This is the peer reviewed version of the following article: De la Cruz Morales K., Alarcón-Angeles G., Merkoçi A.. Nanomaterial-based Sensors for the Study of DNA Interaction with drugs. Electroanalysis, (2019). . : - . 10.1002/elan.201900286, which has been published in final form at https://dx.doi.org/10.1002/elan.201900286. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. Manuscript Click here to access/download;Manuscript;renamed_a05f5.docx Review ELECTROANALYSIS Nanomaterial-based sensors for the study of DNA interaction with drugs K. De la Cruz Moralesa, G. Alarcón-Angelesa, A. Merkoçi b,c* a Universidad Autónoma Metropolitana-Xochimilco, Departamento de Sistemas Biológicos, C.P. 04960, D.F. México Affiliation a b Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and BIST, Campus UAB, Bellaterra, 08193 Barcelona, Spain c ICREA – Catalan Institution for Research and Advanced Studies, Barcelona, 08010, Spain * arben.merkoci@icn2.cat Received: ((will be filled in by the editorial staff)) Accepted: ((will be filled in by the editorial staff)) Abstract The interaction of drugs with DNA has been searched thoroughly giving rise to an endless number of findings of undoubted importance, such as a prompt alert to harmful substances, ability to explain most of the biological mechanisms, or provision of important clues in targeted development of novel chemotherapeutics. The existence of some drugs that induce oxidative damage is an increasing point of concern as they can cause cellular death, aging, and are closely related to the development of many diseases. Because of a direct correlation between the response, strength/ nature of the interaction and the pharmaceutical action of DNA-targeted drugs, the electrochemical analysis is based on the signals of DNA before and after the interaction with the DNA-targeted drug. Nowadays, nanoscale materials are used extensively for offering fascinating characteristics that can be used in designing new strategies for drug-DNA interaction detection. This work presents a review of nanomaterials (NMs) for the study of drug-nucleic acid interaction. We summarize types of drug-DNA interactions, electroanalytical techniques for evidencing these interactions and quantification of drug and/or DNA monitoring. Keywords: Nanomaterials, drug-DNA, Biosensing, DNA-electroanalysis, drug-DNA interaction. DOI: 10.1002/elan.((will be filled in by the editorial staff)) 1. Introduction novel chemotherapeutics [5,6]. The process of interaction involves small or large molecules that selectively Deoxyribonucleic acid (DNA) represents one of the most recognize each other through different types of molecular important biomolecules because it contains all the interactions, i.e., electrostatic, dipole-dipole, hydrogen genetical material that encodes the information necessary bonding, π-π, and van der Waals interactions. The strength for the development of organisms with just four bases: of these interactions determines the stability of the adenine, guanine, cytosine, and thymine. The structure of resulting supramolecular complexes. The main chemical DNA is arranged in so manner that bases are protected driving forces, for both the stability of double-stranded from chemical modification by the environment. DNA (dsDNA) and DNA-drug recognition, are hydrogen The hydrogen bonding resulting from the base pairing bonding interactions, electrostatic and π-π interactions takes place at the center of the double helix while the between the aromatic rings of base pairs [3,7]. characteristic negative-charged phosphate backbone is on Furthermore, the existence of some drugs that induce the outside of the helix. On the outside of DNA, the spaces between the intertwined strands from two helical grooves oxidative damage is an increasing point of concern as they of different widths are called the major groove and the can cause cellular death, aging, and are closely related to minor groove. Hence, the detection of specific sequences, the development of many diseases, such as the base-pair composition or damage of DNA represents arteriosclerosis, ischemia/reperfusion injury, hepatitis, an important issue. Parkinson's and Alzheimer's diseases, rheumatoid arthritis, The interaction mechanism of drugs with DNA has been brain ischemia, stroke, and even carcinogenesis [8–11]. searched thoroughly during the past decades [1–4], giving This work presents a review of nanomaterials for the rise to an endless number of findings of undoubted study of drug-nucleic acid interaction. We summarize importance, such as a prompt alert to harmful substances, types of drug-DNA interactions, electroanalytical ability to explain most of the biological mechanisms, or techniques for evidencing these interactions, as well as the provision of important clues in targeted development of Review ELECTROANALYSIS use of nanomaterials in DNA biosensing and non-specific manner [12,18]. As a result, the ligands self- quantification of drug and/or DNA monitoring. associate and stack on the anionic DNA backbone to reduce charge-charge repulsion between ligand molecules. 1.2 DNA-drug interactions 1.2.4 Alkylators There are several types of interactions associated with ligands that bind DNA. These include intercalation, Alkylating agents are electrophilic enough to add methyl noncovalent groove binding, covalent binding/cross- or other alkyl groups to DNA bases forming covalent linking, DNA cleavage, nucleoside-analog incorporation adducts which are in fact irreversible [18]. These and oxidative damage [12]. Furthermore, several studies nucleophilic substitution reactions at the DNA bases occur have reported mixed or more than one binding modes for by SN1 (nucleophilic substitution 1, the rate-determining various small molecules interacting with DNA. This step is unimolecular) and SN2 (nucleophilic substitution 2, the rate-determining step is bimolecular mechanisms. The property of mixed binding mode can be linked to their most reactive sites in DNA are N(7) and N(3) in guanine mechanism of action and therapeutic efficiency. and adenine, respectively, as both are nucleophilic and more exposed in the grooves of the DNA duplex [5]. DNA 1.2.1. Intercalation alkylating agents have played an important part in cancer chemotherapy since the introduction of nitrogen mustards Intercalation is the most common mode of interaction by more than fifty years ago. Examples of these drugs include which small molecules may bind to DNA. Each drug has bis(benzimidazoles), pyrrolizidines and unique binding site preferences, for example, some prefer pyrrolobenzodiazepines [19], see Figure 1. to intercalate between a 5’-purine–pyrimidine-3’ base step rather than a 5’-pyrimidine–purine-3’ base step (or vice versa), whereas others have no preference of order, but 1.2.5 DNA cleavage prefer certain base-pair sequences [13]. Intercalators, Strand-breaking drugs induce DNA cleavage when the which contain planar heterocycle groups, can slide and covalent sugar-phosphate linkage between nucleotides is stack between the base pairs of DNA and stabilize the cut. Cleavage may be exonucleolytic, removing the end duplex without disrupting base pairing or forming covalent nucleotide, or endonucleotide, splitting the strand in two bonds. Moreover, the intercalation of a drug lengthens, [5,13]. Bleomycines are probably the most representative stiffens and unwinds double helix in DNA[5]. Examples antibiotics of this category since their discovery in 1966; of drugs that intercalate into DNA are acridine, enediyne antibiotics, such as calicheamicins, esperamicins anthracycline antibiotics, echinomycin [14]. and neocarzinostatins, can cleave double-stranded DNA as well [13]. 1.2.2 Groove binding Small molecules, which can bind to nucleic acid grooves, 1.2.6 Oxidative damage have the potential to achieve higher selectivity against Drug-induced oxidative stress is considered a drug-related desired targets compared to DNA intercalators [15]. The side effect and is implicated as a mechanism of toxicity in double-helix structure of DNA displays the major and many tissues and organ systems. The extent to which minor grooves having different width and depth, and one mechanisms of drug-induced oxidative stress have been end of the base pair is exposed to the major groove while characterized varies [10]. Oxidation of nucleic acids the other is exposed to the minor groove [13]. Is reported belongs to one of the most common damages to DNA. that, “the interaction drug-DNA is the sum DNA- Oxidative damage occurs through the formation of radicals intercalation and groove binding assisted by the formation and not radical species, such as ROS (reactive oxygen of additional molecular interactions with the nucleobases” species), nitrogen (RNS), or sulfur (RSS) species. To date, [16]. Drugs that bind to the minor groove are distamycin, more than 20 modifications to purine bases, which are the esperamicin, chromomycin and mitracycin [17] (see most easily oxidized, have been described [20]. Drugs that Figure 1). induce oxidative damage include anthracyclines, analgesics such as paracetamol, and antibiotics such as 1.2.3 External binding albendazole [21–23]. This kind of interaction occurs when some ligands are capable of forming electrostatic interactions with the negatively-charged nucleic sugar-phosphate structure in a Review ELECTROANALYSIS A B size of the binding sites [16]. It should also be possible to obtain kinetic data from current and potential measurements [18]. In recent years, much progress has been achieved in understanding the electrochemical and interfacial behaviors of nucleic acids (NAs) at electrodes, to the extent that researchers can now fine-tune the NA surface C D state and electrical properties at an almost atomic level [25]. Electrochemical methods are well suited for DNA analysis/diagnostics because electrochemical reactions give an electronic signal directly, so there is no need for expensive signal transduction equipment. Moreover, because immobilized probe sequences can be readily confined to a variety of electrode substrates, detection can be accomplished with an inexpensive electrochemical analyzer. Sensitive electrochemical signaling strategies, Fig. 1. DNA binding view from the three-dimensional structure based on the direct or catalyzed oxidation of DNA bases, of different complexes of the drug, A: Distamicyn-DNA duplex as well as the redox reactions of reporter molecules or in the minor Groove. B. Actinomycin-DNA duplex. C. enzymes recruited to the electrode surface by specific Esperamicin A1-DNA duplex, in the minor Groove of double- DNA probe target interactions and by charge transport stranded. D. Cisplatin-DNA intra-strand adduct. From reference [5] with permission from Elsevier. reactions mediated by the π-stacked base pairs, have all been demonstrated [26]. Electrochemical methods can be used for the detection 2. Electroanalytical techniques used to evidence drug- of: (a) DNA strand breaks and base damage; and (b) DNA interactions electroactive substances that specifically interact with DNA (covalently and/or noncovalently) [27]. Currently, there is a wide variety of strategies devoted to depicting drug-DNA interactions, which will continue to grow. Techniques range from the classic UV-Vis 2.1. Electrochemical detection (UltraViolet-Visible) spectrophotometry to the powerful Since the electroactivity of purine and pyrimidine bases tandem HPLC-MS. Needless to say, there is an urgent was found by Emil Palecek in 1958, working procedures need for rapid, high throughput, continuous, and cost- utilize this special detection principle. Bases have effective techniques for analysis of the interaction between electroactive properties, and they can be reduced and/or genes, proteins, and drugs to speed up drug discovery and oxidized; other components of nucleic acids such as sugar drug approval processes. It is particularly true for high and phosphate groups are electroinactive. All common throughput screening of trace molecules and natural nucleobases are known to undergo electrochemical products, where the amount of model DNA and/or drug oxidation at carbon electrodes, and protonation of base might be low or limited to weak absorption spectra, the residues is involved in the electrode process. overlap of electronic transition with that of DNA or weak Electrochemical analysis of the DNA can, thus, be or no absorption spectra [24]. performed without introducing labels into the DNA Electrochemistry is one of the most affordable recognition element (label-free techniques) and even without introducing any additional reagent into the techniques that has gained popularity over the last decades measuring system (reagent-less techniques)[28]. as it provides a useful complement to the standard methods Nevertheless, the electrochemical reduction and oxidation of investigation. Electrochemical methods are based on of nucleobases are irreversible and thus do not allow changes in the current and potential peak in a reusability of biosensors because of the destruction of the voltammogram (as a result of the change in the formal sample. The vast majority of label-free DNA biosensors potential or the change in the electron transfer rate) that employ oxidation of guanine. This choice is dictated by (a) can be attributed to the interaction among the species the relatively low redox potential of guanine and (b) the surrounding the electrode [2,6,12]. Information that can be fact that guanine is the most frequent target for many DNA provided based on the analysis of the current peaks and damaging species and its chemical modification is often potential include evidence for the interaction mechanism, accompanied by a loss of the guanine peak [29]. nature of the redox species formed, binding constant and Review ELECTROANALYSIS 2.1.1 Voltammetry However, Square Wave Voltammetry is considered as one of the most advanced voltammetric techniques, which Voltammetric ―especially cyclic voltammetry (CV), unifies the advantages of pulse techniques (enhanced differential pulse voltammetry (DPV), and square wave sensitivity), cyclic voltammetry (insight into the electrode voltammetry (SWV)― and chronopotentiometric (CP) mechanism) and impedance techniques (kinetic techniques are the most frequently used detection modes. information of very fast electrode processes)[43]. The Together with them, electrochemical impedance analysis in DPV is quite similar to CV and DPV, where spectroscopy (EIS) is becoming popular in DNA-based changes in the current peak and potential are parameters biosensors. that are associated with the event. Drugs such as Cyclic voltammetry is the most widely used technique wedelolactone, 6-thioguanine or oxidative damage for acquiring qualitative information about induced to DNA have been probed by using SWV [44,45]. electrochemical reactions. The power of cyclic voltammetry results from its ability to rapidly provide considerable information on the thermodynamics of the 2.1.2. Electrochemical Impedance Spectroscopy (EIS) redox process, on the kinetics of the heterogeneous The impedance measurement of the electrical interfaces electron-transfer reactions and coupled chemical reactions has been widely used in recent years for investigation of or adsorption processes. Cyclic voltammetry consists of the interactions of nucleic acids and their components with scanning linearly the potential of a stationary working the electrode surface. This technique offers several electrode (in an unstirred solution) using a triangular advantages over chronoamperometry and cyclic potential waveform. During the potential sweep, the voltammetry because the effects of solution resistance, potentiostat measures the current resulting from the double layer charging, and currents due to diffusion or to applied potential [30]. other processes occurring in the monolayer, are observed CV is widely used for the evaluation of mode of action more explicitly. Electrochemical impedance spectroscopy and binding strength of drug-DNA interaction. The peak (EIS) technique can provide information on the impedance potential and peak current of the compound change in the changes before and after the biointeraction process at the presence of DNA if the compound interacts with it. The electrode surface, which is mainly based on the change of variation in the peak current can be exploited for the the charge transfer resistance using a redox couple [46,47]. The EIS experiment is conducted using a sinusoidal determination of binding constant and binding size, electrochemical perturbation (potential or current) to the whereas the shift in peak potential can be used to ascertain sample that covers a wide range of frequencies. Therefore, the mode of interaction (18,31). Drugs that have been EIS allows the measurement of several electrochemical studied using CV and related techniques are nogalamycin, reactions that take place at very different rates and efavirenz, flutamide, anthracyclines, cisplatin, etopside provides a more thorough understanding of an and nevirapine [32–37]. electrochemical system than any other electrochemical On the other hand, differential-pulse voltammetry is an technique [48,49]. According to previous studies, EIS has extremely useful technique for measurement of trace been successfully applied for the detection of the levels of organic and inorganic species. In differential- interaction of topotecan, fluorouracil, benzaldehyde, and pulse voltammetry, fixed magnitude pulses, superimposed doxorubicin with DNA [50–53]. on a linear potential ramp, are applied to the working electrode at a time just before the end of the drop. The first 2.3 Voltammetric parameters involved in drug-DNA current is instrumentally subtracted from the second, and interaction this current difference is plotted versus the applied potential. The differential pulse operation results in a very There is a direct correlation between the response, effective correction of the charging background current strength/ nature of the interaction and the pharmaceutical [30]. DPV is probably the most used voltammetric action of the DNA targeted drugs. The electrochemical technique in drug-DNA interactions as it achieves high analysis is based on the signals of DNA before and after sensitivity. The analysis in DPV is quite similar to CV; the interaction with the DNA-targeted drug. The changes changes in the current peak and potential are parameters in the signals of an electroactive drug before and after the that are associated with the event. Examples of drugs that interaction with DNA also give an idea about what have been tested to interact with DNA using DPV are happens when these two molecules interact in the solution topotecan, codeine, gemcitabine, dextromethorphan and or at the surface [2,6,12]. The principle is based on the fact dacarbazine [38–42]. that the electrode detects the change in the DNA molecule. When an interaction occurs, changes in the current and Review ELECTROANALYSIS position of the voltammogram are visible for both DNA electrochemical signals, are manifested by either positive and drug (as long as the molecule is electroactive), because (for intercalative binding) or negative potential shifts (in of the altered availability of its electroactive sites. If the case of primarily electrostatic interaction) [5], see modification of electroactive base residues occurs, their Figure 2C. Interactions of intercalators or cationic groove intrinsic oxidation may be lost or may also be improved binders with the DNA recognition layer result in because of perturbations in the dsDNA structure. Some accumulation of these substances at the DNA-modified products of DNA base damage or bulky electroactive electrode leading to enhancement of the binder DNA adducts yield new electrochemical signals not electrochemical signals. This structure selectivity of some observed in the unmodified DNA. This is the case of one electroactive noncovalent or covalent DNA binders can be of the most abundant products of oxidative DNA damage, utilized in redox marker-based methods of electrochemical 8-oxoguanine, having lower redox potential than guanine, DNA structural probing. The redox indicators usually see Figure 2A. Formation of noncovalent DNA complexes show their electrochemical response at potentials different with small molecules can in principle be detected via enough from the nucleobases, which is often reversible. changes in electrochemical behavior of both the binders Some of the indicators interact with DNA through and the DNA. Upon binding to large molecules of DNA, electrostatic forces, while others are present “free” in the apparent diffusion coefficients of the binder decrease, and solution phase. Special kind of redox indicators are the altered mass transport of these species influences its intercalators. Due to the accumulation within the electrochemical signals [54]. Peak potentials of the binders immobilized dsDNA layer, the bound indicator exhibits an may also be changed upon the complex formation, increased voltammetric response. Commonly, redox- depending on the binding mode (see Figure 2B). Different indicator drugs are anticancer agents [55]. binding modes, specifically influenced by their A B (a) i G ox ii i ii iii iii 0.4 1.2 0.4 1.2 0.4 1.2 E (V) E (V) E (V) (b) iv II Ru2+ HMDE CE 3 G ox Ru3+ e- i ii i -1.2 -1.6 0.8 1.2 G ox E (V) E(V) CNo interaction D A 2.0 G DNA I (µA) D 1.0 MTZ Current μA 0.0 0.2 1.2 Interaction 2.0 Drug-DNA I (µA) OxoA OxoG 1.0 0.0 Potential (V) 0.2 1.2 E (V) Fig. 2. A. Principles of electrochemical detection of covalent DNA damages. (a) Chemical modification of nucleobases ). (b) Base lesions. B. Principles of electrochemical detection of noncovalent DNA interactions with small molecules C. Schematic representation of the change in the intercalator redox signal derived upon the interaction with DNA. From references [54] with permission from Wiley. Review ELECTROANALYSIS 2.5 Drug adsorbed on the working electrode 2.4. The approach of the drug-DNA interaction To prepare the drug-modified electrode, the targeted drug is immobilized on the electrode surface. The main Different electrochemical approaches have been carried requirement for this strategy is the electroactivity of the out for the analysis of nucleic acids after the discovery of drug so that the electrochemical signals of the drug can be nucleic acid electroactivity at the beginning of the 1960s. monitored and then the changes in these signals after Direct DNA electrochemistry, DNA specific redox interaction with DNA can be observed [60]. reporter electrochemistry, electrochemical amplifications Bulk electrodes with millimeter dimensions such as gold using nanoparticles, polymer-modified electrodes, etc. are (Au), Glassy Carbon Electrode (GCE), carbon paste, were different approaches that have been developed for the early used for implementation in bioanalysis; to date, they electrochemical DNA detection [56,57]. The highly are still widely used for the initial development of assays, specific molecular recognition ability of complementary including DNA-drug interaction, primarily due to the ease nucleic base pairs has been widely applied in the design of of their preparation and functionalization. Progressively, various sensing systems for the detection of target nucleic smaller microscale electrodes were developed, and it was acids [58,59]. For drug-DNA interactions, there are three noted that these devices displayed unusual kinetic main ways in which these studies can be performed. behavior when redox reactions were monitored in solution. Subsequently, it was discovered that ultramicroelectrodes 2.4.1. Interaction in the solution strongly benefited from radial diffusion and in this manner enhanced electrochemical reaction kinetics. Encouraged For detection of interaction in the solution, drug and DNA by these results, the pace of research into new electrode are placed in the same solution, and after some given time materials increased rapidly, and the use of microelectrodes of interaction, the changes in the electrochemical signals was recognized as an important advance [62]. of anticancer drug-DNA complex are compared to the signal obtained with DNA or drug alone [60]. 3. Nanomaterials 2.4.2 DNA adsorbed on the working electrode Nowadays it is recognized that nanomaterials have been produced and used by humans for hundreds of years. The immobilization of biomolecules via supramolecular or However, the understanding of certain materials as coordinative interactions has achieved wide acceptance in nanostructured materials is relatively recent. The recent years in binding biological species to surfaces. multidisciplinary nature of nanotechnology is the These two approaches, related to electrostatic interaction, distinguishing characteristic of this technology, in the π–π stacking, entrapment in polymers, or van der Waals manner that it has a notable impact on different areas forces between the nanomaterial and the biological entity, including health, energy, environment in addition to other preserve all specific properties of both. It is because industries. Publication of nanotechnology research and nanomaterials have the ability to reduce the distance review articles has increased in the previous years, and the between the redox site of immobilized protein or DNA and number of groups and laboratories engaged in the study of the working electrode surface since the rate of electron fundamental science, engineering and applications of transfer is inversely dependent on the exponential distance nanostructured materials has grown almost exponentially between them [59]. [63-67]. Invention of Adsorptive Transfer Stripping (AdTS) A nanomaterial (NM) can be defined as a material voltammetry of DNA (based on DNA immobilization/ having at least one characteristic length scale in the 1–100 adsorption onto the electrode surface followed by medium nm range, and with at least one property being exchange and measurement in a blank background considerably different from that of the bulk counterpart, as electrolyte) was the first step toward the development of a result of the nanoscale dimensions (68-70). electrochemical DNA biosensors. Upon exposure of the Nanomaterials (NMs) can also be larger objects that sensor to different chemical or physical agents (including contain nanoscale structures, either internally or on their species causing DNA damage or otherwise interacting surfaces (nanostructured materials), see Figure 4. with the anchored DNA molecules), changes in the anchored DNA recognition layer may take place, which can subsequently be electrochemically detected. 3.1 Classification Review ELECTROANALYSIS The concept of dimensionality is very useful in the grinding, engine exhaust, and smoke, or are synthesized by classification of nanomaterials. According to Siegel, physical, chemical, biological or hybrid methods [67,70]. nanostructured materials are categorized based on their Morphological characteristics to be considered include dimensionality. Zero-dimensional (0-D) nanomaterials are flatness, sphericity, and aspect ratio, which divide them in smaller than 100 nm in all directions (e.g., nanoparticles, low and high aspect ratio particles. The high aspect ratio nanopores), while one-dimensional (1-D) nanomaterials nanoparticles involve nanowires, nanohelices, are smaller than 100 nm in 2 directions only (e.g., nanozigzags, nanopillars, nanotubes, or nanobelts. The nanorods, nanowires, nanotubes), and two-dimensional (2- low aspect ratio nanoparticles are spherical, helical, pillar- D) nanomaterials are smaller than 100 nm only in the like, pyramidal, and cubic, among others [64,70,71,77], thickness direction (e.g., thin films, nanoplates). However, see Figure 3A. one should keep in mind that the critical length scale is not According to their uniformity, nanoparticles can be necessarily 100 nm, as it depends on the material and classified as isometric and inhomogeneous. From the point property under consideration [67,68,69,71,72]. For carbon of view of their agglomeration status, nanoparticles can be atoms, the main representative materials according to this dispersed or agglomerated. Their agglomeration state classification are: buckyballs (linear or 0-D), nanotubes depends on their electromagnetic properties, such as (1-D), graphene (plane or 2-D), and diamond and graphite surface charge and magnetism [78]. When in a liquid, their (tetrahedral or 3-D), [72–74], see Figure 3. agglomeration depends on their surface morphology and Nanoparticles (NPs) and nanoscale materials (NSMs) functionalization which can confer them either can also be classified as natural or synthetic, based on their hydrophobicity or hydrophilicity [79,80], see Figure 3A. origin. Natural nanomaterials are produced in nature either Nanoparticles can be made of a single material, compact by biological species or through anthropogenic activities. or hollow. Nanomaterials can also be comprised of two or The production of artificial surfaces with exclusive micro more materials and exist like coatings, encapsulated, and nanoscale templates and properties for technological barcode, or mixed. Most current NPs and NSMs can be applications are readily available from natural sources organized into four material-based categories: carbon- based nanomaterials, inorganic-based nanomaterials, (70,75,76). Synthetic (engineered) nanomaterials are organic-based nanomaterials and composite-based produced by several methods including mechanical nanomaterials [81,82], see Figure 3Aa. A B a b c d Fig. 3., A. Nanomaterials with different morphologies: (a) nonporous Pd NPs (0D) (b) Graphene nanosheets (2D) From reference [64,72] with permission from Springer Nature Publishing, (c) urchin-like ZnO nanowires (3D) From reference [75] with permission from Wiley, (d) WO3 nanowire network (3D), From reference [76] with permission from Wiley. B. Crystal structures of different allotropes of carbon. (Left to right) Diamond and Graphite (3D); Graphene (2D); Nanotubes (1D); and Buckyballs (0D). From references [71] with permission from Elsevier. Review ELECTROANALYSIS scale due to the huge surface energy. Self-purification is an intrinsic thermodynamic property of nanostructures and 3.2. Nanomaterials properties nanomaterials. Any heat treatment increases the diffusion Similar to quantum mechanisms, on the nanometer scale, of impurities, intrinsic structural defects, and dislocations, materials or structures may possess new physical and one can easily push them to the nearby surface. properties or exhibit new physical phenomena not seen in Increased perfection would have a visible impact on the their bulk components. Such differences may include chemical and physical properties [82]. altered chemical or biological activity; increased structural integrity; or altered electrical, optical, or magnetic 3.3. Nanomaterials in DNA biosensing properties [84]. The most obvious effect associated with the reduction of Nanomaterials are being explored for applications in scale is the much larger specific surface area or surface different disciplines including physics, medicine, area per unit mass. Surface atoms are less stable than bulk biomedicine, and chemistry to develop miniaturized atoms, which means that the surface of nanomaterials is devices [87–91]. By realizing the extraordinary properties more reactive than their bulk counterparts [85]. As the size of nanomaterials such as their high surface area, tuning of particles decreases towards the molecular level, their property in optical emission, electrical and magnetic behavior is more like that of a vapor; as a consequence, properties, etc., these can be exploited in bioengineering nanometer size particles will have much higher mobility ranging from drug delivery to biosensors [75,92–94]. The than particles in the micrometer scale [77]. Nanomaterials intelligent use of such nano-objects led to clearly enhanced may have a significantly lower melting point or phase performances with increased sensitivities and lowered transition temperature and appreciably reduced lattice detection limits of several orders of magnitude. constants, due to a huge fraction of surface atoms in the In recent years, nucleic acids have been extensively used total amount of atoms. Another property that NMs possess into a wide range of biosensors and bioanalytical assays, is related to the gravitational settling velocity which is due to their wide range of physical, chemical and proportional to their diameter. As a result, nanoparticles biological activities and tremendous molecular recognition suspended in the atmosphere may be deposited onto potential [95,96]. Functionally, integrating DNA and other surfaces much more slowly than larger particles. Indeed, nucleic acids with nanoparticles in all their different deposition of nanoparticles will be independent of physicochemical forms has produced a rich variety of orientation and will occur as they become trapped in the hybrid bio-nanocomposites which, in many cases, display boundary layer on all surfaces [77]. NMs tend to the unique or augmented properties due to the synergistic aggregation due to their specific surface area and volume activity of both components [97]. effects. One of the most fascinating and useful aspects of nanomaterials is their optical properties. The optical 3.4 Interactions DNA-nanomaterials properties of nanomaterials depend on parameters such as feature size, shape, surface characteristics, and other variables including doping and interaction with the 3.4.1 Carbon based surrounding environment or other nanostructures. Likewise, the shape can have a dramatic influence on the Carbon has long been applied as an electrochemical optical properties of metal nanostructures [86]. sensing interface owing to its versatility and unique Mechanical properties of nanomaterials may reach the electrochemical properties. Procedures in electroanalysis theoretical strength, which is one or two orders of strongly depend on material aspects such as chemical and physical properties of electrode surfaces, the effects of the magnitude higher than that of single crystals in the bulk applied potential, adsorption, and coatings applied to the form. The enhancement in mechanical strength is simply a electrode surface to enhance detection [72,73], see Figure result of the reduced probability of defects. Electrical 4A. Carbon-based nanostructures, such as carbon conductivity decreases with a reduced dimension due to nanofibers, carbon nanotubes, and mesoporous carbons, increased surface scattering. However, the electrical have been extensively used in the fabrication of modified conductivity of nanomaterials could also be enhanced electrodes for applications in both analytical and industrial appreciably due to the better ordering in the electrochemistry. In addition to their low price, they microstructure. Magnetic properties of nanostructured exhibit suitable electrocatalytic activity for a variety of materials are distinctly different from those of bulk redox reactions, a broad potential window, and relatively materials. Ferromagnetism of bulk materials disappears inert electrochemistry [98]. and transfers to superparamagnetism in the nanometer Review ELECTROANALYSIS Carbon nanotubes (CNTs) are certainly the most studied into 4 main types, sorted in ascending order of complexity: nanomaterials ever, considering the cumulated number of nanoparticles that increase the loading of electroactive papers devoted to them since the 1990s. Because of their species; nanoparticles that act as ultramicroelectrode reactivity properties, carbon nanotubes may also exhibit arrays for the electrolysis of large amounts of substrate; strong interactions with biological systems [99]. CNTs nanoparticles that are used as catalysts to deposit have a large surface area, small diameter, and high electrochemically detectable species; and nanoparticles curvature, which allow them to effectively interact with that mediate the deposition of electrocatalysts [113]. biomolecules through van der Waals, π-π stacking and Currently, many DNA sensing systems have been hydrophobic interactions [100], see Figure 4B. These integrated with Au NPs to enhance the detection limit and properties can also be used to facilitate the surface sensitivity [114]. modification of CNTs to increase their solubility in Electron transport properties of nanowires are very aqueous media or modulate the covalent attachment of important for electrical and electronic applications as well functional groups for biomedical application [101,102]. as for understanding the unique one-dimensional carrier Recently, graphene has attracted wide research interest transport mechanism. It has been noticed that the wire because of its superior electrical conductivity and diameter, wire surface condition, crystal structure and its excellent mechanical strength [103,104]. Since its quality, chemical composition, crystallographic discovery, graphene oxide (GO) has been used for orientation along the wire axis, etc., are important adsorbing many biomolecules because of its substantial parameters, all of which influence the electron transport surface-to-volume ratio to interface with biomolecules, mechanism of nanowires. Biological macromolecules, especially DNA [105,106], see Figure 5C. GO is a loosely such as proteins and nucleic acids, are typically charged in defined material, and the oxygen content can vary quite a aqueous solution and, as such, can be detected readily by lot depending on the preparation conditions [107]. The nanowire sensors when appropriate receptors are linked to adsorption affinity of DNA is likely to depend on the the nanowire active surface. Silicon nanowires and DNA oxygen content. Another related material is called reduced modified gold nanowires have been used to construct GO (rGO), which is prepared by chemically reducing GO DNA sensors [115]. The improved sensitivity attributed to to decrease its oxygen content. GO has poor electrical these materials strongly suggests that the three- conductivity due to its extensively disrupted π- π dimensional architectures of the nanowires facilitate the conjugation system; however, rGO has an intermediate electrocatalytic reaction because of enhanced diffusion conductivity and still retains the ability to disperse in water occurring around these structures. It means that unique [61,108]. effects at the nanoscale underlie the utility of nanowires for DNA biosensing [116,117]. Nanocomposites usually refer to dispersion-type 3.4.2 Nanoparticles-based coatings wherein one of the components is nanosized but The excellent conductivity, high surface area, and catalytic remains dispersed in a matrix [118]. Nanocomposites and redox properties make Au nanoparticles excellent properties are determined by component properties, material for electrochemical labels in nucleic acid composition, structure, and interfacial interactions as a detection, with numerous configurations being explored; result of improvement in the sensitivity and LOD (lower they provide an efficient tool for immobilizing DNA on limit of detection). Nanocomposites have also been electrodes as well as a label to signal the hybridization investigated as a means to create a suitable event [109,110]. The functionalization of AuNPs is microenvironment for the stabilization of biomolecules generally done through the Au–S bond, using [119]. biomolecules modified with thiol groups [111]. The association of nanomaterials such as noble metal Applications of nanoparticles in electrochemical signal nanoparticles with graphene sheets has become a hot topic amplification are mainly based on two types of and achieved remarkable results. The graphene mechanism: 1) nanoparticles that increase the loading of nanocomposites have been tested very widely [120]. the electrochemically detectable species since every Decoration of graphene by other carbon nanomaterials has nanoparticle contains thousands of atoms that can be given rise to new physical properties through cooperative electrochemically detected and 2) nanoparticles that and synergetic effects, and the functionalized composites catalyze the electrolysis of a large quantity of substrate, have found more applications in electrochemical sensing which enhances the electrochemical signal [112]. The fields where they were previously less competitive [121]. strategies that utilize nanoparticle labels in Metal NP/ graphene nanocomposites can not only act as electrochemical affinity biosensors can be sub-categorized excellent electrochemical sensing materials but also Review ELECTROANALYSIS provide an ideal matrix for the immobilization of enzyme retroviral [3,4,128-173]. Upon discovery of the advantages and DNA, enabling the design of highly selective and that nanomaterials could bring to analytical science, the sensitive electrochemical biosensors. Metaloporphyrins first researches regarding the DNA-drug interaction were and metalphthalocyanines (MPcs) can noncovalently bind done in solution. Later, more sophisticated methods to graphene via strong π-interactions offering stable, making use of complex architectures and immobilization functional matrixes, used in DNA sensing [122]. Polymers techniques to construct DNA biosensors were reported such as chitosan form nanocomposites with graphene (Table 1 y 2). Furthermore, these biosensors showed oxide, carbon nanotubes and AuNPs and have been clinical application to be used in the detection of drugs in employed for the development of DNA based biological matrixes. electrochemical biosensor for diagnosis [123–125]. In 2006, Zhongze Gu et al. [128] reported a new nanocomposite produced by the combination of A (polylactide) PLA nanofibers, fabricated by electrospinning, with titanium dioxide (TiO2) nanoparticles. They found that the combination could facilitate the interaction of daunorubicin with DNA in solution better than those with free nanocomposites or just nano TiO2 or PLA nanofibers so that much more significant decrease in the peak current occurs when both :Nucleoside nanomaterials are applied. This observation suggests the B C :Phosphate group II efficient accumulation or self-assembly of daunorubicin I and/or DNA molecules on the nanoscaled surface of the new nano TiO2-PLA polymer nanocomposites due to the relatively large surface area and unique properties of this Fully-hybridized dsDNA Sigle-stranded DNA new blending nanocomposites. III Similarly, in 2007, Wang et al. [129] explored the utilization of nano-titanium dioxide TiO2/GCE modified electrodes to enhance the detection of doxorubicin and its Partially-hybridized DNA biorecognition by DNA in solution upon applying UV Fig. 4.A. Schematic illustration. A. The multitude of possibilities irradiation. They explained that under UV irradiation, in terms of the combination of physical properties, surface some active radicals, such as hydrogen peroxide, hydroxyl chemistry, and functionalization which will be further modified radicals, and other ROS (Reactive Oxigen Species), are by the biological environment From references [79] with generated in such a manner that they could approach the permission from The Royal Society of Chemistry. B. The model doxorubicin and make the oxidation of doxorubicin easier. of DNA interaction with CNT. C. The interaction between graphene and (I) fully hybridized dsDNA, (II) ssDNA (single As such, more doxorubicin molecules could be readily stranded DNA), and (III) partially hybridized DNA. From self-assembled onto the surface of the nano-TiO2- references [126,127] with permission from Elsevier. modified electrode through electrostatic interaction, see Figure 5A. Moreover, they carried out experiments where they found that the surface of the electrode became much 3.5. Analysis of the drug-DNA interaction using more hydrophilic after the UV, while the corresponding nanomaterials change of contact angle values of doxorubicin takes place in the presence of DNA. In recent years, nanoscale materials are used extensively for offering fascinating characteristics which can be used in designing new strategies for drug-DNA interaction 3.5.1 Analysis of the drug-DNA interaction in a DNA detection. These nanoparticles provide the researchers biosensor with more efficient techniques for interfacing DNA recognition with electrochemical signal transduction. The One general advantage of all nanomaterials is that the recent electrochemical drug-DNA binding studies based high specific surface enables the immobilization of an on nanomaterials are summarized in Table 1; the types of enhanced amount of biomolecule units [73,99,130–133]. drugs that have been analyzed are anticancer agents However, one of the constant challenges is the antibiotics, antihistaminic, antivirals, nonsteroidal anti- immobilization strategy used to conjugate the bio-specific inflammatory drugs, neurotransmitters, vitamins and entity intimately on such nanomaterials. Since most of the Review ELECTROANALYSIS nanoparticles carry charges, they can electrostatically helix of the dsDNA that supports the bCNTs, making adsorb biomolecules with different charges. possible the sensitive quantification of PMZ. Since 1991, carbon nanotubes (CNTs) have received Bagheryan et al. in 2016 [169], reported a screen-printed great attention due to their outstanding properties; graphite electrode (SPE) modified with synthesized SBA- however, they still surprise us with their unlimited N-propylpipyrazine-N-(2-mercaptopropane-1-one) (SBA- applications in different disciplines. In 2012, Brahman and NPPNSH) mesoporous structure to investigate the G colleagues [137] designed an MWCNT (Multiwalled quadruplex DNA (G4DNA) formation and stabilization. Carbon Nanotubes) paste electrode and developed a Human telomeric DNA typically consists of many tandem method which served to monitor the interaction of the repeats of the guanine-rich sequences d(TTAGGG) termed fluoroquinolone antibiotic, gatifloxacin (GTF) with DNA, an intermolecular G-quadruplex structure (Table 1). in solution and on DNA modified electrode. They Telomeres are highly repetitive sequences at the end of successfully constructed a DNA biosensor and found that chromosomes that are essential for genome stability. This GTF is oxidized at both electrodes, but the peak current structure plays an important role in the protection, decreases and peak potential shifts to more positive values stabilization, and replication of chromosome ends and is at the DNA-modified electrode in comparison with the thereby an active target for therapeutic purposes. It is bare electrode. Furthermore, they constructed a plausible that G-quadruplex structures in certain regions of pharmacosensor by modifying the working electrode with the genome also contribute to the inhibition of the activity GTF that allowed monitoring the decrement in the current of telomerase, a cancer-specific reverse transcriptase that of guanine and adenine. The analysis of the interaction was is activated in 80–90% of tumors. It has been reported that also made in solution, and the determination of the small molecules targeting the G-quadruplex structure constant binding and the ratio of the complex formed was through various binding modes can inhibit telomerase achieved using CV. The applicability of the proposed activity and interfere with telomere extension by blocking method was further extended to in vitro determination of the elongation step catalyzed by telomerase. In this regard, the drug in biological fluids such as serum, plasma, and choosing a ligand with a good affinity with G-quadruplex urine with DPV. to either stabilize this structure or induce the folding of this Primo et al. in 2014 [155], reported a study that single-stranded DNA is a good strategy for cancer therapy, represents a new alternative to build supramolecular as it could be a potential anticancer agent. architectures for biosensing. A GCE modified with Differential pulse voltammetry was used to examine the bamboo-like multi-walled carbon nanotubes (bCNT) stability and formation of G4DNA in the presence of dispersed in double-stranded calf-thymus DNA (dsDNA), positive and negative G4DNA binding ligands (Table 1). showed bioaffinity properties of the dsDNA that supports After the addition of ligand, another oxidation peak was the bCNT at GCE/bCNTdsDNA towards promethazine observed and increased by increasing ligand concentration (PMZ), despite the drastic conditions for preparing the at Epa=1.044 and 1.094 V due to the oxidation of guanine dispersion of bCNT in dsDNA. After comparing dsDNA in the G-quadruplex, with similar results to those observed and ssDNA, an increment in the oxidation current of PMZ in the presence of K+. In the second phase of the study, the about 19.7 times higher than at GCE and considerably competition of the complementary strand of the G- higher than the one obtained at GCE/bCNT-ssDNA was quadruplex structure with the drug TMPyP4, for binding observed. To demonstrate the nature of the interactions, to G-quadruplex was investigated in the presence and the effect of the ionic strength was evaluated; the results absence of K+ ion concentration. This study revealed that showed a decrease in the current peak of the drug upon after adding K+ or positive G4DNA-binding ligand, a new addition of NaCl evidencing that the main interaction peak was observed in higher potential due to oxidation of mode between PMZ and ssDNA was electrostatic, see guanine residuals in the G-quadruplex structure. This data Table 2. They suggested that although electrostatics plays revealed interesting information about the stabilization of some role in the interaction between PMZ and bCNT- the ligand and the G-quadruplex: 1) TMPyP4 has dsDNA due to the presence of the phosphate groups, in the stabilized G-quadruplex structure and interacted with it in case of dsDNA van der Waals interactions between the a good manner, 2) the ligand can also induce the DNA bases and PMZ, independent of the salt complementary strand to form the G4DNA structure and concentration of the medium, are the main factors so reduce the stability of dsDNA, 3) the formation of the responsible for the PMZ oxidation current. They G-quadruplex structure is stabilized by K+ cations as demonstrated that PMZ could be accumulated not only due observed in the DPV signals by the appearance of a new to the adsorptive effect of bCNTs but also to the oxidation peak in higher potential due to resistance to preconcentration mainly by intercalation within the double oxidation. Furthermore, the described method provides a Review ELECTROANALYSIS simple way for selecting a G quadruplex-binding ligand drug concentration in biological fluids is important as and can be used for selecting antitumor ligands and many side effects are related to this kind of compounds. therapeutic agents. Laboratory biosensors used for screening chemotherapeutic drugs and the assessment of DNA modification/damage caused by these drugs are currently 3.5.2 Analysis of the drug-DNA interaction in a being developed (see Table 2). In 2018, Hatamluyi et al. pharmacosensor [151], constructed a biosensor made of polyamidoamine In 2018, Shervedani et al. [167] published an interesting (PAMAM) dendrimer/reduced graphene oxide paper as a prove concept, where they reported the nanocomposite, using a modified pencil graphite electrode preparation of a pharmacosensor by immobilizing (PAMAM/RGO/PGE) for determination of Rituxan at low methotrexate (MTX) anticancer drug in three different concentrations, see Figure 5B. In this study, they ways: a) covalent bonding using EDC/NHS as the organic optimized the conditions to favor the interaction of the activators to form the amide bond, b) electrografting of drug and took advantage of the linear decrease in the MTX diazonium salt and c) noncovalent bonding, being oxidation peak current of guanine with the increase in electrografting the best method to immobilize the drug. To Rituxan to quantify the drug in human serum samples. evaluate the interaction ability of the immobilized MTX Furthermore, they calculated the binding constant of the with biological species they used calf thymus DNA Rituxan-DNA complex. In a similar study, Karimi-Maleh (ctDNA), mouse 4T1 breast tumor and Human foreskin et al. [156] used didanosine as a drug model to probe that fibroblast (hFF) cells as models of the primary the DNA biosensor fabricated by using a pencil graphite intracellular target of anticancer drugs, for cancer and normal cells, respectively. The interaction of MTX with electrode modified with conductive materials such as DNA was performed using DPV by means of the decrease polypyrrole (PPy) and reduced graphene oxide (rGO) in the current peak of the drug, which is electroactive. (PGE/PPy/rGO), see Figure 5C. Then, the interaction of Further information about the nature of the interaction was the drug with DNA was identified through decreases in the achieved with UV-Vis, which were electrostatic. oxidation currents of guanine and adenine by differential Interaction of GC-GNs-MTX(ER) and GC-GNs/MTX pulse voltammetric (DPV) method. However, the main systems with 4T1 cancer cells and hFF cells was traced by purpose of the novel biosensor was the measurement of EIS; the Rct was increased by a factor of ~4.5 and 5.3 upon didanosine in real samples. According to the obtained data, incubation. The authors concluded that the system showed the detection limit was found to be lower than in HPLC. a high affinity of the constructed systems for 4T1 cancer Another example was proposed by Ilkhani et al. [146], cells. who monitored the doxorubicin-DNA reactivity with a self-assembled monolayer protected gold-disk electrode 3.6. Quantification of drug and/or DNA monitoring (AuDE); this electrode was coated with a reduced graphene oxide (rGO) and decorated with plasmonic gold- Using the drug–DNA interaction, useful analytical coated Fe2Ni@Au magnetic nanoparticles, functionalized information can be obtained from the increased DNA with a sequence of the breast cancer gene BRCA1, see (guanine) response, in a manner that one can determine Figure 5D. They found differences in the electrochemical several drugs concentration using an electrochemical signals of a redox probe (Fe 2+), attributed to the approach in complex biological matrices [47,60, 134-164]. intercalation of doxorubicin with DNA using CV and Widely used anti-cancer treatments involving DPV, see Figure 5E chemotherapeutic drugs result in cancer cell damage due . to their strong interaction with DNA and monitoring of the Review ELECTROANALYSIS A B a Detection of Rituxan (RTX) b + EDC+NHS By ds-DNAPAMAM/RGO/PGE Current / μA PAMAM RGO PAMAM/RGO dendrimer (G4) nanocomposite ds-DNA absortion Potential /V DNA Drug nanocomposite PAMAM/RGO/PGE ds-DNAPAMAM/RGO/PGE C PGE PPy/rGO/PGE ds-DNA/PPy/rGO/PGE D Immobilization of Electropolymerization ds-DNA + 1 2 3 4 NH NH NH NH NH NH NH NH NH NH NH NH AuDE@CYS NH NH NH NH Pyrrole: NH 1. rGO; 2. AuNP or Fe2Ni@Au ; 3.pDNA ; 4.tDNA Graphene DNA: : Fig. 5. Schematic representation of biosensors used to detect DNA-drug interaction A. Electrochemical detection of daunorubicin binding to DNA at GCE (I) in the absence and (II) presence of the blends of nano TiO2 and PLA nanofibers. B. Fabrication of dsDNA/PAMAM/RGO/PGE biosensor and the detection of Rituxan. From references [128,138] with permission from Wiley. C. Surface amplification of pencil graphite electrode with polypyrrole and reduced graphene oxide for fabrication of a guanine/adenine DNA based electrochemical biosensors for determination of didanosine anticancer drug E. Construction of SERS/electrochemical nanobiosensor for testing doxorubicin interactions with DNA: gold disk electrode (AuDE) substrate protected with a Self-assembled monolaye (SAM) of cysteamine (CYS) and reduced graphene oxide (rGO) with covalently attached AuNPs or Fe 2Ni@Au NPs functionalized with dsDNA. From references [139,146] with permission from Elsevier Review ELECTROANALYSIS In 2011, Elahi et al. [161] constructed a biosensor based genotoxicity of diclofenac was reduced after on the physisorption of dsDNA on a polypyrrole nanofiber photodegradation. (PPyox) film that was electrochemically deposited onto a Lin et al. in 2017 [134] provided a method to prepare a Pt electrode. Then the interaction of DNA with salicylic modified glassy carbon electrode (GCE) using reduced acid (SA) and acetylsalicylic acid (ASA) was studied on graphene oxide-Nafion and dsDNA in order to produce a the electrode surface with DPV. Binding of DNA to the Pt- dsDNA/RGO-Nafion/GCE sensor to quantify Clenbuterol modified working electrode occurred because of the (CLB), b2 agonist typically employed in bronchial electrostatic attraction of the delocalized positive charges treatment, especially for the therapy of chronic illnesses. It of the oxidized polymer and the negative charges of DNA is illegally added to the feed for pigs and cattle to improve phosphate groups. The guanine oxidation currents from the yield of lean animals in some developing countries as the DNA/PPyox electrode decreased upon the addition of a kind of adrenal neural stimulant that promotes protein SA and ASA and over time, indicating the interaction of synthesis, which can accelerate the transformation and DNA with both drugs. A linear relationship was observed decomposition of fat. The dsDNA/RGO-Nafion/GCE between the decrease in the current of guanine oxidation produced more intense DPV peak currents for both and the concentration of both drugs. guanine and adenine. The experimental results showed They found interesting data with regard to the affinity of that the anodic peak currents of both guanine and adenine the drugs for DNA: a) the slope of the ASA reaction is decreased with the increase in immersion time and their smaller than that of the SA reaction, and the peak current anodic peak potentials shifted negatively. They suggested of guanine oxidation vanished and diminished to zero, that the electrochemical oxidation product of CLB is faster for the SA–DNA complex than for the ASA–DNA positively charged and interacts very readily with the complex, indicating faster interaction kinetics of SA negatively charged phosphonic groups of the backbone of compared to ASA with DNA, b) SA interacts at lower DNA. The dsDNA was surrounded by the oxidation concentrations compared with ASA, c) the binding product of CLB with a positive charge, so a decline of the constant, Kd, of SA for DNA was higher than ASA. oxidation peak currents for guanine and adenine was Finally, from the comparison of the structures of SA and detected. It was found that there is a proportional ASA, they hypothesized that the salicylate backbone plays relationship between the decrease in the anodic peak an important role in the DNA–ligand interaction, currents of the adenine and the concentration of added suggesting that the presence of an extra acetyl group in CLB, thus providing a good method to determine CLB ASA causes a slower interaction that may be due to steric indirectly (Table 2). hindrance of the acetyl group in the binding of salicylate Tahernejad-Javazmi et al. in 2018 [144] reported the backbone to the minor groove. construction of a biosensor by the modification of GCE Wei et al. in 2012 [163], investigated the interaction of with Au-NPs/rGO/dsDNA to monitor the interaction of diclofenac with DNA and took advantage of this to dasatinib with DNA and its determination in biological construct a graphene-based biosensor to monitor matrixes. They found that the guanine oxidation peak photodegradation (UV/H2O2) of the drug. Diclofenac, as a current decreased in the presence of dasatinib and the typical emerging pharmaceutical contaminant in the oxidation peak potential shifted to more positive potentials environment, has been the focus of many reports. Upon due to intercalation of this anticancer drug in major or increasing the concentration of diclofenac, they found that minor grooves of DNA. Also, they figured it out that this the peak current of DNA-GO/GCE decreased, while the reduction in current could be selected as a suitable factor peak potential shifted positively. It means that it could be relative to the concentration of dasatinib in the solution attributed to the fact that diclofenac intercalated between and saw that the biosensor was suitable for analysis of the base pairs of DNA inhibiting the electrochemical dasatinib in tablet and urine samples by the standard oxidation of DNA on the electrode (see Table 1 and 2). addition method. The peak current of guanine was found to linearly decrease Furthermore, they found that the temperature has an with the increase of diclofenac concentration; this important effect on the reaction speed. The guanine signal behavior was useful to evaluate the applicability of the reduces by moving the temperature from 10 °C towards DNA-GO/GCE biosensor as an analytical tool. The 25 °C due to the faster movement of dasatinib towards response of the sensor to photodegraded diclofenac was AuNPs/rGO/GC surface and better interaction with the increase in the current peak of guanine with prolonged dsDNA. Further increase in temperature (to 30 °C) was not degradation time, indicating that diclofenac was degraded suitable because a weak interaction between dsDNA and by the UV/H2O2 process. The result also implied that the dasatinib was detected. Finally, the binding constant was calculated (Table 2). Review ELECTROANALYSIS Table 1. Electrochemical drug-DNA binding studies based on nanomaterials (Part I) CLASS BINDING RESPONSE OF SUGGESTED DRUG TECHNIQUE RESPONSE OF THE DRUG REF. (EFFECT/USE) CONSTANT DNA MECHANISM Decrease in the oxidation current of β2 Agonist Clenbuterol DPV, EIS NM NM G and A and Electrostatic [134] negative potentials shifts Slight shift of the peak potential β-Adrenoreceptor Isoproterenol DPV, CV NM NM Increase in the Intercalation [135] oxidation current of G Decrease in the DPV NM NM oxidation current of Intercalation [136] Ciprofloxacin G and A CV NM Decrease in the peak current NM Intercalation [165] Antibiotic/ Decrease in the peak current Decrease in the Antimicrobial oxidation current of Gatifloxacin CV/DPV 3.820 L.mol-1 peak potential shifts to more Intercalation [137] G while A oxidation positive current increases Decrease in the peak current and a positive shift Electrostatic and Sulfadiazine CV 2.87 E3 L.mol-1 NM [138] intercalation in the peak potential Decrease in the 6-Tioguanine DPV, EIS NM Decrease in the peak current oxidation current of Stacking [140] Anticancer G Decrease in the peak current and Amsacrine DPV NM NM Intercalation [141] peak shift NM: not mention, CV: cyclic voltammetry, DPV: differential pulse voltammetry and, EIS: electrochemical impedance spectroscopy, PBS: Phosphate-buffered saline Review ELECTROANALYSIS Table 1. Electrochemical drug-DNA binding studies based on nanomaterials (Part II) CLASS BINDING RESPONSE OF THE SUGGESTED DRUG TECHNIQUE RESPONSE OF DNA REF. (EFFECT/USE) CONSTANT DRUG MECHANISM cis-diammine/ Binding to guanine Decrease in the oxidation dichloroplatinum II DPV, EIS NM No electroactive bases in the skeleton [142] current of G oxaliplatin of DNA Decrease in the oxidation GN7 reacting with Cisplatin DPV NM No electroactive [143] current of G cisplatin Decrease in the oxidation Dasatinib DPV 2.25 E5 L.mol-1 NM current of G and A and Intercalation [144] positive potentials shifts Decrease in the peak Decrease in the oxidation DPV, ESI NM Intercalation [47] Daunorubicin current current of G and A CV, DPV NM NM NM NM [128] Decrease in the peak Anticancer DPV NM NM Intercalation [146] Doxorubicin current DPV NM NM NM NM [129] CV NM NM NM NM [166] Methotrexate Decrease in the peak Electrostatic &/or DPV, EIS NM NM [167] current hydrogen bonding Decrease in the oxidation Mytomicin C DPV, EIS NM NM NM [168] current of G NM: not mention, CV: cyclic voltammetry, DPV: differential pulse voltammetry and, EIS: electrochemical impedance spectroscopy, PBS: Phosphate-buffered saline, ABS: Acetate buffer solution,:, G: guanine, A: adenine. Review ELECTROANALYSIS Table 1. Electrochemical drug-DNA binding studies based on nanomaterials (Part III) BINDING RESPONSE OF RESPONSE OF SUGGESTED CLASS (EFFECT/USE) DRUG TECHNIQUE REF. CONSTANT THE DRUG DNA MECHANISM Decrease in the peak Decrease in the Mytomicin C DPV NM Cross-linking [150] current oxidation current of G Decrease in the Rituxan DPV 1.33 E-6L.mol-1 No electroactive Groove binding [151] oxidation current of G Anticancer Decrease in the 1.85 E5 L.mol-1 NM Groove binding [152] oxidation current of G Spermidine DPV Electrostatic interaction, Decrease in the minor groove 4.08 E5 L.mol-1 NM [153] oxidation current of G binding TMPyP4 DPV NM NM NM NM [169] Decrease in the peak Topotecan DPV NM NM Intercalation [170] current and peak shift Increase in the peak antihistaminic Promethazine DPV NM NM Electrostatic [155] current Decrease in the Antiviral & retroviral Didanosine DPV NM NM oxidation current of G NM [156] and A NM: not mention, dsDNA: double-stranded DNA, , DPV: differential pulse voltammetry, PBS: Phosphate-buffered saline, ABS: Acetate buffer solution, G: guanine, A: adenine. . Review ELECTROANALYSIS Table 1. Electrochemical drug-DNA binding studies based on nanomaterials (Part IV) BINDING RESPONSE OF RESPONSE OF SUGGESTED CLASS (EFFECT/USE) DRUG TECHNIQUE REF. CONSTANT THE DRUG DNA MECHANISM Decrease in the peak current CV, SWV 2.0 E4 L.mol-1 NM intercalation [157] And positive peak Antiviral & retroviral Ganciclovir potential shift Disappearance of the Decrease in the EIS/ DPV NM [171] peak oxidation current of G Decrease in the peak Endocrine disruptor Bisphenol A DPV NM NM current and positive intercalative [158] peak potential shift Neuroleptic and Promethazine and Increase in the peak CV NM NM Intercalation [159] antidepressant chlorpromazine current SA 1.15 E-4 L.mol-1 Decrease in the NSAIDs/analgesic ASA, AS CV NM Electrostatic [161] oxidation current of G ASA: 7.46 10-5 L.mol- 1 Codeine and Increase in the peak Codeine: Electrostatic DPV NM NM [162] morphine current morphine: Intercalation NSAIDs/analgesic PBS: 6.51 E3 L.mol-1, Decrease in the peak Decrease in the Intercalative and Diclofenac DPV current and positive peak [163] oxidation current of G electrostatic CAS: 4.71 E4 L.mol-1 potential shift Decrease in the peak Vitamine Vitamine B1 DPV, CV 5.097 L.mol-1 current and negative peak MN Electrostatic [172] potential shift NM: not mention CV: cyclic voltammetry, DPV: differential pulse voltammetry and, EIS: electrochemical impedance spectroscopy, PBS: Phosphate-buffered saline, ABS: Acetate buffer solution, BRS: Britton- Robison solution, ASA: acetyl salicylic acid, SA: salicylic acid, NSAIDs: Nonsteroidal Anti-Inflammatory Drugs,CAS: Citric acid solution. Review ELECTROANALYSIS Table 2. Analytic parameters of Electrochemical DNA biosensors to detect drugs (Part I) AMOUNT CLASS LINEAR DRUG ELECTRODE OF IMMOBILIZED TECHNIQUE MEDIUM TIME (s) LOD REF. (EFFECT/USE) RANGE DNA dsDNA/rGO- 1000 µg/mL calf β2 Agonist Clenbuterol DPV, EIS pH 4.4 ABS 120 0.5 µM- 4.0 µM 0.32 µM [134] Nafion/GCE thymus dsDNA GCE/AuNP/PANI/MW 150000 µg/mL β-Adrenoreceptor Isoproterenol DPV, CV pH 7 PBS NM 20 μM -80 μM NM [135] CNT/DNA genomic DNA PGE/PPy/SWCNTs/DN 30 µg/mL Salmon 0.008 mM –30.0 Ciprofloxacin DPV pH 7 300 4.0 nM [136] A sperm dsDNA mM serum: 4.4 nM, Gatifloxacin MWCNT/DNA/ 100 µg/mL dsDNA CV/DPV pH 5 ABS 120 23 µM – 230 µM plasma: 2.5 nM, [137] Antibiotic/ urine 1.2 nM Antimicrobial 0.12 µM-0.51 µM 400 µg/mL calf Sulfadiazine MWCNT/DNA/GCE CV pH 6.5 RBS 240 and 2.4 µM -13.98 0.12 µM [138] thymus dsDNA µM 6- PPy/MWCNTs/ 20.0 µg/mL Salmon DPV pH 4.8 ABS NM 0.2 μM −100 μM 0.08 μM [139] Mercaptopurine DNA/PGE sperm dsDNA Anticancer 2 mM-10 mM and 1 6-Tioguanine SWCNT-PGEs/DNA 25 µg/mL dsDNA DPV, EIS pH 4.8 ABS 420 0.25 mM [140] mM-2.5 mM 10000 µg/L salmon Amsacrine GPE DPV pH 7 PBS 300 0.7 µM-100 µM 0.3 µM. [141] sperm dsDNA NM: not mention, CV: cyclic voltammetry, DPV: differential pulse voltammetry and, EIS: electrochemical impedance spectroscopy, PBS: Phosphate-buffered saline, MWCNT: multiwalled carbon nanotubes SWCNTs: single-walled carbon nanotube, G: guanine, A: adenine, GCE: glassy carbon electrode, , PPy: poly pyrrole, rGO: reduced graphene oxide, PGE: pencil graphite electrode, dsDNA: double-stranded DNA, NPs: nanoparticles, , GPE: graphene paste electrode ASA: acetyl salicylic acid, SA: salicylic acid, NSAIDs: Nonsteroidal Anti-Inflammatory Drugs, CV: cyclic voltammetry, DPV: differential pulse voltammetryand, EIS: electrochemical impedance spectroscopy, NM: Not Mention, dsDNA: double-stranded DNA, ssDNA: single-stranded DNA, CFME: carbon fiber microcylinder electrodes, PPy: Poly Pyrrole, GO: graphene oxide, GCE: Glassy Carbon Electrode, rGO: reduced Graphene Oxide, MWCNT: Multiwalled Carbon Nanotubes, CNT: Carbon Nanotube, CPE: Carbon Pencil Electrode, PANI: polyaniline, GE: graphite electrode, NP: Nanoparticle Review ELECTROANALYSIS Table 2. Analytic parameters of Electrochemical DNA biosensors to detect drugs (Part II) CLASS AMOUNT OF TIME LINEAR DRUG ELECTRODE TECHNIQUE MEDIUM LOD REF. (EFFECT/USE) IMMOBILIZED DNA (s) RANGE cis-diammine- 50 µg/mL calf thymus pH 4.5 dichloroplatinum SWCNTs-GE/DNA DPV, EIS 1800 NM NM [142] dsDNA ABS II oxaliplatin 10000 µg/mL fish sperm pH 4.7 G: 10- 100 mM G: 0.3 mM A: Cisplatin GCE/ERGO/DNA DPV 180 [143] dsDNA ABS A: 1- 100 mM 0.2 mM 50.0 µg/mL salmon pH 4.8 0.03 μM -5.5 Dasatinib Au-NPs/rGO/dsDNA/GCE DPV 240 ~9.0 nM [144] dsDNA ABS μM pH 4.8 Daunorubicin SWCNT/DNA/PGE 1500 µg/mL DNA DPV, ESI 1800 NM 184 nM [47] ABS DNA:5 DNA 2.71 µg/mL-30 pH=4.80 µg/mL rGO-PGEs/DNA 26 µg/mL of fsDNA DPV, ESI 60 µg/mL [145] ABS Anticancer Daunorubicin: Daunorubicin: 1 µM -6 µM 0.55 µM Doxorubicin AuDE/SAM/rGO/Fe2Ni@Au/dsDNA NM ssDNA DPV pH 7.4 PBS NM 55 µM-9.2 mM 14 µM [146] 8.2 nM-19 nM Calf thymus DNA DNA DNA: 8.6 Ag-nano/MWCNTs-COOH/DNA/GCE DPV pH 7 PBS NM 1.7 nM. [147] (NM) µg/mL-43 µg/mL pH 4.6 0.055 µM - Methotrexate GCE/GO/DNA 0.1 mol/L herring sperm DPV NM 7.63 nM [148] ABS 2.23 µM 120000 µg/mL dsDNA pH 4.8 57 mM to 344 Mitomicyn C GO/DNA/PGEs DPV 3600 25 µM [149] calf thymus ABS mM DPV: differential pulse voltammetryand,CV: cyclic voltammetry, EIS: electrochemical impedance spectroscopy, NM: Not Mention, PBS: Phosphate-buffered saline, ABS: acetate: buffer solution, PBS: phosphate buffer solution, SWCNT: single walled Carbon Nanotubes, GE: graphite electrode, G: Guanine, A:Adenine, dsDNA: double-stranded DNA, ssDNA: single-stranded DNA, GCE: Glassy Carbon Electrode, SAM: self-assembled monolayer, GO: graphene oxide, MTX: methotrexate, ER: electrografting, EDC: 1-ethyl-3(3-dimethylamino)-propyl) carbodiimide, NHS: N-hydroxysuccinimide, CNT: Carbon Nanotube, NPs: Nanoparticles, rGO: reduced Graphene Oxide, ERGO: electroreduced graphene oxide, PGE: Pencil Graphite Electrode, CHIT: chitosan, GNs: graphene nanosheets, PAMAM: Polyamidoamine dendrimers. Review ELECTROANALYSIS Table 2 Analytic parameters of Electrochemical DNA biosensors to detect drugs (Part III) AMOUNT OF CLASS LINEAR DRUG ELECTRODE IMMOBILIZED TECHNIQUE MEDIUM TIME (s) LOD REF. (EFFECT/USE) RANGE DNA 50 µg/mL calf DNA: Mytomicin C CHIT/CNT/DNA/PGE DPV pH 4.8 ABS 1800 DNA: 6.85 ug/mL [150] thymus dsDNA 0 and 60 µg/mL 7.0 – 100 µg/mL salmon Rituxan PAMAM/rGO/ DPV pH 4.8 ABS 2100 60.0 mM and 60.0 0.56 mM [151] sperm dsDNA -300.0 mM Au/Mixed azide and hydroxy- 1000 µg/mL Calf pH 7.4 Tris- Anticancer NM 1.6 µM -70.4 µM 0.72 µM [152] terminated SAMs/DNA thymus dsDNA HCl BS Spermidine DPV DNA/PPy-NFF / >10 µg pH 7.4 PBS 260 1.0 µM -15.0 µM 0.8 µM [153] (Pt) disk 10 µg/mL salmon- Tamoxifen GPE/DNA DPV pH 4.9 ABS 300 0.8 µM- 85 µM 0.1 µM [154] sperm (dsDNA ) 100 µg/mL dsDNA antihistaminic Promethazine GCE/bCN-dsDNA/DNA DPV pH 5 ABS 180 0.1 µM -6.0 µM 23 nM [155] calf thymus dsDNA PGE/PPy/rGO/ Didanosine NM DPV pH 4.8 ABS NM 0.02 μM -50 µM 8 nM [156] DNA Antiviral & retroviral 1000 µg/mL calf Ganciclovir Fe3O4/cMWCNTs/DNA/GCE CV, SWV pH 7.4 PBS NM 80-53 000 mM 20 nM [157] thymus dsDNA pH 7.0 Tris- Endocrine disruptor Bisphenol A dsDNA/CNT/TiO2/CPE 1 µM ssDNA DPV NM 0.1 μM -60 µM 0.56 µM [158] BS CV: cyclic voltammetry, DPV: differential pulse voltammetryand, SWV: square wave voltammetry, NM: Not Mention, dsDNA: double-stranded DNA, ABS: acetate buffer solution, PBS: phosphate buffer solution, PPy: Poly Pyrrole, GCE: Glassy Carbon Electrode, MWCNT: Multiwalled Carbon Nanotubes, CNT: Carbon Nanotube, CPE: Carbon Pencil Electrode, GE: graphite electrode, NP: Nanoparticle, NFF: nanofiber film, PGE: Pencil Graphite Electrode, bCN: bamboo carbon nanotube, GE: graphite electrode, NSAIDs: Nonsteroidal Anti-Inflammatory Drugs, ASA: acetyl salicylic acid, SA: salicylic acid, GPE: graphene paste electrode. Review ELECTROANALYSIS Table 2. Analytic parameters of Electrochemical DNA biosensors to detect drugs (Part VI) AMOUNT OF CLASS LINEAR DRUG ELECTRODE IMMOBILIZED TECHNIQUE MEDIUM TIME (s) LOD REF. (EFFECT/USE) RANGE DNA Promethazine Neuroleptic and 2000 µg/mL salmon 20 -160 µM DNA/AuNP/Cysteamine/GE CV pH 6.0 PBS 300 10 µM [159] antidepressant dsDNA 10 µM-120 µM Chlorpromazine 7 µM Dopamine 2 -500 µM 0.2 µM 100 µg/mL calf neurotransmitters DNA/nano Au/CFME DPV pH 7 PBS NM [160] thymus dsDNA Serotonine 0.8 -200 µM 0.8 µM SA 0.1-2 µM 8.62 E-1 µM Calf thymus dsDNA DNA/PPyox/Pt electrode CV pH 7.2 PBS NM [161] (NM) ASA 0.05 - 1 mM 5.2410-6 µM 1.67 – Codeine 0.137 µM 133.62 μM 1000 µg/mL ds NSAIDs/analgesic dsDNA/MWCNTs–PDDA/PGE DPV pH 7 BRS 600 [162] salmon sperm Morphine 1.75–147 μM 0.015 μM 500 µg/mL herring Diclofenac DNA/GO/GCE DPV pH 7.0 PBS NM 1 -130 μM NM [163] sperm dsDNA Progesterone disruptor Mifepristone DNA/AuNPs/ITO NM DPV pH 7 PBS NM 0.4 -6 µM 0.2 µM [164] NSAIDs: Nonsteroidal Anti-Inflammatory Drugs, DPV: differential pulse voltammetry, CV: cyclic voltammetry, NM: Not Mention, dsDNA: double-stranded DNA, ssDNA: single-stranded DNA, BRS: Britton-Robison solution, PBS: phosphate buffer saline, ABS: acetate buffer solution, GO: graphene oxide, GCE: Glassy Carbon Electrode, MWCNT: Multiwalled Carbon Nanotubes, CPE: Carbon Pencil Electrode, PDDA: Poly(diallyldimethylammonium chloride), CFME: carbon fiber microcylinder electrodes, PPy: Poly Pyrrole, MWCNT: Multiwalled Carbon Nanotubes, PGE: Pencil Graphite Electrode, PDDA: Poly(diallyldimethylammonium chloride), NPs: Nanoparticles Review ELECTROANALYSIS The ICN2 is funded by the CERCA programme / Generalitat de Catalunya. ICN2 acknowledges the support 5. Conclusions of the Spanish MINECO for the Project MAT2017-87202- P and through the Severo Ochoa Centers of Excellence This review summarizes the recent advances in the Program under Grant SEV2201320295. KDCM thank the application of nanomaterials for the detection of drug- DNA interactions using electrochemical techniques. CONACyT for the support through the scholarship Electrochemical techniques such as DPV and CV are the #817447. most commonly used to evaluate the interaction in spite of the high sensitivity and simplicity. These types of analysis 4. References are based on the changes in the peak current and peak potential of DNA signal: positive shifts of the peak [1] T.I. Abdullin, I.I. Nikitina, O.V. Bondar’. Russ. J. potential were observed in the binding via hydrophobic Electrochem. 2008, 44, 1345–1349. interactions, while electrostatic interactions led to negative [2] V.C. Diculescu, A-M Chiorcea-Paquim, A.M. shifts. 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