.2

Drugs

12

3.3

Metal ions and their complexes

19

3.4

Pollutants

23

3.5

Free radicals

26

3.6

Radiation

29

Conclusion

30

Acknowledgements

31

References

32

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3 Highlights



Design of DNA-electrochemical biosensors that use DNA direct electrochemistry.



AFM and voltammetric characterization of new immobilisation procedures described.



Self-assembled nanostructures of DNA, G-quadruplex, and i-motif presented.



Application for label-free detection of DNA interactions and damage.



Interaction with proteins, drugs, metals, pollutants, radicals, and radiation, revised.

Abstract As carrier of genetic information, DNA is one of the most important intracellular targets that undergo modification and damage upon interaction with endogenous and exogenous factors. DNA is an excellent biomaterial for the construction of new devices, in nanotechnology and biosensor technology, for evaluation of DNA interaction with a broad range of chemical compounds and biomolecules, essential from a biological and a medical point of view. This review discusses recent advances on the design and applications of DNAelectrochemical biosensors that use DNA direct electrochemistry as a detection platform. AFM and voltammetric characterization of new bottom up immobilisation procedures of selfassembled nanostructures based on DNA single- and double-stranded, G-quadruplex, and i-motif configurations are presented, relevant for the development of new DNA-electrochemical biosensor devices. The applications of DNA-electrochemical biosensors, for the label-free detection of interactions with proteins, pharmaceutical compounds, metal ions and metal complexes, pollutants, free radicals, and electromagnetic radiation, were revisited.

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4 Keywords: DNA-electrochemical biosensor; voltammetry; G-quadruplex; i-motif; DNA oxidative damage; protein; drug; metal ion; pollutant; free radical; radiation.

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5 1. Introduction

DNA is a stable, low-cost and easily adaptable molecule, being an excellent building block for the construction of new devices in nanotechnology and biosensor technology. DNAbased biosensors have been successfully used in numerous applications, such as, investigation and evaluation of DNA-drug interaction mechanisms, detection of DNA base damage in clinical diagnosis, rapid monitoring of metals or pollutant agents in the environment, direct monitoring hybridization processes or label-free detection of specific DNA sequences and proteins. A DNA-electrochemical biosensor is formed by an electrode (the electrochemical transducer) with a DNA probe immobilized on its surface (the biological recognition element) and is used to detect DNA-binding molecules (the analyte) that interact and induce changes in the DNA structure and electrochemical properties, which are further translated into an electrical signal (Scheme 1). Electrochemical methods offer rapid detection, great sensitivity, and low cost. A DNA-electrochemical biosensor build in this way can either be label-free, by directly monitoring the changes in the DNA bases oxidation peaks before and after the interaction with the analyte, or use different amplification strategies [1-3]. In this review, we will discuss recent advances on the design and applications of DNAelectrochemical biosensors that use DNA direct electrochemistry as a detection platform. This type of DNA-electrochemical based biosensors are highly sensitive, up to femtomoles of analyte, label-free and allowed the use of different electrode substrates, which made them suitable for inexpensive miniaturization for clinical diagnosis and on-site environmental monitoring.

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6 2

Development of DNA-electrochemical biosensors

Understanding the redox behaviour of the DNA probe is critical for the design and successful application of label-free DNA-electrochemical biosensors. Electrochemical studies of DNA bases, purines: adenine (A) and guanine (G), and pyrimidines: thymine (T) and cytosine (C) (Scheme 2A), their nucleosides, and nucleotides, as well as of native or denaturated DNA probes were performed at mercury and carbon electrodes [4-7]. Cyclic voltammograms of nucleic acids at dropping mercury electrode showed one cathodic peak, due to irreversible reduction of C and A residues, while the reduction of G residues occurred at very negative potentials, and only the oxidation peak of the G residues reduction product was detected in the reverse scan [4,5]. At carbon electrodes, the voltammetric studies showed that DNA bases, nucleosides and nucleotides are all electroactive (Fig. 1), and their oxidation is pH dependent [6,7]. The voltammetric detection of the G and A oxidation products, 8-oxoguanine (8-oxoG) and 2,8dihydroxyadenine (2,8-DHA), biomarkers of DNA oxidative stress, allowed direct detection of DNA oxidative damage after interaction with the analyte. Differential pulse (DP) voltammograms recorded at a glassy carbon electrode (GCE) in DNA solutions showed two anodic peaks, corresponding to the oxidation of guanosine (dGuo) and adenosine (dAdo) residues in dsDNA (Fig. 2A) [8-10]. The difference obtained for single- (ssDNA) versus double(dsDNA) stranded DNA oxidation peak currents was correlated with the greater difficulty for the transfer of electrons from the inside of the double-helix to the electrode surface, when compared with a single-helix with residues in closer proximity to the electrode surface (Fig. 2). These results represented the sensing strategy of many label-free DNA-electrochemical sensors.

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7 The development of DNA-electrochemical biosensors involved the immobilization of DNA at the electrode surface. This process influences the characteristics of the DNA probe, the accessibility of the chemical compounds to the DNA, the sensor response and its performance. The development of new immobilisation methodologies, based on controlled bottom-up selfassembling of nucleic acid nanostructures, starting with either long chain nucleic acid or custom synthetic short oligodeoxynucleotide (ODN) sequences is a current concern in DNAelectrochemical biosensor technology. Studies of DNA adsorption were first conducted on mercury [4,10,11] and later on carbon electrodes [8-10], and a smaller adsorption was always observed for dsDNA compared with for ssDNA. Ellipsometry and spectroscopic techniques, such as surface enhanced Raman spectroscopy, have been used to investigate the adsorption of DNA onto electrode surfaces [10]. More recently, atomic force microscopy (AFM) was employed to resolve at nanoscale the surface morphological structure of nucleic acid molecules and to understand the nature of the DNAelectrode surface interactions [12-22]. Both dsDNA and ssDNA showed tendency to spontaneously self-assemble onto carbon electrodes, forming thin, two-dimensional network films, whose characteristics depended on DNA concentration, pH and immobilization procedure (Fig. 2B-E). Adsorption under low positive applied potential, not sufficient to oxidise the DNA bases, leaded to more robust and stable DNA films (Fig. 2C, E). The knowledge of the morphology of adsorbed DNA on electrode surfaces explained the non-specific adsorption on the DNA-electrochemical biosensor surface. DNA-electrochemical biosensors with a low degree of non-specific binding required deposition of multilayers of DNA probe which can be achieved for a higher DNA concentration. Many DNA biophysical properties, such as its conformational flexibility and ability to self-assemble through hydrogen bonds, are influenced by the DNA base sequence, length,

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8 concentration, pH and ionic strength. In order to determine the optimum conditions for the nucleic acid immobilization on carbon electrodes, the influence of the sequence composition on the DNA self-assembly was investigated. The adsorption and redox behaviour of homo-ODNs d(A)10, d(T)10 and d(C)10, were studied, by AFM at a highly oriented pyrolytic graphite (HOPG) surface and by DP voltammetry at a GCE [23]. The combination of AFM and voltammetry revealed strong correlations between the degree of surface coverage, the base composition of the ODN molecules, and the ODN secondary structure which is directly influenced by the solution concentration and pH. Homo-ODNs can adopt different configurations, ranging from single to quadruple helical structures (Scheme 2D), since bases of the same type self-associate in a variety of arrangements (Scheme 2C), different from the Watson Crick base-pairs (Scheme 2B). Under physiological pH, d(A)10, d(C)10 and d(T)10 self-assembled at the surface of carbon electrodes as network films with knobby appearance, due to the aggregation and coiling of the single-strands. In mild acid pH solutions, d(A)10 double-helical conformations (Scheme 2D-left) were observed, by AFM as network films with lower surface coverage, and detected by DP voltammetry through the decrease of the A oxidation peak current. In the same conditions, d(C)10 formed i-motifs (Scheme 2D-right), observed by AFM as spherical aggregates. G-rich DNA can form G-quadruplex (GQ) structures and have been the subject of numerous applications, covering areas from structural biology to medical chemistry, supramolecular chemistry, nanotechnology and biosensor technology. They are considered cancerspecific molecular targets for anticancer drugs, since the GQ stabilisation by small organic molecules can lead to telomerase inhibition and telomere dysfunction in cancer cells. The redox behaviour of DNA sequences able to self-assemble into GQ configurations was studied only recently [24-27]. The first report on the electrochemical oxidation of GQs concerned the investigation of two, different length, thrombin-binding aptamer (TBA) sequences,

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9 d(G2T2G2TGTG2T2G2) and d(G3T2G3TGT3T2G3), using DP voltammetry at a GCE and AFM at a HOPG surface [24]. In Na+ containing solutions, the oxidation of both TBA sequences showed one anodic peak corresponding to the oxidation of G residues in the TBA single-strands. Upon addition of K+ ions, both sequences fold into GQs, causing the decrease of G oxidation peak and occurrence of a new GQ peak at a higher potential, due to the oxidation of G residues in the GQs. The process of GQ formation is directly influenced by the ODN sequence and concentration, pH and presence of monovalent cations (Na+ vs. K+). This was determined using 10-mer ODNs that contain only one block of 8–10 guanines, d(G)10, d(TG9) and d(TG8T) [23,2628], and expected to form parallel tetra-molecular GQ structures (Scheme 2D-middle). DP voltammetry allowed the detection of the association of single-strands into GQs and G-based nanostructures, in freshly prepared solutions, at concentrations 10 times lower than usually detected using other techniques currently employed to study the formation of GQs. Singlestranded ODNs were detected only in Na+ ions containing solutions for short incubation times. The GQ structures were formed slowly in Na+ ions, after a long incubation time, and faster in K+ ions, after a short incubation time. The formation of higher-order nanostructures, due to the presence of a long contiguous G region, and the influence of the T residues at the 5’ and 3’ molecular ends was clarified. For increased d(G)10 concentrations, long G-nanowires were formed, demonstrating the potential of G-rich DNA sequences as a scaffold for nanotechnological applications. The d(TG4T) telomeric repeat sequence of the free-living ciliate protozoa Tetrahymena forms tetra-molecular GQ structures, and is considered a simpler model of biologically relevant GQs, being used to obtain high resolution data on drug-DNA interactions. The well-known conformation of the d(TG4T)-GQ and its extraordinary stiffness have made the d(TG4T) sequence a good candidate for the development of novel devices, with medical and nanotechnological

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10 applications. AFM and DP voltammetry showed d(TG4T) single-strands self-assembling into GQs, very fast in K+ and slowly in Na+ ions containing solution (Scheme 3 and Fig. 3) [29]. The optimum K+ ions concentration for the formation of d(TG4T)-GQs was similar to the healthy cells intracellular K+ ions concentration. In the presence of Na+ ions, d(TG4T) also formed short nanowires and nanostructured films that were never observed in K+ ions containing solution, suggesting that rapid formation of stable GQs in the presence of K+ is relevant for the good function of cells. Synthetic polynucleotides poly(dG) and poly(G) are widely prevalent in the human and other genomes at both DNA and RNA levels, and were used as models to determine the interaction of drugs with G-rich segments of DNA. AFM and DP voltammetric studies showed the poly(G) single-strands self-assembling into short GQ regions at low incubation time, while large poly(G)-GQ aggregates with low adsorption were formed after high incubation times in the presence of monovalent Na+ or K+ ions [30]. The DP voltammetry in freshly prepared poly(G) solutions showed only the G oxidation peak, due to the oxidation of G residues in the poly(G) single-strand. Increasing the incubation time, the G oxidation peak decreased and disappeared, and a GQ residues oxidation peak in the poly(G)-GQ morphology appeared, at a higher oxidation potential, dependent on the incubation time, presenting a maximum after 10 days incubation, and reaching a steady value after ~ 17 days incubation. The recent advances in the DNA electrochemical characterisation and immobilisation of short and long DNA sequences have determined in further detail the key factors in controlling the distribution, size and shape of highly ordered two- and three-dimensional DNA nanostructures on the electrode surface. The bottom up self-assemble of diverse DNA nanostructures allows different applications. The long chains dsDNA, ssDNA, and the purine homo-polynucleotide poly(A) and poly(G), as well as ODNs in single- and double-stranded and GQ configurations, can

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11 be used for screening cancer therapeutic agents. The perfectly aligned G-nanowires may represent building blocks of molecular nanowires for nanoelectronics, and the G-based superstructures and frayed G-nanowires with slipped-strands can work as a nucleation platform for the addition of subsequent strands and the formation of larger structures.

3

Applications of DNA-electrochemical biosensors

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

3.1

Proteins The detection and quantification of proteins play an essential role in fundamental research

and clinical applications. Aptamers are small nucleic acid sequences (DNA or RNA) selected in vitro from large combinatorial pools to bind to specific targets. Due to their high affinity for a series of biomolecules, they are largely used in biosensor development. The structure-activity relationship of

the

complex

formed

between

two

different

length

TBA

aptamer

sequences,

d(G2T2G2TGTG2T2G2) and d(G3T2G3TGT3T2G3), and the serine protease thrombin was evaluated, by DP voltammetry at a GCE and AFM at a HOPG surface, and the interaction mechanism was established [25,26]. The effects on the interaction with thrombin of TBA primary and secondary structures, as well as of its folding properties in the presence of alkaline metals were investigated. Single-stranded TBA sequences coiled around thrombin, leading to the formation of a robust TBA-thrombin complex that maintained the thrombin symmetry and

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12 conformation, therefore, the thrombin oxidation peaks within the TBA-thrombin complex occurred at more positive potentials, than in the case of free thrombin. In the presence of K+ ions, the aptamers folded into GQs that facilitated the interaction with thrombin. TBA-thrombin complexes adsorbed on the carbon electrode with the TBA in contact with the surface and the thrombin on top, far from the surface, thus thrombin being less accessible to oxidation and also leading to the occurrence of thrombin oxidation peaks at more positive potentials. Monoclonal antibodies (mAb) have earned special attention due to their specific and effective therapeutic properties and have become one of the most promising strategies for cancer treatment. The study of the interaction between mAbs and dsDNA has great importance to predict its action mechanism as a genotoxic anticancer drug and to understand its biological activity and toxicity in vivo. Bevacizumab (BEVA) is a recombinant humanized IgG1 monoclonal antibody that targets vascular endothelial growth factor A, being effective in treatment of several types of cancer, such as colon, lung, kidney, ovarian and brain cancers. BEVA interaction with dsDNA was studied by voltammetry and gel-electrophoresis in incubated samples and using a dsDNAelectrochemical biosensor [31]. The voltammetric results at a DNA electrochemical biosensor revealed a decrease and disappearance of the dsDNA oxidation peaks with increasing incubation time, showing that BEVA binds to dsDNA but no DNA oxidative damage was detected (Fig. 4). BEVA also undergoes structural modification upon binding to dsDNA, and BEVA electroactive amino acid residues oxidation peaks were identified. Non denaturing agarose gel-electrophoresis experiments were in agreement with the DP voltammetric results showing the formation of compact BEVA-dsDNA adducts. Rituximab (RTX) is a chimeric human/mouse mAb that targets specifically the CD20 antigen, a receptor expressed on the majority of malignant B-cells (more than 80%) and on

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13 normal differentiated B-lymphocytes (pre-B and mature B-lymphocytes). RTX was the first Food and Drug Administration (FDA) approved genetically engineered mAb for use in indolent B-cell non-Hodgkin’s lymphoma (b-NHL), and is currently used in both indolent and aggressive BNHLs, B-cell chronic lymphocytic leukemia and some autoimmune disease. RTX interaction with dsDNA was investigated by DP voltammetry, in incubated samples and using a multilayer DNA-electrochemical biosensor, and gel electrophoresis [32]. The dsDNA-RTX interaction promoted a strong condensation of the dsDNA helical structure, followed in DP voltammetry by the dGuo oxidation peak current decrease, dAdo oxidation peak disappearance, and the occurrence of the oxidation peaks of free G and A bases released from the DNA, but no DNA base oxidative damage was detected.

3.2

Drugs Protein kinases are enzymes responsible for phosphorylation processes. Several classes of

kinases have been recognised as targets for the development of small inhibiting molecules for anticancer therapy. In vitro studies demonstrated that kinase inhibitors and/or their metabolites can increase the amount of DNA damage [33] and showed that some of these compounds retained a genotoxic activity either through intercalation into the DNA, or formation of alkalilabile sites and/or DNA strand breaks. Danusertib is a kinase inhibitor and anti-cancer drug, and its interaction with dsDNA was investigated in bulk solution and using a dsDNA-electrochemical biosensor [34]. It has been shown that the dsDNA-danusertib interaction occurs in two sequential steps. First, danusertib binds electrostatically to dsDNA phosphate backbone through the positively charged piperazine moiety. The second step involves the pyrrolo-pyrazole moiety and leads to small morphological modifications in the dsDNA double helix which were electrochemically characterised through the

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14 changes of dGuo and dAdo oxidation peaks and confirmed by electrophoretic and spectrophotometric measurements. The nitrenium cation radical product of the danusertib amino group oxidation was electrochemically generated in situ on the dsDNA-electrochemical biosensor surface. The danusertib nitrenium cation radical redox product was covalently attached to the C8 of G residues, protecting them and preventing their oxidation (Scheme 4). A dsDNA-danusertib interaction mechanism was proposed and the formation of the danusertib redox nitrenium radical product-guanine adduct explained. The kinase inhibitor lapatinib (LPT) is a new active drug for breast cancer and other solid tumours. The electro-oxidation mechanism of LPT at a GCE was studied using various voltammetric techniques, and the effect of pH and scan rate on lapatinib signal was investigated [35]. LPT exhibited three charge transfer reactions, each involving the transfer of two electrons. The LPT oxidation was compared with model compounds which contained aromatic amine structures, and an electrooxidation pathway was proposed. The dsDNA-LPT interaction using a DNA-electrochemical biosensor and by spectroscopic techniques was studied. The binding constant (K) between LPT and DNA was calculated as 6.03 × 105 M−1 by electrochemistry, 4.20 × 105 M−1 by UV-vis spectrophotometry, and 3.50 × 104 M−1 by fluorescence spectroscopy. Based on electrochemical and spectroscopic methods, it was confirmed that LPT intercalated into the dsDNA helix. Methotrexate (MTX) is an antimetabolite of folic acid that targets the enzyme dehydrofolate reductase and plays a supporting, but essential, role for the synthesis of thymine nucleotide. Recently, it was demonstrated that MTX interfered with the JAK/STAT (Janus Kinase / Signal Transducer and Activator of Transcription) pathway although the action mechanism was not fully understood. Nevertheless, it has been shown that the MTX treatment caused the accumulation of 8-oxoG in cells. The in situ evaluation of the dsDNA-MTX

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15 interaction was performed by DP voltammetry using a DNA-electrochemical biosensor and characterized by AFM at HOPG [36]. The electrochemical experiments in incubated solutions showed that the interaction of MTX with dsDNA leads to modifications to the dsDNA structure in a time-dependent manner (Fig. 5), and AFM images showed a reorganization of the DNA selfassembled network upon MTX binding. The intercalation of MTX in dsDNA leaded to dsDNA unwinding, detected by the increase of the purine residues oxidation peaks. The dsDNAelectrochemical biosensor, and the purine homo-polynucleotide sequences poly(G) and poly(A)electrochemical biosensors, were used to investigate and understand the interaction between MTX and dsDNA. Telomeres and their associated proteins act by protecting the DNA from recombination, degradation and end-to end fusion. The formation of GQ structures at the end of telomeric DNA increases genomic instability. Another class of anticancer drugs, small ligands able to induce and stabilize GQ configurations, with broad therapeutic selectivity, target the telomerase inhibition, and telomere dysfunction in cancer cells. The GQ-targeting acridine derivative BRACO-19 has been an important tool for studying the antitumor activity of acridine heterocyclic compounds. However, BRACO-19 was relatively non GQ-selective, having also significant binding affinity for dsDNA. For this reason, more recently, a series of new triazole-linked acridine ligands, e.g. GL15 and GL7, with enhanced selectivity for human telomeric GQs binding versus dsDNA binding have been designed, synthetized and evaluated. The redox properties of GL15 and GL7 were investigated using cyclic, DP, and square wave voltammetry at a GCE [37], showing a complex, pH-dependent, adsorption-controlled irreversible mechanism. The interaction between dsDNA and GL15 or GL7 was investigated in situ, in incubated solutions and using dsDNA-, poly(G)-, and poly(A)-electrochemical biosensors [37]. It was demonstrated that the interaction is

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16 time-dependent, both GL15 and GL7 binding to dsDNA and causing condensation of dsDNA morphological structure, but no oxidative damage was detected. The interaction of the GQ-targeting triazole-linked acridine ligand GL15 with the Tetrahymena telomeric DNA repeat sequence d(TG4T) and with the poly(G) sequence have been investigated at the single-molecule level, using AFM and voltammetry [38]. GL15 interacted with both sequences, in a time dependent manner, and the GQ formation was detected by AFM via the adsorption of GL15-d(TG4T)-GQ and GL15-poly(G)-GQ small spherical aggregates (Fig. 6A-E) and large GL15-poly(G)-GQ assemblies, and by DP voltammetry via GL15 and G oxidation peak current decrease and disappearance, and the occurrence of a GQ oxidation peak (Fig. 6F, G). The small-molecule complex with the d(TG4T) quadruplex is discrete and approximately globular, whereas the GQ complex with poly(G) is formed at a number of points along the length of the polynucleotide, analogous to beads on a string. These results are consistent with the interaction of triazole-linked acridine derivatives with terminal G-quartets in an individual GQ. The GL15 stabilized and accelerated GQ formation, in both Na+ and K+ ioncontaining solutions, although only K+ promoted the formation of perfectly aligned tetramolecular GQs. Anthracyclines are among the most effective antibiotics used for the treatment of several types of cancers, including leukemia, lymphomas, breast, uterine, ovarian, bladder or lung cancers, but their use is considerably limited by their cardiotoxicity [39,40]. Adriamycin, doxorubicin hydrochloride, is an antibiotic of the family of anthracyclines with a wide spectrum of chemotherapeutic applications and antineoplasic action, but causes very high cardiotoxicity that ranges from a delayed and insidious cardiomyopathy to irreversible heart failure [40-42]. In order to understand the adriamycin in vivo mechanism of action, the oxidation and reduction of adriamycin on GCE was studied. Adriamycin intercalation and in situ interaction with dsDNA

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17 were investigated using a DNA-electrochemical biosensor [40,41]. The dsDNA-adriamycin interaction mechanism showed that adriamycin intercalated onto DNA, disrupted the double helix, and G and 8-oxoG were detected, showing the occurrence of DNA oxidative damage. Idarubicin (IDA), 4-demethoxydaunorubicin, is an anthracycline derivative and widely used in the treatment of leukemia. The electrochemical behaviour of IDA was examined at GCE in different aqueous supporting electrolyte solutions, using cyclic and DP voltammetry [43]. The oxidation process of IDA was found to be a pH dependent, irreversible diffusion controlled mechanism that occurred with the transfer of one proton and one electron. The electroactive center is the hydroxyl group on the aromatic ring which produces a final quinonic product. The diffusion coefficient of IDA was calculated to be DIDA = 7.47 × 10-6 cm2 s-1 in pH = 4.3. The interaction of IDA with dsDNA was investigated using dsDNA-electrochemical biosensors and incubated solutions [43], showing that IDA interacted with DNA causing changes in the DNA morphological structure. DNA damage was detected following the changes in the oxidation peaks of dGuo and dAdo. In addition, poly(G)- and poly(A)-electrochemical biosensors were also used to confirm the interaction between dsDNA and IDA. However, no oxidation peaks of the purine residues oxidation products, 8-oxoG and 2,8-DHA, were observed. Apart from anticancer drug investigation, DNA-electrochemical biosensors were widely used for studying other pharmaceutical compounds. Thalidomide (TD) was developed as a sedative and anti-emetic drug to combat morning sickness during pregnancy, but it was removed from market due to its teratogenic side effects. More recently, TD regained interest due to its potential for treating a number of otherwise intractable inflammatory skin diseases, such as erythema nodosum leprosum, graft versus host disease, weight loss in tuberculosis, aphthous ulcers and human immunodeficiency virus replication in acquired immune deficiency syndrome. The TD interaction with dsDNA was

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18 studied by AFM and DP voltammetry at GCE, UV-vis spectrophotometry and electrophoresis [44]. After incubation of dsDNA with TD, AFM and voltammetry showed modifications of the TD-DNA adsorption and redox behaviour, depending on the TD concentration and incubation time. A model was proposed for the TD-DNA interaction, considering that TD intercalates into the dsDNA, causing unwinding and defects in the dsDNA secondary structure. Moreover, DNA condensation oxidative damage was detected electrochemically by the appearance of the 8-oxoG and/or 2,8-DHA oxidation peaks. Aripiprazole (ARP) is an atypical antipsychotic agent used to treat cognitive deficit symptoms in patients with schizophrenia, which acts through dopamine D2 partial agonism, serotonin 5-HT1A partial agonism and 5-HT2A antagonism. Neurobehavioral effects and genotoxic/mutagenic activities of the ARP were investigated and demonstrated increased DNA strain-break damage in peripheral blood but not in the brain, suggesting a direct effect of ARP on the long-term genomic stability. The dsDNA-ARP interaction was investigated by DP voltammetry and UV-vis spectrophotometry in incubated solutions and using a dsDNAelectrochemical biosensor [45]. The binding constant between dsDNA and ARP in pH = 4.7, K  3 × 105 M−1, was obtained spectrophotometrically. Moreover, the dsDNA-ARP association was confirmed by voltammetry and spectrophotometry in mixed solutions of either poly(G) or poly(A) with ARP. The interaction between ARP and ultraviolet C (UVC) radiation-damaged dsDNA was also investigated using DP voltammetry. UVC is the shortest and highest energy UV with wavelengths less than 290 nm, the most damaging type of UV radiation. However, it is completely filtered by the atmosphere and does not reach the earth's surface. When the UVC radiation was applied to the dsDNA-electrochemical biosensor, the dGuo response increased due to helix structure opening. The damaged dsDNA-electrochemical biosensor was incubated in

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19 ARP and a greater decrease in dGuo signal was observed when compared to the dsDNAelectrochemical biosensor control. Nitrofurantoin (NFT) is an antibacterial drug that acts at the inflammation site produced by various gram-negative and gram-positive bacteria. The dsDNA-NFT interaction was monitored by DP voltammetry at a DNA-electrochemical biosensor fabricated by modifying a GCE with poly(5-amino-2-mercapto-1,3,4-thiadiazole) (PAMT) [46]. The GCE/PAMT/dsDNA electrode was prepared by adsorption of dsDNA upon deposition of PAMT at the GCE surface, and the decrease of the G oxidation peak current was used as an indicator of the NFT-dsDNA binding. The reproducibility, repeatability, stability and applicability of the analysis to pharmaceutical dosage forms in human serum samples, were examined, demonstrating that the GCE/PAMT/dsDNA-electrochemical biosensor could be used for the sensitive, accurate and precise determination of NFT-dsDNA interaction with a detection limit of 0.65 mg L-1. Natural products are the greater contributors to the production of active products, and many are used as drugs. Biflorin is a prenylated οrtho-naphthoquinone, isolated from the roots of Capraria biflora, a perennial shrub distributed in North and South Americas, which demonstrated cytotoxic activity against several tumour cell lines indicating an antitumor therapeutic potential. However, the exact mechanisms underlying its activity still remain unclear. The pharmacoelectrochemistry of biflorin was evaluated by electrochemistry and spectrophotometry in the presence of ssDNA, dsDNA and isolated DNA bases [47]. DNA-biflorin binding constants were obtained by DP voltammetry and fluorimetry. Spectroscopic studies and thermodynamic data had shown that biflorin can intercalate with dsDNA through van der Waals interactions and hydrogen bonds. The effects of biflorin-dsDNA interaction were addressed through a molecular cytogenetic approach, using the comet assay and the chromosome aberration induction evaluation. Biflorin, compared to the negative control, presented approximately 4- and 6-fold

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20 increase in DNA damage. However, biflorin did not significantly induced chromosome aberrations, suggesting that it does not possess clastogenic potential, but cytotoxic potential. The absence of either clastogenic or aneuploidogenic activity of the compound reinforced its safety.

3.3

Metal ions and their complexes Epidemiological studies have shown that occupational and environmental exposure to

specific metals is associated with an increased risk of different cancers and adverse health effects. Transition and post-transition metal ions can interact specifically with DNA inducing partial disordering of the B-form DNA and reduction of base stacking and base pairing. However, consensus over the transition and post-transition metal ions’ direct interaction with dsDNA is still needed. The in situ evaluation of the direct interaction of chromium species with dsDNAelectrochemical biosensors was studied using DP voltammetry at a GCE [48]. The DNA damage was electrochemically detected following the changes in the dGuo and dAdo oxidation peaks. The results obtained revealed the interaction with dsDNA of the Cr(IV) and Cr(V) reactive intermediates of Cr(III) oxidation by O2 dissolved in the solution bound to dsDNA. This interaction leads to different modifications and causes oxidative damage in the DNA structure. Using poly(A) and poly(G)-electrochemical biosensors, it has been shown that the interaction between reactive intermediates Cr(IV) and Cr(V) with DNA causes oxidative damage, and takes place preferentially at G-rich segments, leading to the formation of 8-oxoG, the oxidation product of G residues and a biomarker of DNA oxidative damage. The interaction of Cr(VI) with dsDNA caused breaking of hydrogen bonds, conformational changes, and unfolding of the double helix, which enabled easier access of other oxidative agents to interact with DNA, and the occurrence of DNA oxidative damage.

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21 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 [49]. The AC voltammetric signal sensitive to the formation of DNA strand breaks increased after incubation of the DNA-modified electrode in micromolar solutions of Cr(VI) at potentials sufficiently negative for the Cr(VI) reduction. Damage to DNA in solutions containing Cr(VI) and a chemical reductant (ascorbic acid) was observed only at relatively high chromium concentrations (hundreds of µM). To eliminate interferences of excess Cr(VI) in the measurements of the G electrochemical signals, a magnetoseparation double surface electrochemical technique was introduced. Using this approach, DNA damage in solution was detected for 50-250 µM Cr(VI) upon addition of 1 mM ascorbic acid. The results suggested a more efficient DNA damage at the electrode surface due to continuous production of the reactive chromium species, compared to DNA exposure to chromium being reduced chemically in solution. The evaluation of the interaction of lead, cadmium, nickel and palladium divalent cations with dsDNA, forming a metal-DNA complex, was studied by AFM on HOPG and DP voltammetry at GCE [50,51]. The electrochemical behaviour of these metal-DNA complexes was related to the different adsorption patterns and conformational changes. The dsDNA interaction was specific with each metal cation, inducing structural changes in the B-DNA structure, local denaturation of the double helix and oxidative damage. The AFM images showed an increase of the electrode surface coverage by lead-, cadmium- and nickel-DNA complexes (Fig. 7A, B, D). For cadmium- and nickel-DNA complexes oxidative damage to DNA was electrochemically detected for the concentrations studied (Fig. 7C). Palladium interaction with dsDNA induced condensation of the dsDNA secondary structure, which led to helices aggregation. The voltammetric data for the palladium-DNA complex showed a sharp decrease of the dGuo and dAdo oxidation peak currents, consistent with the AFM results for DNA condensation in the

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22 presence of palladium, but no DNA oxidative damage was detected, for the range of concentrations investigated. The mechanism of interaction of a lipoic acid-palladium complex (LAPd) with dsDNA, as well as the adsorption process and the redox behaviour of LAPd, of its ligand lipoic acid (LA), and of the LAPd-containing dietary supplement, Poly-MVATM, were studied using AFM and voltammetry [52]. In the presence of small concentrations of LAPd, the dsDNA appeared less knotted and bended, and more extended onto HOPG, when compared with the dsDNA adsorbed control. The voltammetric results demonstrated the interaction of both LAPd and Poly-MVATM with dsDNA, but no oxidative damage caused to dsDNA was detected. The LA, LAPd and PolyMVATM adsorption patterns depended on the chemical structures, the dimensions, the solution concentration and the applied potential. The LAPd molecules interacted and adsorbed strongly on HOPG, in comparison with LA, due to the incorporation of palladium into the ligand structure. The application of a negative potential caused the dissociation of the LAPd complex and Pd(0) nanoparticle deposition, whereas the application of a positive potential induced the oxidation of the LAPd complex and the formation of a mixed layer of LA and palladium oxides. The development of new chemotherapeutic agents led to the synthesis of polynuclear metal complexes, a new class of third generation anticancer agents with specific chemical and biological properties designed as alternatives to first-generation agents such as cisplatin. The biogenic polyamines spermidine (Spd) and spermine (Spm) were addressed in several studies of polynuclear metal complexes as potential antineoplastic drugs, due to their important biological activity and affinity for DNA. In this context, the interaction of dsDNA with two polynuclear Pd(II) chelates with Spd and Spm, Pd(II)-Spd and Pd(II)-Spm, as well as with the free ligands Spd and Spm, was studied using AFM, voltammetry, and gel electrophoresis [53]. The AFM and voltammetric results showed that the interaction of Spd and Spm with DNA occurred even for a

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23 low concentration of polyamines and caused no oxidative damage to DNA. The Pd(II)-Spd and Pd(II)-Spm complexes were found to induce greater morphological changes in the dsDNA conformation, when compared with their ligands. The interaction was specific, inducing distortion and local denaturation of the DNA structure with release of some G bases. The DNA strands partially opened give rise to palladium intra- and inter-strand cross-links, leading to the formation of DNA adducts and aggregates, particularly in the case of the Pd(II)-Spd complex. Metal complexes of fungal and plant secondary metabolites are in the centre of interest, especially due to their biological properties including cytotoxicity. The electrochemical behaviour of seven ternary copper(II) complexes of lawsone (2-hydroxy-1,4-naphthoquinone) with additional O-donor (water) and N-donor ligands (pyridine, 2-, 3-, and 4-aminopyridine, 3hydroxypyridine, and 3,5-dimethylpyrazole) using cyclic and DP voltammetry was studied [54]. The ability of these complexes to interact with DNA was also tested using the DNAelectrochemical biosensor on carbon paste electrode. The results indicated that the most simple complex Cu(lawsone)2(H2O)20.5 H2O showed significant prooxidant properties, which contributed to its cytotoxicity. In addition, all complexes evidenced ability to interact with dsDNA, and the interaction mechanisms were discussed. Polyphenols exhibit well-known chemoprotective effects, as well as prooxidant activity, via interactions with metal ions. Current research focuses not only on natural polyphenols but also on synthetically prepared analogues with promising biological activities. Quercetin, in the presence of transition metals, acts as a prooxidant, has mutagenic activity and intercalates into the dsDNA. Quercetin interaction with dsDNA was investigated electrochemically in incubated solutions [55] and using two types of DNA-electrochemical biosensors [56], in order to evaluate the occurrence of DNA damage caused by oxidized quercetin. The results showed that quercetin binds to dsDNA where it can undergo oxidation. The radicals formed during quercetin oxidation

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24 caused hydrogen bond breaks in the dsDNA, giving rise to 8-oxoG, since the dGuo and dAdo nucleotides in contact with the electrode surface were easily oxidized. A mechanism for oxidized quercetin-induced damage to DNA-electrochemical biosensors, prepared after dsDNA immobilization onto the GCE surface, was proposed and the formation of 8-oxoG was explained. The antioxidant and prooxidant properties of a semi-synthetic flavonolignan 7-Ogalloylsilybin (7-GSB) were described, and it has been shown that the presence of a galloyl moiety significantly enhances the antioxidant capacity of 7-GSB compared to that of silybin (SB) [57]. These findings were supported by electrochemistry, DPPH• (2,2-diphenyl-1-picrylhydrazyl radical) scavenging activity, total antioxidant capacity (CL-TAC) and DFT (density functional theory) calculations. A three-step oxidation mechanism of 7-GSB was proposed at pH 7.4 and confirmed by molecular orbital analysis. The Cu(II) complexation of 7-GSB was also studied and the prooxidant effects of the metal-complexes were then tested according to their capacity to induce DNA oxidative modification and cleavage. The results led to the conclusion that 7-Ogalloyl substitution to SB concomitantly enhances antioxidant (reactive oxygen species (ROS) scavenging) capacity and decreases the prooxidant effect/DNA damage after Cu complexation. This multidisciplinary approach provided a comprehensive mechanistic picture of the antioxidant vs. metal-induced prooxidant effects of flavonolignans at the molecular level, under ex vivo conditions.

3.4

Pollutants Pollutants are chemicals purposely or accidentally introduced in the environment by

agricultural and industrial processes, which cause short- or long-term damage in plants and animals. Even if biodegradable, their degradation products may possess toxic potential. From this

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25 point of view is of outmost importance to understand their effects on organism as well as to develop analytical methodologies for their sensitive detection. Cyanobacterial hepatotoxins microcystin-LR (MC-LR) and nodularin (NOD) are among the most commonly reported toxins produced by cyanobacteria [58]. Several previous studies have brought evidence for the possibility of direct induction of dsDNA damage in vitro and in vivo upon interaction with any of these toxins whereas other studies suggested that MC-LR and NOD genotoxicity and carcinogenicity arise mainly from the secondary effects of these toxins chemical degradation products rather than direct toxin-DNA interaction. The interaction between DNA and MC-LR and NOD was investigated using dsDNA-electrochemical biosensors and in incubated solutions and it was confirmed the decrease of the dsDNA oxidation peaks with time. It was shown that MC-LR, NOD and their chemical degradation products, interacted with the dsDNA causing the aggregation of dsDNA strands [59]. The analysis of dsDNA interaction with MC-LR or NOD in incubated solutions, where dsDNA strands are allowed to move freely and adopt the better conformations for and after the interaction, enabled the detection of adenine free residues (Fig. 8). The interaction between DNA and MC-LR and NOD, besides dsDNA aggregation, caused dsDNA abasic sites, which if left unrepaired can lead to mutations during the replication process. A DNA-electrochemical biosensor based on graphene-ionic liquid-Nafion modified pyrolytic graphite electrode (PGE) was developed by layer-by-layer assembly of DNA and horseradish peroxidase (HRP) for the detection of acrylamide (AA) and its product [60]. The PGE/graphene-ionic liquid-Nafion and the construction of the (HRP/DNA)n film were characterized by electrochemical impedance spectroscopy (EIS). Using the G signal as indicator, the DNA damage was detected by DP voltammetry after incubation in AA or AA+H2O2 solutions at 37 °C. The results indicated that, in the presence of H2O2, HRP was activated and catalysed the

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26 transformation of AA to glycidamide, which may form DNA adducts and induce more serious DNA damage than the AA. The DNA damage induced by AA and its products in solution were also investigated by UV–vis spectrophotometry, and similar results were obtained. Hydroquinone is a widely used chemical compound that can be metabolized to benzoquinone which presented haematotoxic, genotoxic and carcinogenic potential. Detection of hydroquinone was performed at a DNA-electrochemical biosensor constructed using chitosan (CTS) and polyaniline (PANI) [61]. The electrochemical behaviour of hydroquinone on the biosensor and its DNA-damaging mechanisms were investigated. The results showed that the DNA redox peak current was remarkably increased after GCE modification by PANI/CTS. The dsDNA damage by hydroquinone was concentration dependent and the G oxidation peak current of

dsDNA

decreased.

The

UV–vis

spectrophotometry

confirmed

that

applying

dsDNA/PANI/CTS/GCE to monitor hydroquinone was accurate and reliable, and that hydroquinone intercalated in dsDNA. Polycyclic aromatic hydrocarbons (PAHs) represent a large group of organic contaminants widely diffused in different ecosystems. Because of their hydrophobic nature, PAHs can easily cross cell membranes and tend to bioaccumulate in lipid tissues. The mutagenic/genotoxic effects of different PAHs have been proved, being classified as potentially carcinogenic by the International Agency for Research on Cancer (IARC). The best studied among the PAHs is benzo[a]pyrene (BaP) and IARC concluded in 1987 that it is a potential human carcinogen. The reactivity of photodegradation products of BaP versus DNA were assessed using genomic and ODN based DNA-electrochemical biosensors [62]. The kinetic of a photooxidation reaction of BaP carried out in controlled conditions using a 6 W UV lamp peaked at 365 nm has been studied using HPLC with fluorimetric detection. The degradation of BaP by both UV and UV/H2O2 exhibited pseudo-first-order reaction kinetics with half-lives ranging from

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27 3.0 to 9.8 h depending on the pH and on the amount of H2O2. The oxidation products of BaP obtained in different conditions were tested with the DNA-electrochemical biosensors prepared after immobilization on graphite or gold screen-printed electrodes. The G residues oxidation peak obtained using chronopotentiometry was used to detect the interaction of the BaP products with DNA. The dose-response curve obtained with BaP incubated was different from that of the parent compound indicating a different type of interaction with DNA. The formation of stable adducts between the G residues and the BaP oxidation products was described. HPLC with mass spectrometry detection of the oxidation products confirmed the presence of chemical species potentially forming adducts with DNA. The data reported demonstrated that DNAelectrochemical biosensors have the potential to be used to monitor remediation processes and to assess the potential toxicity vs. DNA of chemicals forming stable DNA adducts. Another strategy for the detection of BaP involved the layer-by-layer assembling of horseradish peroxidase (HRP) and double-stranded DNA at nafion-solubilized single-wall carbon nanotubes-ionic liquid (SWCNTs-NA-IL) composite film [63]. The biosensor was characterized by cyclic and DP voltammetry, EIS, scanning electron microscopy and computational methods. UV-vis spectrophotometry was also used to investigate DNA damage induced by BaP and its products in solution. The DNA-electrochemical biosensor was investigated separately in BaP, H2O2, and in their mixture. The analysis demonstrated the unwinding of DNA helix and exposure of the bases.

3.5

Free radicals ROS such as superoxide (O2-•), peroxyl (ROO•), and hydroxyl (OH•) radicals are

generated inside cells as products of metabolism, by leakage from mitochondrial respiration, and also under the influence of exogenous agents such as ionizing radiation, quinones, and peroxides.

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28 Excess ROS are responsible for causing DNA oxidative modifications and mutations, which can initiate carcinogenesis and may play a role in the development of several age-correlated degenerative diseases. Under aerobic conditions, transition metal ions caused DNA damage through production of ROS, frequently via Fenton-type reactions. Formation of strand brakes (sb) in covalently closed supercoiled (scDNA) were detected using an electrochemical biosensor based on a scDNA-modified mercury electrode [64]. scDNA anchored at mercury electrode was cleaved by catalytic amounts of Fe/EDTA ions in the absence of chemical reductants when appropriate electrode potential was applied, the process requiring oxygen or hydrogen peroxide. The extent of DNA damage increased with the shift of the electrode potential to negative values, displaying a sharp inflection point matching the potential of Fe(EDTA)]2-/[Fe(EDTA)]1- redox pair. In the absence of transition metal ions, significant DNA damage was observed at potentials sufficiently negative for reduction of dioxygen at the mercury electrode, suggesting cleavage of the surfaceattached scDNA by radical intermediates of oxygen reduction. Damage to DNA immobilized at the surface of GCE modified with silver nanoparticles and covalently attached neutral red conductive polymer was studied in a model system based on the Fenton reagent [65]. The oxidation process resulted in synchronous increase of electron transfer resistance and capacitance measured by EIS. The contribution of each sensor component on the signal was specified and sensitivity estimated against similar surface coatings. The shift of EIS parameters was found to be higher than that of similar biosensors. The DNA-electrochemical biosensor was tested for the evaluation of antioxidant capacity of green tea infusions. Among the electrochemical transducers, the boron-doped diamond electrode (BDDE) presents unique properties. The BDDE behaviour is strongly related to the controlled in situ electrochemical generation (by water discharge) of hydroxyl radicals and their subsequent

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29 reactions [66]. The effect of BDDE surface termination, immediately after cathodic and anodic electrochemical pre-treatment, and the influence of the pre-treatment, in different supporting electrolytes, on the electrochemical oxidation potentials of dsDNA, DNA bases, nucleotides, homopolynucleotides and biomarker 8-oxoG, in aqueous media at different pHs, were investigated [67]. The results demonstrated that the BDDE electrochemical properties were dependent on the surface functional groups. The interaction and adsorption of DNA and its components on the BDDE surface pre-treated cathodically was facilitated due to a BDDE higher conductivity. On the other hand, after anodic pre-treatment a wider potential window of BDDE was obtained enabling the detection of the pyrimidine bases. However, the hydroxyl radicals produced on BDDE during anodic pre-treatment were highly reactive, and consequently the BDDE surface was not completely inert. The in situ interaction and oxidative damage caused by hydroxyl radicals to dsDNA was also investigated using a thick multilayer DNA-electrochemical biosensor prepared onto the oxidized BDDE surface [68]. The BDDE allowed the generation of OH• at approximately + 3.00 V (vs. Ag/AgCl in pH = 4.5 0.1 M acetate buffer) [69] in agreement with the reaction: BDD + H2O  BDD(OH•) + H+ + eThe DNA-electrochemical biosensor on the BDDE enabled preconcentration of the OH• electrogenerated at the BDDE surface. Controlling the applied potential, different concentrations of OH• were electrochemically generated in situ on the BDDE surface. After monitoring the modification of the oxidation peak currents of the purine deoxynucleoside residues, it was found that OH• oxidatively damaged the immobilized dsDNA on the BDDE surface, leading to modifications in the dsDNA structure, exposing more purine residues to the electrode surface and facilitating their oxidation (Fig. 9). The dsDNA structural modifications were confirmed by

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30 electrophoresis and the DP voltammetric results demonstrated the occurrence of the 8-oxoG oxidation peak, due to the occurrence of DNA oxidative damage.

3.6

Radiation The interaction of electromagnetic radiation with living organisms has been studied for a

long time but is still an actual topic of research. The irradiation outcomes on living cells or tissues is difficult to analyse, since many biochemical processes are taking place at the same time, competing with the radiation effects [70]. Ionizing radiation can change the structure of biomolecules, creating potentially harmful effects. Although radiation has the potential to damage a multitude of biomolecules inside a cell, the structure of most concern is DNA. Radiation can be absorbed directly by DNA, leading to ionization of both the bases and sugar in a mechanism described as the direct effect of generating single and tandem DNA damage. Contrary, approximately 65% of the DNA damage is caused by the indirect effect of free radicals such as hydroxyl radicals that are formed from the radiolysis of surrounding water molecules and that successively attack DNA [71]. The interaction of  radiation with poly(G), poly(A), poly(T), poly(C), ssDNA and dsDNA was first studied by DP voltammetry at a GCE in aqueous solutions. A DNA-electrochemical biosensor was used for the detection of DNA damage by UV-C radiation and reactive oxygen species produced by the Fenton type reaction model as well as mineral water samples with additives [72]. Among different detection strategies, square wave voltammetry of DNA bases was used to characterize the time changes on the dsDNA structure. It has been shown that the G residues intrinsic response presented a method for the detection of dsDNA helix changes and single-strand breaks that depended on the incubation time in the cleavage medium.

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31 A similar approach has been used in the presence of CdTe quantum dots (QDs) [73]. In this report, the sensor was a GCE modified with a layer of dsDNA and another layer of CdTe QDs. It has been demonstrated that the size of the QDs exerted a significant effect on the rate of the degradation of dsDNA by UV-C light, and even by visible light. Time-dependent structural changes of DNA included unwinding of the double helix indicated by the increase of the redox response of the G moiety, due to easy electron exchange with the electrode surface when compared to the original double helix. The effects of QDs were verified for salmon sperm DNA and calf thymus DNA, and corroborated by experiments in which DNA solutions were irradiated in the presence of QDs.

4

Conclusion

The development of DNA-electrochemical biosensors, that use direct electrochemistry of DNA as a detection platform, designed from engineered DNA structures that self-assembled in unusual but biologically relevant structures, such as G-quadruplexes and i-motifs, were revisited. The influence of DNA sequence composition on DNA self-assembling properties and the understanding of the key factors in controlling the distribution, size and shape of highly ordered two- and three-dimensional DNA nanostructures on the electrode surface were referred. Combining the characteristics of DNA probes with the capacity of direct and label-free electrochemical detection has allowed applications in many different fields, from the investigation and evaluation of DNA oxidative damage and interaction mechanisms with pharmaceutical compounds, such as anticancer kinase inhibitors, anthracyclines, anti-cancer drugs, antipsychotic and antifungal drugs, to rapid monitoring metals, pollutant agents, and free radicals from homogenous redox reactions.

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32 The electrochemical and AFM investigation has been of great relevance to explain many biological mechanisms, and for nano- and biosensor technology applications of the DNAelectrochemical biosensors.

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

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E. Katz, B. Willner, I. Willner, Amplified electrochemical and photoelectrochemical analysis of DNA. In: Perspectives in bioanalysis, Vol. 1, Electrochemistry of nucleic acids and proteins – Towards electrochemical sensors for genomicss and proteomics, E. Palecek, F. Scheller, J. Wang (Eds.), Elsevier, Amsterdam, 2005, pp. 195-246, and references therein.

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A.M. Oliveira Brett, S.H.P. Serrano, J.A.P. Piedade, Comprehensive Chemical Kinetics, Applications of Kinetic Modelling, R. G. Compton (Ed.), Elsevier, Oxford, UK, (1999), Vol. 37, Ch. 3, p. 91, and references therein.

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A.M. Oliveira Brett, V.C. Diculescu, A.M. Chiorcea Paquim, S.H.P. Serrano, Chapter 20 DNA-electrochemical biosensors for investigating DNA damage, in: S. Alegret and A. Merkoçi (Eds.), Comprehensive Analytical Chemistry, Electrochemical Sensor Analysis, Elsevier, 2007, pp. 413-437, and references therein.

[10] A.M. Oliveira-Brett, Electrochemistry for probing DNA damage, in Encyclopedia of Sensors, C. Grimes (Ed.), American Scientific Publishers, 2006, Vol. 3, pp. 301-314, and references therein. [11] V. Brabec, E. Palecek, Interactions of nucleic acids with electrically charged surfaces: Part IV. Local changes in the structure of DNA adsorbed on mercury electrode in the vicinity of zero charge, J. Electroanal. Chem. 88 (1978) 373-385. [12] 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 surface characterisation, Electrochem. Commun. 5 (2003) 178-183. [13] A.M. Oliveira Brett, A.M. Chiorcea, Atomic force microscopy of DNA immobilized onto a highly oriented pyrolytic graphite electrode surface, Langmuir 19 (2003) 3830-3839. [14] A.M. Chiorcea, A.M. Oliveira Brett, Atomic force microscopy characterisation of an electrochemical DNA-biosensor, Bioelectrochemistry 63 (2004) 229-232. [15] A.M. Chiorcea Paquim, V. Diculescu, A.T.S. Oretskaya, A.M. Oliveira Brett, AFM and electroanalytical studies of synthetic oligonucleotide hybridization, Biosens. Bioelectron. 20 (2004) 933-944. [16] A.M. Oliveira Brett, A.M. Chiorcea, DNA imaged on a HOPG electrode surface by AFM with controlled potential, Bioelectrochemistry 66 (2005) 117-124.

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35 [17] A.M. Oliveira Brett, A.M. Chiorcea Paquim, V. Diculescu, T.S. Oretskaya, Synthetic oligonucleotides: AFM characterisation and electroanalytical studies, Bioelectrochemistry 67 (2005) 181-190. [18] V.C. Diculescu, A.M. Chiorcea Paquim, A.M. Oliveira Brett, Electrochemical DNA sensors for detection of DNA damage, Sensors 5 (2005) 377-393. [19] A.M. Chiorcea Paquim, T.S. Oretskaya, A.M. Oliveira Brett, Adsorption of synthetic homo– and hetero–oligodeoxynucleotides onto highly oriented pyrolytic graphite: atomic force microscopy characterization, Biophys. Chem. 121 (2006) 131-141. [20] A.M. Chiorcea Paquim, T.S. Oretskaya, A.M. Oliveira Brett, Atomic force microscopy characterization of synthetic pyrimidinic oligodeoxynucleotides adsorbed onto an HOPG electrode under applied potential, Electrochim. Acta, 51 (2006) 5037-5045. [21] A.M. Oliveira Brett, A.M. Chiorcea Paquim, V.C. Diculescu, J.A.P. Piedade, Electrochemistry of nanoscale DNA surface films on carbon, Med. Eng. Phys. 28 (2006) 963-970. [22] A.M. Chiorcea Paquim, J.A.P. Piedade, R. Wombacher, A. Jäschke, A.M. Oliveira Brett, Atomic force microscopy and anodic voltammetry characterization of a 49-mer DielsAlderase ribozyme, Anal. Chem. 78 (2006) 8256- 8264. [23] A.M. Chiorcea Paquim, P.V. Santos, A.M. Oliveira Brett, Atomic force microscopy and voltammetric characterisation of synthetic homo-oligodeoxynucleotides, Electrochim. Acta 110 (2013) 599-607. [24] V.C. Diculescu, A.M. Chiorcea Paquim, R. Eritja, A.M. Oliveira Brett, Thrombine Binding Aptamer Quadruplex Formation: AFM and Voltammetric Characterization, Journal of Nucleic Acids, (2010) Article ID 841932, 8 pages, doi:10.4061/2010/841932.

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36 [25] V.C. Diculescu, A.M. Chiorcea Paquim, R. Eritja, A.M. Oliveira Brett, Evaluation of the structure-activity relationship of thrombin with thrombin binding aptamers by voltammetry and atomic force microscopy, J. Electroanal. Chem. 656 (2011) 159-166. [26] A.M. Chiorcea Paquim, P. Santos, V.C. Diculescu, R. Eritja, A.M. Oliveira Brett, Electrochemical characterization of guanine quadruplexes, in: G.P. Spada (Ed.) Guanine quartets-structure and application, RCS Publishing, 2013, pp. 100-109. [27] A.M. Chiorcea Paquim, A.M. Oliveira Brett, Redox behavior of G-quadruplexes, Electrochim. Acta 126 (2014) 162-170. [28] A.M. Chiorcea Paquim, P.V. Santos, R. Eritja, A.M. Oliveira Brett, Self-assembled Gquadruplex nanostructures: AFM and voltammetric characterization, Phys. Chem. Chem. Phys. 15 (2013) 9117-9124. [29] A.D.R. Pontinha, A.M. Chiorcea Paquim, R. Eritja, A.M. Oliveira Brett, Quadruplex Nanostructures of d(TGGGGT): Influence of Sodium and Potassium Ions, Anal. Chem. 86 (2014) 5851-5857. [30] A.M. Chiorcea Paquim, A.D.R. Pontinha, A.M. Oliveira Brett, Time-dependent polyguanylic acid structural modifications, Electrochem. Commun. 45 (2014) 71-74. [31] L.I.N. Tomé, N.V. Marques, V.C. Diculescu, A.M. Oliveira-Brett, In situ dsDNAbevacizumab anticancer monoclonal antibody interaction electrochemical evaluation, Anal. Chim. Acta (2015) doi:10.1016/j.aca.2015.09.049. [32] I.B. Santarino, S.C.B. Oliveira, A.M. Oliveira-Brett, In Situ Evaluation of the Anticancer Antibody Rituximab-dsDNA Interaction Using a DNA-Electrochemical Biosensor, Electroanalysis 26 (2014) 1304-1311.

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37 [33] V.C. Diculescu, M. Vivan, A.M. Oliveira Brett, Voltammetric Behavior of Antileukemia Drug Glivec. Part III - In Situ DNA Oxidative Damage by the Glivec Electrochemical Metabolite, Electroanalysis 18 (2006) 1963-1970. [34] V.C. Diculescu, A.M. Oliveira Brett, In situ electrochemical evaluation of dsDNA interaction with anticancer drug danusertib nitrenium radical metabolite using the DNAelectrochemical biosensor, Bioelectrochem. (2015) doi:10.1016/j.bioelechem.2015.10.004. [35] B. Dogan-Topal, B. Bozal-Palabiyik, S.A. Ozkan, B. Uslu, Investigation of anticancer drug lapatinib and its interaction with dsDNA by electrochemical and spectroscopic techniques, Sensor. Actuator. B-Chem. 194 (2014) 185-194. [36] A.D.R. Pontinha, S.M.A. Jorge, A.-M. Chiorcea Paquim, V.C. Diculescu, A.M. OliveiraBrett, In situ evaluation of anticancer drug methotrexate-DNA interaction using a DNAelectrochemical biosensor and AFM characterization, Phys. Chem. Chem. Phys. 13 (2011) 5227-5234. [37] A.D.R. Pontinha, S. Sparapani, S. Neidle, A.M. Oliveira-Brett, Triazole-acridine conjugates: Redox mechanisms and in situ electrochemical evaluation of interaction with double-stranded DNA, Bioelectrochem. 89 (2013) 50-56. [38] A.M. Chiorcea Paquim, A.D.R. Pontinha, R. Eritja, G. Lucarelli, S. Sparapani, S. Neidle, A.M. Oliveira Brett, Atomic force microscopy and voltammetric investigation of quadruplex formation between a triazole-acridine conjugate and guanine-containing repeat DNA sequences, Anal. Chem. 87 (2015) 6141-6149. [39] V.C. Diculescu, A.M. Oliveira-Brett, DNA-electrochemical Biosensors and Oxidative Damage to DNA: Application to Cancer, in: V.R. Preedy, V.B. Patel (Eds.), Biosensors and Cancer, CRC Press, London, 2012, pp. 187-210.

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38 [40] A.M. Oliveira-Brett, M. Vivan, I.R. Fernandes, J.A.P. Piedade, Electrochemical detection of in situ adriamycin oxidative damage to DNA, Talanta 56 (2002) 959-970. [41] J.A. Piedade, I.R. Fernandes, A.M. Oliveira Brett, Electrochemical sensing of DNAadriamycin interactions, Bioelectrochem. 56 (2002) 81-83. [42] A.M. Oliveira-Brett, J.A.P. Piedade, A.-M. Chiorcea, Anodic voltammetry and AFM imaging of picomoles of adriamycin adsorbed onto carbon surfaces, J. Electroanal. Chem. 538-539 (2002) 267-276. [43] H.E.S. Kara, Redox mechanism of anticancer drug idarubicin and in-situ evaluation of interaction with DNA using an electrochemical biosensor, Bioelectrochem. 99 (2014) 1723. [44] S.C.B. Oliveira, A.M. Chiorcea Paquim, S.M. Ribeiro, A.T.P. Melo, M. Vivan, A.M. Oliveira Brett, In situ electrochemical and AFM study of thalidomide-DNA interaction, Bioelectrochem. 76 (2009) 201-207. [45] S. Kurbanoglu, B. Dogan-Topal, L. Hlavat, J. Labuda, S.A. Ozkan, B. Uslu, Electrochemical investigation of an interaction of the antidepressant drug aripiprazole with original and damaged calf thymus dsDNA, Electrochim. Acta 169 (2015) 233-240. [46] G. Aydogdu, G. Gunendi, D.K. Zeybek, B. Zeybek, S. Pekyardimci, A novel electrochemical DNA biosensor based on poly-(5-amino-2-mercapto-1,3,4-thiadiazole) modified glassy carbon electrode for the determination of nitrofurantoin, Sensor. Actuat. BChem. 197 (2014) 211-219. [47] M.C. de Vasconcellos, C. de Oliveira Costa, E.G. da Silva Terto, M.A.F.B. de Moura, C.C. de Vasconcelos, T.L.Go. de Lemos, L.V. Costa-Lotufo, R.C. Montenegro, M.O.F. Goulart, Electrochemical, spectroscopic and pharmacological approaches toward the understanding

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39 of

biflorin

DNA

damage

effects,

J.

Electroanal.

Chem.

(2015)

doi:10.1016/j.jelechem.2015.09.040. [48] S.C.B. Oliveira, A.M. Oliveira-Brett, In situ evaluation of chromium-DNA damage using a DNA-electrochemical biosensor, Anal. Bioanal. Chem. 398 (2010) 1633-1641. [49] J. Vacek, T. Mozga, K. Cahova, H. Pivonkova, M. Fojta, Electrochemical sensing of chromium-induced

DNA

damage:

DNA

strand

breakage

by

intermediates

of

Chromium(VI) electrochemical reduction, Electroanalysis 19 (2007) 2093-2102. [50] A.-M. Chiorcea-Paquim, O. Corduneanu, S.C.B. Oliveira, V.C. Diculescu, A.M. OliveiraBrett, Electrochemical and AFM evaluation of hazard compounds-DNA interaction, Electrochim. Acta 54 (2009) 1978-1985. [51] S.C.B. Oliveira, O. Corduneanu, A.M. Oliveira-Brett, In situ evaluation of heavy metalDNA interactions using an electrochemical DNA biosensor, Bioelectrochem. 72 (2008) 5358. [52] O. Corduneanu, A.-M. Chiorcea-Paquim, M. Garnett, A.M. Oliveira-Brett, Lipoic acidpalladium complex interaction with DNA, voltammetric and AFM characterization, Talanta 77 (2009) 1843-1853. [53] O. Corduneanu, A.-M. Chiorcea-Paquim, V. Diculescu, S.M. Fiuza, M.P.M. Marques, A. M. Oliveira-Brett, DNA Interaction with Palladium Chelates of Biogenic Polyamines Using Atomic Force Microscopy and Voltammetric Characterization, Anal. Chem. 82 (2010) 1245-1252. [54] P. Babula, J. Vanco, L. Krejcova, D. Hynek, J. Sochor, V. Adam, L. Trnkova, J. Hubalek, R. Kizek, Voltammetric Characterization of Lawsone-Copper(II) Ternary Complexes and Their Interactions with dsDNA, Int. J. Electrochem. Sc. 7 (2012) 7349-7366.

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40 [55] A.M. Oliveira-Brett, V.C. Diculescu, Electrochemical study of quercetin-DNA interactions. Part I - Analysis in incubated solutions, Bioelectrochem. 64 (2004) 133-141. [56] A.M. Oliveira-Brett, V.C. Diculescu, Electrochemical study of quercetin-DNA interactions. Part II - In situ sensing with DNA-biosensors, Bioelectrochem. 64 (2004) 143-150. [57] J. Vacek, M. Zatloukalova, T. Desmier, V. Nezhodova, J. Hrbac, M. Kubala, V. Kren, J. Ulrichova, P. Trouillas, Antioxidant, metal-binding and DNA-damaging properties of flavonolignans: A joint experimental and computational highlight based on 7-Ogalloylsilybin, Chem.-Biol. Interact. 205 (2013) 173-180. [58] I.C. Lopes, P.V. F. Santos, V.C. Diculescu, F.M. P. Peixoto, M.C. U. Araújo, A.A. Tanaka, A.M.

Oliveira-Brett,

Microcystin-LR

and

chemically

degraded

microcystin-LR

electrochemical oxidation, Analyst 137 (2012) 1904-1912. [59] P.V.F. Santos, I.C. Lopes, V.C. Diculescu, A.M. Oliveira-Brett, DNA-cyanobacterial hepatotoxins microcystin-LR and nodularin interaction electrochemical evaluation, Electroanalysis 24 (2012) 547-553. [60] Y. Qiu, X. Qu, J. Dong, S. Ai, R. Han, Electrochemical detection of DNA damage induced by acrylamide and its metabolite at the graphene-ionic liquid-Nafion modified pyrolytic graphite electrode, J. Hazard. Mater. 190 (2011) 480-485. [61] W. Tanga, M. Zhang, W. Li, X. Zeng, An electrochemical sensor based on polyaniline for monitoring hydroquinone and its damage on DNA, Talanta 127 (2014) 262-268. [62] M. Del Carlo, M. Di Marcello, M. Giuliani, M. Sergi, A. Pepe, D. Compagnone, Detection of benzo(a)pyrene photodegradation products using DNA electrochemical sensors, Biosens. Bioelectron. 31 (2012) 270-276.

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41 [63] 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 vitro detection of DNA damage, Int. J. Biol. Macromol. 79 (2015) 1004-1010. [64] M. Fojta, T. Kubicarova, E. Palecek, Electrode potential-modulated cleavage of surfaceconfined DNA by hydroxyl radicals detected by an electrochemical biosensor, Biosens. Bioelectron. 15 (2000) 107-115. [65] Y. Kuzin, A. Porfireva, V. Stepanova, V. Evtugyn, I. Stoikov, G. Evtugyn, T. Hianik, Impedimetric Detection of DNA Damage with the Sensor Based on Silver Nanoparticles and Neutral Red, Electroanalysis 27 (2015) doi: 10.1002/elan.201500312. [66] S.C.B. Oliveira, A.M. Oliveira-Brett, Voltammetric and electrochemical impedance spectroscopy characterization of a cathodic and anodic pre-treated boron doped diamond electrode, Electrochim. Acta 55 (2010) 4599-4605. [67] S.C.B. Oliveira, A.M. Oliveira Brett, Boron doped diamond electrode pre-treatments effect on the electrochemical oxidation of dsDNA, DNA bases, nucleotides, homopolynucleotides and biomarker 8-oxoguanine, J. Electroanal.Chem. 648 (2010) 60-66. [68] S.C.B. Oliveira, A.M. Oliveira-Brett, In situ DNA oxidative damage by electrochemically generated hydroxyl free radicals on a boron-doped diamond electrode surface, Langmuir 28 (2012) 4896-4901. [69] T.A. Enache, A.-M. Chiorcea-Paquim, O. Fatibello-Filho, A.M. Oliveira-Brett, Hydroxyl radicals electrochemically generated in situ on a boron-doped diamond electrode, Electrochem. Commun. 11 (2009) 1342-1345. [70] A. Deppman, J.O. Echeimberg, A.N. Gouveia, J.D.T. Arruda-Neto, F.M. Milian, N. Added, M.E. Camargo, F. Guzman, O.A.M. Helene, V.P. Likhachev, O. Rodriguez, A.C.G.

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42 Schenberg, V. Vanin, E.J. Vicente, Radiation interaction with DNA, Braz. J. Phys. 34 (2004) 958-961. [71] J.A.P. Piedade, P.S.C. Oliveira, M.C. Lopes, A.M. Oliveira-Brett, Voltammetric determination of  radiation-induced DNA damage, Anal. Biochem. 355 (2006) 39-49. [72] L. Hlavata, K. Benikova, V. Vyskocil, J. Labuda, Evaluation of damage to DNA induced by UV-C radiation and chemical agents using electrochemical biosensor based on low molecular weight DNA and screen-printed carbon electrode, Electrochim. Acta 71 (2012) 134-139. [73] L. Hlavata, I. Striesova, T. Ignat, J. Blaskovisova, B. Ruttkay-Nedecky, P. Kopel, V. Adam, R. Kizek, J. Labuda, An electrochemical DNA-based biosensor to study the effects of CdTe quantum dots on UV-induced damage of DNA, Microchim. Acta 182 (2015) 1715-1722.

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43 Schemes and Figures

Scheme 1 - DNA electrochemical biosensor principle of operation.

Scheme 2 - (A) Chemical structure of DNA bases, (B) Watson-Crick base-pairing (C) HomoODNs base-pairing and (D) Schematic representation of the DNA double-strand, G-quadruplex and i-motif configurations. [Adapted from Ref. [23] with permission].

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

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

Figure 1 - DP voltammograms baseline corrected recorded in a 20 M equimolar mixture of guanine (G), adenine (A), thymine (T) and cytosine (C) in pH = 7.4 with: (a) 1.5 mm, (b) 7 m diameter GCE. [From Ref. [7] with permission].

Figure 2 - (A) DP voltammograms base line corrected obtained with the GCE in solutions of 60 g mL-1 (••••) ssDNA and (▬) dsDNA. (B-E) AFM images of an HOPG electrode modified by free adsorption and adsorption at + 300 mV, vs. AgQRE, from solutions of (B) 60 g mL-1 ssDNA, (C) 1.0 g mL-1 ssDNA, and (D, E) 60 g mL-1 dsDNA [Adapted from Refs. [13,16] with permission].

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

Figure 4 - DP voltammograms baseline corrected, in 0.1 M phosphate buffer pH 7.0 and 100 g mL-1 dsDNA (

) before and (A) after incubation in solution with

100 g mL-1 BEVA during (▬) 0 and (•••) 48 h, and (B, C) after incubation in solution with (•••) 10 and (▬) 500 g mL-1 BEVA during (B) 0 and (C) 48 h. [From Ref. [31] with permission].

Figure 5 - DP voltammograms, base line corrected, in pH 4.5 0.1M acetate buffer with dsDNAelectrochemical biosensors (

) before and after incubation during (▬) 5, () 10

and (•••) 20 min in a solution of 100µM MTX. [From Ref. [36] with permission].

Figure 6 - GL15-d(TG4T) complex after 42 days incubation. AFM images and cross-section profiles through the white dotted lines: (A, B, C) d(TG4T) control and (D, E) GL15d(TG4T), in the presence of (A, B, D) Na+ and (C, E) K+ ions. DP voltammograms baseline corrected: (▬) d(TG4T) control, (

) GL15 control and (

) GL15-

d(TG4T), in the presence of (G) Na+ and (F) K+ ions. [From Ref. [38] with permission].

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45 Figure 7 - AFM images: (A) dsDNA and (B) Ni-DNA complex. (C) DP voltammograms in buffer of an immobilised thin-layer film on GCE of: () dsDNA from control solution after 24 h preparation, and Ni-DNA complex from a solution incubated with 2 mM Ni2+ during (

) 12 h and (▬) 24 h. (D) Cross-section profile through white

line in the image B. [From Ref. [50] with permission].

Figure 8 - DP voltammograms of 50 g ml-1 dsDNA solution in pH = 4.5 0.1 M acetate buffer ( ) before and after incubation with 30 μM MC-LR during () 0, (▬) 6 and (

)

24 h. [Adapted from Ref. [59] with permission].

Figure 9 - DP voltammograms in pH = 4.5 0.1 M acetate buffer with a thick multi-layer dsDNABDDE biosensor: (

) control and (

) first and (▬) 4th scans after applying + 3.0

V during 2 h to the BDDE surface causing electrogeneration of hydroxyl radicals. [Adapted from Ref. [68] with permission].

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46

Scheme 1

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47

Scheme 2

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48

Scheme 3

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49

Scheme 4

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50

Figure 1

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Figure 2

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Figure 3

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53

Figure 4

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54

Figure 5

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55

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

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Figure 7

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57

Figure 8

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58

Figure 9

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