electrochemical dna biosensors based on dna-drug interactions

Review

Electrochemical DNA Biosensors Based on DNA-DrugInteractionsArzum Erdem,* Mehmet Ozsoz*

Department of Analytical Chemistry, Faculty of Pharmacy, Ege University, 35100 Bornova-Izmir, Turkeye-mail: [email protected] or [email protected]

Received: January 10, 2002Final Version: February 21, 2002

AbstractThe world of drug designing is ever changing. The investigations of drug-DNA interactions would provide newcompounds to be tested for an effect on a biochemical target, and also to be used as promising hybridization indicatorsfor the design of DNA biosensors, which will further become DNA microchip systems. An overview is reported hereabout DNA biosensors based primarily on drugs interacting with DNA and shows how to determine this interactionelectrochemically, the quantification of drug and/or DNA, and the promising applications of these drugs as DNAhybridization indicator. The applications of these electrochemical DNA biosensors are described and discussed.

Keywords: DNA interactions, drug interactions, Electrochemical DNA biosensors, DNA

1. Introduction

After discovery of electroactivity in nucleic acids at thebeginning of the sixties [1], many electrochemical ap-proaches have been performed for analyzing or quantifica-tion of nucleic acids. There has been an increase in the use ofnucleic acids as tools in the recognition and monitoring ofmany compounds of analytical interest. Nucleic acid layerscombined with electrochemical or optical transducersproduce a new kind of affinity biosensors for smallmolecular weight molecules. The activity in this directionis related upon the explanation on molecular interactionsbetween the surface linked DNA and the target pollutantsor drugs, in order to develop devices for rapid screening ofthese compounds [2 ± 6].The interaction of DNA with drugs is among the

important aspects of biological studies in drug discoveryand pharmaceutical development processes. A variety oftechniques from molecular biology have been used inresearch laboratories to study the effects of proteins anddrugs on gene expression, such as the gelmobility shift assay,filter binding assay, DNA foot-printing assay, and fluores-cence-based assays. However, most of these methods areindirect and discontinuous, and require various labelingstrategies. Moreover, the turnover time and cost of theconventional assays place a limit on the applicability oflarge-scale screening of drug candidates [7].There are several types of interactions associated with

ligands that bind DNA. These include intercalation, non-covalent groove binding, covalent binding / cross linking,DNA cleaving, nucleoside-analog incorporation. Conse-quences of these binding interactions involve changes toboth the DNA and drug molecules to accommodatecomplex formation. In many cases, changes to the structure

of the DNA duplex result in altered thermodynamicstability and are manifested as changes in the functionalproperties of the DNA [8].The interaction of drugs with DNA occurs principally by

three different ways [8, 9]:

1) Through control of transcription factors and polymer-ases. In this case, the drugs interact with proteins thatbind to DNA.

2) Through RNA binding to DNA double helix to formnucleic acid triple helical structures or RNA hybrid-ization (sequence specific binding) to exposed DNAsingle strand regions forming DNA-RNA hybrids thatmay interfere with transcriptional activity.

3) Small aromatic ligand molecules that bind to DNAdouble helical structures. These bindings to DNA occuras follows;

a) Electrostatic interaction with the negative-chargednucleic sugar-phosphate structure, which is generallynonspecific.

b) Intercalation of planar aromatic ring systems betweenbase pairs. Planar organic molecules containing severalaromatic condensed rings often bind DNA in an inter-calative mode; e.g., some antitumor antibiotics (dauno-mycin, doxorubicin, echinomycin, bleomycin, etc.).

c) Groove binding interaction: The drug interacts with twogrooves (minor and major) of DNA double helix.± Minor groove binding causes intimate contacts withthe walls of the groove, and numerous hydrogenbonding and electrostatic interactions with the basesand the phosphate backbone; e.g., Mithramycin,netropsin, etc.

± Major groove binding causes hydrogen bonding to theDNA, formingaDNAtriplehelix; e.g.,Norfloxacin etc.

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Electroanalysis 2002, 14, No. 14 ¹ 2002 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim 1040-0397/02/1407-0965 $ 17.50+.50/0

Fig. 1. Chemical structures of some DNA targetted drugs: a) Daunomycin (DM); b) Mitomycin C (MC); c) Promethazine (PRM); d)Epirubicin (EPR) and e) Echinomycin (ECH).

966 A. Erdem, M. Ozsoz

Electroanalysis 2002, 14, No. 14

There is a direct correlation between the response, strength /nature of the interaction and the pharmaceutical action ofthe DNA targeted drugs. Observing the electrochemicalsignals of DNA before and after the interaction with theDNA-targeted drug can provide evidence for the interac-tion mechanism. The changes in the signals of an electro-active drug before and after the interaction with DNA alsogives an idea about what happens when these twomoleculesinteract in the solution or at the surface. Aromatic moieties

of intercalative molecules (e.g., the chemical structures ofsome electroactive anticancer drugs have been illustrated inFigure 1) penetrate into the DNA double-helix betweenadjacent base pairs, being bound predominantly by stackinginteractions. Side groups of the molecules can make addi-tional contacts (via hydrogen bonds or electrostatic inter-actions) with edges of base pairs or sugar-phosphate back-bone in major or minor groove, or at the surface-phosphatebackbone in major or minor groove, or at the surface of the

Table 1. Summary of the recent electrochemical investigations of interactions between anticancer drugs and/or other DNA-targetedmolecules and DNA.

Drug Technique Response of drug Response of DNA Electrode References

Doxorubicin Adsorptive transferstripping alternativecurrent voltammetry,differential pulsevoltammetry

Decrease in thevoltammetric current ofthe drug due to its in-tercalation

± Hanging mercury dropelectrode

[18, 29]

Daunomycin Chronopotentiometricstripping analysis,cyclic voltammetry

Decrease in thevoltammetric currentof the drug due to itsintercalation

Almost no changes inguanine signal

Screen printed carbonelectrode, carbonpaste electrode, rotat-ing disk electrode

[19, 23]

Mitomycin C Cyclic voltammetry Decrease in the voltam-metric current of theacid activated mitomy-cin C

Decrease in the gua-nine oxidation signal

Hanging mercury dropelectrode

[24, 35]

Mitoxantrone Differential pulsevoltammetry, cyclicvoltammetry, square-wave voltammetry

Irreversible and pHdependent electroche-mical oxidation of thedrug

No specific interactionwith guanine or ade-nine bases

Glassy carbon elec-trode, carbon pasteelectrode

[25, 26]

Metronidazole Differential pulsevoltammetry

Shift in the peak poten-tial of drug

± Glassy carbon elec-trode, mercury thinfilm electrode

[28]

[Pt(dien)¥ (H2O)]2�

Differential pulsevoltammetry

± Decrease in the gua-nine oxidation signal

Paraffin-wax impreg-nated spectroscopicgraphite electrode

[30]

PromethazinePhenothiazineChlorpromazineThioridazineProchlorpera-zine

Potentiometric strippinganalysis, differentialpulse voltammetry

Increase in thevoltammetric current ofthe drug due to interca-lation

Decrease in the gua-nine oxidation signal

Carbon paste elec-trode

[31, 32]

Carboplatin Differential pulse vol-tammetry

± Increase in the oxida-tion signals of adeninedue to the covalentbinding of drug toDNA by cross linking

Glassy carbon elec-trode

[33]

Anthramycin Cyclic voltammetry,differential pulse vol-tammetry

Decrease in the drugsignal upon covalentlinkage

Decrease in the gua-nine oxidation signaldue to the covalentlinkage between theguanine and the iminoform of the drug

Hanging mercury dropelectrode

[34]

Mifepristone Differential pulsevoltammetry

Increase in the drugsignal due to its inter-calation

± Indium tin oxideelectrode

[36]

Epirubicin Cyclic voltammetry,differential pulse vol-tammetry

Decrease in the drugsignal due to its inter-calation

± Carbon paste elec-trode, glassy carbonelectrode

[38, 40]

967Electrochemical DNA Biosensors Based on DNA-Drug Interactions


DNA double-helix. When the groove-binding moleculesand intercalators are compared, it is clear that groove-binders display significantly greater interaction with DNAthan the intercalators [9].Significant progresses for drug targeting have been made

over the past few years in studies of drug-DNA interactionsand structure-based design strategies have yielded newDNA-binding agents with clinical promise [10 ± 17].There is an urgent need for rapid, high throughput,

continuous, and cost-effective techniques for analysis of theinteraction between genes, proteins and drugs in order tospeed up drug discovery and drug approval processes.Recent advances in automated DNA synthesis and theconvenient site-specific labeling of synthetic oligonucleo-tides with advances in microelectronics, have generated anumber of attempts at the development of novel biosensordevices for the analysis of specific gene sequences and forthe study of nucleic acid-ligand interactions [7].DNA biosensor (genosensor or gene-based biosensor),

normally employs immobilized DNA probes as the recog-nition element andmeasures specific binding processes suchas the formation of DNA-DNA and DNA-RNA hybrids,and the interactions between proteins or ligand moleculeswith DNA at the sensor surface [5].Typically, the design of a genosensor involves the follow-

ing steps: 1) modification of the sensor surface to create anactivated layer for the attachment of the DNA probe; 2)immobilization of the probe molecules onto the surface,preferably with controlled packing density and orientation;

and 3) detection of target gene sequence by DNA hybrid-ization at the sensor-liquid interface. For the study of ligand-DNA / RNA bindings and for drug-screening, the sensordesign will involve: 1) The formation of a duplex DNA orRNA layer with specific sequence on the sensor surface,either by direct immobilization of duplex DNA or byhybridization with an immobilized ssDNA probe, 2) manip-ulation of the duplex DNA by specific enzymes to create aDNA template with desired length and conformation; and3) monitoring the kinetics of protein or drug-binding withthe DNA template [7].In recent years, there has been a growing interest in the

electrochemical investigations of interactions betweenanticancer drugs and other DNA-targeted molecules andDNA [18-45] (summarized also in Table 1). Electrochemis-try offers great advantages over the existing devices basedon optical schemes, because electrochemical ones providerapid, simple and low-cost point-of-care detection ofspecific drugs. Electrochemical measurement systems areunique and suitable for fabrication of compact devices. Forthe electrochemical detection of the interactions betweendrug and DNA, the drug should be redox active; i.e., itshould either be oxidizable or reducible. These investiga-tions are mainly based on the differences in the electro-chemical behaviors of the drugs in the absence, andpresence, of double-stranded DNA (dsDNA): 1) thechanges as a dramatic decrease/ increase at the oxidation /reduction peak current of the drug which selectively bindswith dsDNA, and 2) the changes as a dramatic decrease/

Scheme 1. Schematic representation for designing electrochemical DNA biosensors based on DNA-drug interaction: A) Interaction atthe working electrode surface (CPE, HMDE, GCE, SPE etc.), a) DNA-modified electrode, b) drug-modified electrode. B) Interaction insolution phase. After the interaction step, the following steps were performed: washing and electrochemical measurements by usingvoltammetric techniques (DPV, CV, AdSTV, PSA, SWV). A representative increase or decrease of the voltammetric signal(s) of thedrug and/or DNA before and/or after the interaction is shown. During all electrochemical procedures and other steps, a three-electrodesystem consisting of working electrode, reference electrode (an Ag/AgCl or SCE), and auxiliary electrode (Pt wire) was used.



increase at oxidation / reduction peak current of theelectroactive DNA bases such as guanine or adenine; andalthough it has not been used as a detection approach, i.e., 3)the shifts of the formal potentials of the redox couple causedby the intercalation of nucleic acid-binding molecules intodsDNA.This review will focus on the developments in the design

of electrochemical DNA biosensors based on DNA-druginteractions. It is hoped that an overview of the currentstatus of DNA biosensor development based on DNA-druginteractions will discuss insights into the applications andfuture prospects in genomic pharmaceutical science.

2. Discussion

2.1. The Differences in the Electrochemical Behaviors ofDrug and/or Electroactive DNA Bases

The investigations due to interaction between DNA anddrugs are mainly based on the differences in the electro-chemical behaviors of the nucleic acid-binding molecules inthe absence or presence of double-strandedDNA; includingthe observed oxidation or reduction peak current of drugand/or that of guanine oxidation peak current resulting fromthe dramatic decrease, or the shifts of the formal potentialsof the redox couple caused by the intercalation of nucleicacid binding molecules into DNA double helix. Theseinvestigations were performed by using different electrodesfor the detection of interaction between different drugs andDNA at solution and/or in solution phase (see Scheme 1).At the first step, the electrodes are usually electrochemicallypretreated. If a DNA modified electrode was to beprepared, thenDNAwould be immobilized at the electrodesurface. The immobilization of DNA could be achieved bycovalent attachment, electrostatic attraction or in manydifferent ways, that were covered in numerous reviews. Theelectrochemical signals of guanine and adenine are meas-ured before the interaction. After the preparation of a freshDNA modified electrode, it would then be immersed intothe drug solution and after some given time of interaction,the differences in the intrinsic signals of DNA would beobserved. If a drug modified electrode was to be prepared,then drug would be immobilized at the electrode surface.The electrochemical signals of drugwould bemonitored andthen the changes in these signals after interactionwithDNAwould be observed. For the detection of interaction in thesolution, drug and DNAwould be put in the same solutionand after some given time of interaction, the changes in theelectrochemical signals of drug-DNA complex would becomparedwith the signals obtainedwithDNAor drug alonein the solution.Typical electrochemical responses in Scheme1 demonstrate the increase or decrease in the DNA and/ordrug responses associated with the binding event; beforeand after interaction between DNA and drug.Plambeck et al. [18] described a method based on the

electrochemical characteristics of both free and intercala-tively bound drugs such as some anthracyclines derivatives

by using mercury drop electrode. They reported that theassociation constants did not vary significantly with poten-tial when the conditioning potential ranges from � 0.4 to� 1.0 V (aganist SCE), nor does the addition of 3%acetonitrile alter their values, although it was observedsignificantly, a decrease as much as 30 % in these values. Itwas also reported that the association constant calculatedfor doxorubicin is significantly higher than that for daunor-ubicin. One of important conclusions reported was that thevalues obtainedwith solution rather than electrodemethodsalso tend to be consistently higher andmay refer to DNA ofa considerably more accessible to anthracyclines configu-ration than that on electrode surface.Wang et al. [19] have shown the interaction of anthracy-

cline antibiotic and antitumor drug daunomycin (DM) withdsDNA studied in solution and at the electrode surface bymeans of cyclic voltammetry and particularly by consant-current chronopotentiometric stripping analysis (CPSA)with carbon paste electrodes (CPE).The DM core, rings B and C, lies between base pairs, the

aminosugar which is attached to ring A lies in the minorgroove and ring D protrudes into the major groove. Theabove hexamer has two DM molecules intercalated be-tween the two guanine-cytosine pairs at the duplex ends [9].It was shown that as a result of intercalation of this drug

between the base pairs in dsDNA, the CPSA daunomycinpeak decreased [19] and a new more positive shoulderappeared at the potential as (�0.79) ± (�0.81) V. Thisshoulder was attributed to the oxidation of the drugintercalated in DNA. It was also reported that under thesame conditions, almost no changes in the oxidation peak ofguanine residues were observed. At very low DM concen-trations and/or at very short interaction time intervals atleast some molecules of DM bound to this face of DNA arenot accessible for electrooxidation. This is due to theirhiding in the interior of the DNA duplex or their binding tothe face ofDNAopposite to its contactwith theCPE surfaceeither by partial intercalation or binding at theDNAsurfacebeing too far from the electrode to undergo oxidation. Dueto the strong adsorption of DNA, RNA and other bioma-cromolecules to mercury and carbon electrode surface [20 ±22], it was tested if DM was strongly bound to CPE surfaceand this made it possible to define the interaction betweensurface confinedDManddsDNAin solution.Consequently,they observed the more well separated peak instead ofshoulder and the highest decrease at theDMpeak, when theimmersion time of DM-modified CPE in the DNA solutionwas increased; i.e., 10 min.Another study based on DM-DNA interaction was

reported by Chu et al. [23]. The cyclic voltammetricbehavior of DM in aqueous medium on addition of DNAhave been studied and utilized for quantitation of DNA byusing rotating disk electrode. The decrease in peak currentof DM caused by the addition of DNA to a certainconcentration of DM solution can be employed to quanti-tatively determine the concentration of DNA. The DNAconcentration level of 525 �g/mL was selected to examinethe relative standard deviation (n�8) calculated as 4.9%. In



analytical application studies, the apparent diffusion coef-ficients of DM and DM-DNA adduct were calculatedrespectively as 8.77� 10�6 and 6.20� 10�7 cm2s�1. It can beseen that the apparent diffusion coefficient of DM-DNAadduct is much smaller than that of DM, showing thatdramatic decrease in the peak current of DM-DNA adductis caused by the decrease in the apparent diffusioncoefficient of DM after binding to large DNA.Clinically applied antitumor agent, Mitomycin C (MC)×s

interaction with single strandedDNA (ssDNA) detected bychanges of guanine (peak G) residues was studied byMarinet al. [24]. They showed that acid-activated MC binds toDNA at the hanging mercury drop electrode (HMDE)surface as detected by the decrease of guanine signal andappearance of redox couple at approximately � 0.4 V byusing transfer stripping cyclic voltammetry (TSCV) andhence, they concluded acid-activated MC was covalentlybound to guanine residues in DNA at pH 3.9 with theaccumulation time as 5 min by observing a strong decreaseof guanine signal. Besides, they reported that the quinonegroup in the occured DNA-MC adduct was reversiblyreduced at the HMDE resulting with a 10 fold higher and50 mV more negative cathodic peak.Oliveira Brett et al. [25] reported a procedure for the

determination of the interaction between anthraquinonedrug, mitoxantrone (MTX) and dsDNA or ssDNA at highconcentration levels. The interactionwas studied in aqueousmedium or on electrode surface by using glassy carbonelectrode (GCE) in connection with using square-wavevoltammetry (SWV) and differential pulse voltammetry(DPV). Its intercalation moiety was determined by thesignificant changes onoxidation signal ofMTX, guanine andadenine signal after interaction between MTX and DNA.The report of Erdem et al. [26] is in good agreementregarding the observed decrease at MTX signal afterinteraction at lower concentration level of dsDNA orssDNA at CPE surface.The mechanism of interaction of in situ nitroimidazole

reduction derivatives with DNA was observed by OliveiraBrett et al. [27]. The comparison of the voltammetricbehavior of metronidazole at a DNA-modified GCE, amercury thin filmelectrode andGCEwas also reported [28].The results from this comparative study encouraged us tostudy the interaction of metronidazole with DNA and toquantify this interaction using the DNA modified glassycarbon electrode with possible preconcentration.Fojta et al. [29] studied on conformational changes of

DNA due to binding of DNA intercalators such asanticancer agent, Doxorubicin (DXR) by using adsorptivetransfer stripping alternative current voltammetry atHMDE as showing that less than 40% and 30% decreaseat DXR peak was observed respectively consisting of 30 �g/mL concentration level of ssDNA and dsDNA.Interaction between some platinum complexes, potent

anticancer agents and DNA was studied by using DPV atwax impregnated graphite electrode at DPV [30]. Theyreported that the paraffin-wax impregnated spectroscopicgraphite electrode (PWISGE) transducer displays an ana-

lytically useful response for submicromolar levels of [Pt-(dien)(H2O)]2� at short accumulation times as 2 ± 10 minbased on diminution of the guanine oxidation signal.

2.2. The Quantification of Drug and/or DNA Monitoring

In some literature [31 ± 38], the quantification of drug ordrug monitoring based on DNA-drug interactions wasreported; such investigations may facilitate the develop-ment of fast and accuratemethods for these drugs interactedon DNA, especially for antineoplastic drugs in cancertherapy or in the planning new developments for cancertherapy. In minimum sample volume, a direct, simple andrapid determination for drug or DNA-drug adduct byelectrochemical methods in connection with separationmethods such as high performance liquid chromatographyand capillary electrophoresis can be of special interest inpharmaceutical preparations and in biological fluids at lowconcentrations.A DNA modified electrode for trace measurements of

some phenothiazine drugs possessing neuroleptic and anti-depressive actions by using PSA at CPE surface wasreported by Wang et al. [31]. In this study, the DNA-phenothiazine intercalative association is utilized for de-signing a highly sensitive electrochemical biosensor forpromethazine, chlorpromazine and phenothiazine andhence, this DNA modified CPE permits the measurementsin nanomolar concentration levels of these phenothiazinesin short accumulation times. The sensitivity calculated wasreported as 0.542 for promethazine, 0.083 for phenothiazine,0.251 for chlorpromazine and hence, the trend in sensitivitywas concluded as promethazine � chlorpromazine �phenothiazine: this sensitivity appears to be related to thestrength of the DNA-drug interaction. The ability of thedrug tobind to theDNAsurface layer increases uponaddingthe side chain to the intercalating ring system. To facilitatesuch drug monitoring at DNA modified electrodes, theyused dsDNA immobilized layer on microfabricated screenprinted electrodes for promethazine and concluded thatconvenient quantitation as nanomolar concentration levelindicating sharp, well-defined PSA peaks at 3 min accumu-lation time. The sensitivity for some phenothiazine drugssuch as, chlorpromazine, thioridazine and prochlorperazine,obtained by using DPVand DC technique in Vanickova etal. study [32] is comparable to that of PSA by Wang et al.[31]. It was also shown that a decrease in the DPV signalassociated with oxidation of DNA guanine residue wasobserved after interaction of thioridazine with DNA in theblank solution. In addition, a high working stability as 3months period stored at 40 �C for a bulk phase modifiedbiosensor was reported.In a study performed by Oliveira Brett et al. [33], the

analytical qualification of carboplatin in serum samplesfrom women patients with ovarian cancer undergoingtreatment with this drug, was described by using a DNA-modified GCE. These electrochemical results clearly dem-onstrated that, for low concentrations, carboplatin interacts



preferentially with adenine rather than guanine groups inthe DNA. They reported as its binding to DNA occurredcovalently it seemed quite clear that it could be possible todevelop an indirect analytical method to determine plati-num compounds with antitumor activity by measuring thisinteraction. For analytical applications of a DNA-modifiedGCE, after conditioning step, stable oxidation currentswereobserved for the peaks corresponding to guanine andadenine when electrode was placed in ssDNA solution.These experiments were done as carboplatin was added tothe solution containing ssDNA. There was a decreaseobserved in the oxidation current of adeninewith increasingthe concentration of carboplatin in solution while theguanine oxidation currents only decrease slightly. Theresponse range for carboplatin×s determination in serumsamples by the standard addition method using ssDNAsolutions was found in the range 6.5� 10�5 M to 1.5� 10�3M and the detection limit in serum samples was calculatedas 5.7� 10�6 M.Teijeiro et al. [34] studied DNA-interactions and quanti-

tative analysis of antineoplastic antibiotic, anthramycin,which is similar to other antibiotics, like MC and Ecteinas-cidin 743, by using differential pulse polarography inaqueous buffered media at HMDE. It was reported thatanthramycin produced covalent adducts with native DNA.This adduct was formed between the amino exocyclic groupof guanine and C(11) of anthramycin by covalent linkageand hence, the presence of the anthramycin at the N(2)position of guanine bases included an increase of stiffness onthe double helix. When the adduct was formed, the redoxbehavior of guanine bases was also affected. In conclusion,due to the smaller accessibility of the electroactive groupN(7)�C(8) of guanine in the adduct than that in the DNA,there was a decrease at the guanine signal. This result basedon adecrease in the guanine signal shows, simply and clearly,that the possibility for detecting modifications in the DNAstructure due to interaction between DNA and anthramy-cin. In addition, one of the other important reported aspectsof this study is the detection limit. The detection limit at ananomolar level for anthramycin such as 70 nM in 4%methanol buffered media gives the opportunity to developa useful technique to control doses for this drug frompatients.The quantification in real sample of some drugs inter-

acted with DNA as alone or their mixture in presence ofother drugs as interferences is also an important aspect fordeveloping a new DNA biosensor based on DNA-druginteraction. In contrast to the previous work ofMarin et al.[24], they developed this procedure [35] for direct deter-mination ofMCwith an excess of 5-fluorouracil or cisplatinin urine without any cleaning up step, except urine dilution(1 : 3) with background electrolyte. Due to any differenceobserved at stripping voltamograms by using HMDE atthe urine samples spiked with MC alone, MC and 5-fluorouracil or MC and cisplatin, this procedure wasdescribed as sufficiently sensitive to be a potential use inng/mL concentration level of MC for clinical analysisapplications.

Another quantification for a drug based on a DNA-modified electrode was performed by Xu et al. [36] at nano-goldmodified indium tin oxide (ITO) electrode forMifepri-stone (MF) determination at �Mconcentration level. It wasreported that a novel approach was developed by a self-assembly technique modified with (3-aminopropyl) trime-thoxysilane. These results for trace determination of MFresulted in a simple, stable and repeatable approach forfurther drug quantification based on DNA-modified elec-trodes.The interaction between some nonelectroactive quinazo-

line derivatives with DNAwas investigated by Labuda et al.[37]. Five morpholino-quinazoline derivatives such as;9-bromo-5-morpholino-tetrazolo[1,5-c]quinazoline; 6-chloro-2-morpholino-4-(4�-nitroanilino) quinazoline; 2-morpholi-no-4-(4�-bromoanilino)quinazoline; 6-bromo-2-morpholi-no-4-anilinoquinazoline; 6-bromo-2-morpholino-4-(4�-ni-troanilino) quinazoline which have biological activity inthe pharmaceutical industry and medicine, were quantifiedvoltammetrically using a competition with tris(o-phenan-throline)cobalt (III) (Co(phen)3�3 � redox marker for theaccumulation at dsDNAmodified SPEs in connection PSAof guanine signals. It was reported that in concentrationrange from 5� 10�7 to 4� 10�6 mol/L with calculateddetection limit 2� 10�7 mol/L, the voltammetric determi-nationof quinazoline derivatives canbeused in the presenceof 5� 10�7 mol/L Co(phen)3�3 marker based on DNAmodified SPEs. After the comparison of these results withthe ones of fluorometric andbiological test, it was concludedthat there was no effect of the quinazolines on the DNAcomplex with the fluorescent thiazole orange derivativeTO-PRO-3 within the quinazolines concentration range of10�7 to 10�5 mol/L and similar results were obtained in thecell cycle analysis by flow cytometry.Xia et al. [38] reported a method based on CV in aqueous

medium containing Pharmorubicin; Epirubicin (EPR) uponaddition of dsDNA and utilized this method for thedetermination of dsDNA by using GCE and the bindingconstant and the binding site size calculated from regressionlineswere reported as 7.46� 10�4 M�1 and 1.85, respectively.The proposed method was reported as being performedsuccessfully under no interference effect of any substances;first, for determination of interaction between EPR andDNA in presence of Tween-20 or bovine serum albumin(BSA); and finally, for quantification of DNA under theinterference effect of some amino-acids and antigens inhuman serum such as �-lysine, ��-histidine, ��-arginine,�-glutamic acid, C-reactive protein (CRP), carcinoem-bryonic antigen (CEA) and prolactin (PRL).

2.3. Intercalator Drugs Used for DNA HybridizationBiosensors

Most of recent reports forDNAhybridization biosensors byusing redox-active compounds like intercalator drugs suchas DM [39, 41 ± 43], Epirubicin (EPR) [40], and Echinomy-cin (ECH) [44] due to their electrochemical behavior with



dsDNA and ssDNA in different approaches. A quantitativeunderstanding of such factors that determine recognition ofDNA sites would be valuable in the development of newtools for biotechnology based on faster and more simpleDNA hybridization.In one of the early studies about DNA hybridization

biosensors, it was shown that DM was oxidized at a lowerpotential showing high current densities and this intercala-tor could be expected to amplify signals froma small amountof formed hybrids on the basal plane pyrolytic graphiteelectrode (BPPG) by using linear sweep voltammetry(LSV) [39]. Hashimato et al. [39] reported that the anodicpeak potentials of DM shifted tomore positive values in thecase of dsDNA modified electrode compared with ssDNAmodified one. The use ofDMresulted in the detection of thetargeted gene of 10±8 g/mL in the hybridization buffersolution.Erdem et al. [40] used the DNA-modified CPE in

combination with CV and especially DPV to obtaininformation about the interaction of EPR with dsDNAand ssDNAandobserved changes in theEPR signals causedby the intercalation of EPR into dsDNA. It was concludedthat EPR could be used as a promising indicator at a �Mconcentration level.A new approach for electrochemical detection of single-

base mismatches in DNA based upon charge transportthrough DNA films on gold surfaces as coupling the redoxreactions of intercalated species such asDM,was reported byKelley et al. [41, 42]. It was clarified by Kelley et al. [41] thatthe site of intercalation ofDMwas controlled in the duplexesby incorporating a single guanine-cytosine base step inotherwise adenine-thymine or inosine-cytosine sequences;as DM requires the N(2) atom of guanine for covalent cross-linking, the intercalator is constrained to these positions. Thisresulting assay gave an opportunity to differentiate betweencomplementary versus mismatched duplexes and henceallows the detection of point mutations in oligonucleotides.In these studiesofKelley et al. [41, 42], themechanismofDMbinding to DNA was explained as site-specifically cross-linking to guanine residues in DNA duplex.Marrazza et al. [43] developed a new procedure for

detecting genetic polymorphisms of Apolipoprotein E(apoE) in human blood samples. The procedure was basedon coupling of DNA electrochemical sensors with PCR-amplified DNA extracted from human blood, using DM asan electrochemical indicator. PCR amplified products wereimmobilized on graphite SPEs and hence, the hybridizationreaction on the electrode surfacewasmonitored byPSA in ashort time with reproducible and comparable results with amethod based on polyacrylamide gel electrophoresis. Con-sequently, DM is reported as an indicator of the hybrid-ization and gives a clear signal increase if the DNAhybridization takes place at SPEs surface. The resultsreported in literatures [41 ± 43] shows that DM can be avery promising hybridization indicator for future screeningof genotyping approaches.In a report by Jelen et al. [44], a bis-intercalator anticancer

drug, ECH was introduced as an electrochemically active

drug. The interaction of ECH with dsDNA attached toHMDE resulted in high signals of ECH. However with astrong binding of ECH to dsDNA, there was almost nosignal with ssDNA due to very weak binding of ECH tossDNA in solution. Thus, ECH was reported as a goodcandidate for a redox indicator for electrochemical DNAhybridization sensors.

2.4. The Determination of New Drug Targeting to DNA

The investigation reported here can form a theoretic guidefor the design of new anticancer drugs and chemicaltreatments of tumor and virus. In a study by Erdem et al.[45], the electrochemical interaction of a compound synthe-sized as an alkylating anticancer agent; 4,4�-dihydroxychalcone (DHC)withDNAwas investigated at CPE surfaceand in solution phase.The major cytotoxic and mutagenic effects of the alkylat-

ing agents are believed to result from their interactions withDNA. The major mechanism of cytotoxicity is believed tooccur as a consequence of a damage to DNA. These agentsusually have other sites of action and often react with othercellular targets, such as membranes. Alkylating agents formpositively charged carbonium ions, which react with nucle-ophyllic groups, such as SH, PO4 and NH3 on nucleic acids,proteins and smaller molecules. The N(7) of guanine isespecially susceptible to alkylation of DNA, cellular dam-age may occur from single-strand breakage and cross -linking of DNA, thus interfering with cell division [46].Kamei et al. [47] studied chalcone or 1,3-diphenyl-2-propen-1-one,which is considered to be a precursor of all flavonoids,for its antitumor growth activity in vitro and in vivo. Theirresults suggested that the chalcone induced abnormal DNAsynthesis and mitosis in the cultured cells. The activities ofthese compounds were attributed, in part, to alkylatingability of olefinic groups conjugated with a carbonylfunction to guanine bases in DNA [48].Erdem et al. [45] performed this study for clarifying the

interaction between DHC and DNA and reported that thevoltammetric signal of the drug decreases when it interactswith the dsDNAdue to the ability ofDHC to attack guaninebases inDNAresulting in the cross-linking of theDNA.Theresults obtained with the interaction of DHC and DNA insolution are in good agreement with the results obtainedwith the surface confinedDNA: the signal ofDHCobtainedwith ssDNA modified CPE was higher than the signalobtained with the dsDNAmodified CPE. In case of ssDNA,the high voltammetric signal ofDHCwas observed, becauseDHC could only bind to the one strand of ssDNA and thealkylating process could not be accomplished. The inter-action of DHCwith DNAmodified at the electrode surfacedid not differ from those occurring in the solution. It istherefore probable that interaction of DHCwith DNAmaytake place even in vivo. The differences in the way of druginteraction with DNA in solution and at surfaces might beimportant for the efficiency of the administered drug.Electrochemical methods offer great promise for exploring



suchbehavior differences of drugswithDNAin solution andat the electrode surface.

3. Conclusions and Future Perspectives

The extraordinary pace and scale of developments in thefield of genomics has forced a paradigm shift in the mannerwith which the pharmaceutical industry approaches thediscovery and development of new drug compounds. Thecompletion of the first draft of the human genome hasmadeit possible to foresee major steps forward in our under-standing of the molecular basis of disease, both from attackby external pathogens and internally from variations withinthe human genome resulting in a plethora of newmoleculartherapeutic targets for drug design and discovery [49].An understanding of the structural orientations, kinetics

and thermodynamics associated with these complexes ispivotal to design and development of novel ™next-gener-ation∫ chemotherapeutic agents. Elucidation of the forcesthat drive the thermodynamics and kinetics propertiesassociated with complex formation coupled with structuraland chemical features of the DNA binding ligands providesnew insight into rational drug design. These studies can playa key role in developing novel chemotherapeutic agents thatcould be pivotal in targeting specific genes and therebyprovide selective control of gene expression [8].Prospects for continued advances in this area are ex-

cellent, with rational design strategies poised to yield morenew compounds of interest. Electrochemical DNA biosen-sors are experimentally convenient and sensitive so thatthey require only a small amount of materials. In principle,they can be applied to a wide range of intercalating drugs,provided they bear an electrochemically active moiety.Electrochemical methods thus offer great promise forexploring such difference in solution and at surface inter-actions and to the study of DNA-drug interactions ingeneral.It is hoped that continued development through com-

bined efforts in microelectronics, surface/ interface chem-istry,molecular biology, and analytical chemistry will lead tothe establishment of genosensor technology as a majorcomponent of analytical biochemistry [7]. These investiga-tions are very valuable for probing the mechanism of theinteraction between drugs and DNA, especially for anti-cancer drugs as establishing the convenient methods toeffectively choose specific anticancer drugs to effectivetreatment on cancer.Immediate applications will include directly quantifying

DNA samples for use in sequencing or polymerase chainreactions (PCR), or pharmaceutical testing and qualitycontrol. Eventually, they could be applied to producingcredit card-sized sensor arrays for clinical applications suchas detection of pathogenic bacteria, tumors, and geneticdisease, or for forensics [50].Genoelectronics, the molecular interfacing approach into

regulating DNA interactions, and also into exploiting DNArecognition events is another important coming perspective.

This affecting drug discovery perspective can bring us theterm as ™DNA microarray in drug discovery and develop-ment∫ to measure the expression patterns of thousands ofgenes in parallel, generating clues to gene function that canhelp to identify appropriate targets for therapeutic inter-vention, and to monitor changes in gene expression inresponse to drug treatments [51].Understanding the role of genetic polymorphisms, espe-

cially single nucleotide polymorphisms (SNP), in the drugresponse will facilitate drug efficacy and decrease adverseeffects by helping scientists to tailor medication to apatient×s genetic makeup. One of the coming perspectivesis ™Pharmacogenomics∫ that should profoundly change thevision of companies developing and marketing medicinesand will create predictive and diagnostic tests [52].A quantitative understanding of such factors that deter-

mine recognition of DNA sites would be valuable in therational design of new DNA targeted molecules forapplication in chemotherapy and in the development ofnew tools for the point-of-care tests and diagnosis based onDNA.

4. Acknowledgements

Authors acknowledge the financial support from TUBI-TAK (Project number: TBAG-1871) and Ege UniversityScience and Technology Research and Application Center(EBILTEM)Project number: 2000/BIL/031. A. E. acknowl-edges the scientific scholarship from Highly Skilled YoungScientist Programme of The Turkish Academy of Sciences(TUBA-GEBIP).

5. References

[1] E. Palecek, Nature, 1960, 188, 656.[2] J. Wang, Chem. Eur. J. 1999, 6, 1681.[3] J. Wang, Nucl. Acids Res. 2000, 28, 3011.[4] E. Palecek, M. Fojta, Anal. Chem. 2001, 73, 75A.[5] M. I. Pividori, A. Merkoci, S. Alegret, Biosens. Bioelectron.

2000, 15, 291.[6] G. Chiti, G. Marrazza, M. Mascini, Anal. Chim. Acta 2001,

427, 155.[7] M. Yang, M. E. McGovern, M. Thompson, Anal. Chim. Acta

1997, 346, 259.[8] D. E. Graves, L. M. Velea, Curr. Org. Chem. 2000, 4, 915.[9] G. M. Blackburn, M. J. Gait, Nucleic Acids in Chemistry and

Biology IRL Press, New York, 1990, Ch. 8, p. 297 ± 332.[10] J. B. Chaires, Curr. Opin. Struct. Biol. 1998, 8, 314.[11] P. E. Nielsen, Curr. Med. Chem. 2001, 8, 545.[12] A. S. Lubbe, C. Alexiou, C. Bergemann, J. Surgical Res. 2001,

95, 200.[13] C. Bailly, P. Colson, C. Houssier, Biochem. Biophys. Res.

Commun. 1998, 243, 844.[14] T. R. Krugh, Curr. Opin. Struct. Biol. 1994, 4, 351.[15] A. Vaquero, J. Portugal, FEBS Lett. 1997, 420, 156.[16] G. Borchard, Adv. Drug Deliv. Rev. 2001, 52, 145.[17] M. F. Taylor, K. Wiederholt, F. Sverdrup,Drug Discov. Today

1999, 4, 562.



[18] J. A. Plambeck, W. L. Lown, J. Electrochem. Soc.: Electro-chem. Sci. Technol. 1984, 131, 2556.

[19] J. Wang, M. Ozsoz, X. Cai, G. Rivas, H. Shiraishi, D. H.Grant, M. Chicharro, J. Fernandes, E. Palecek, Bioelectro-chem. Bioenerg. 1998, 45, 33.

[20] X. Cai, G. Rivas, P. A. M. Farias, H. Shiraishi, J. Wang, M.Fojta, E. Palecek, Bioelectrochem. Bioenerg. 1996, 40, 41.

[21] E. Palecek, Electroanalysis 1996, 8, 7.[22] C. Teijeiro, K. Nejedly, E. Palecek, J. Biomol. Struc. Dyn.

1993, 11, 313.[23] X. Chu, G.-L. Shen, J.-H. Jiang, T.-F. Kang, B. Xiong, R.-Q.

Yu, Anal. Chim. Acta, 1998, 373, 29.[24] D. Marin, P. Perez, C. Teijeiro, E. Palecek, Biophysical Chem.

1998, 75, 87.[25] A. M. Oliveira Brett, T. R. A. Macedo, D. Raimundo, M. H.

Marques, S. H. P. Serrano, Biosens. Bioelectron. 1998, 13,861 .

[26] A. Erdem, M. Ozsoz, Turk, J. Chem. 2001, 25, 469.[27] A. M. Oliviera Brett, S. H. P. Serrano, I. Gutz, M. A. La-

Scalea, M. L. Cruz, Electroanalysis 1997, 9, 1132.[28] A. M. Oliviera Brett, S. H. P. Serrano, I. G. R. Gutz, M. A.

La-Scalea, Electroanalysis 1997, 9, 110.[29] M. Fojta, L. Havran, J. Fulneckova, T. Kubicarova, Electro-

analysis 2000, 12, 926.[30] V. Brabec, Electrochim. Acta 2000, 45, 2929.[31] J. Wang, G. Rivas, X. Cai, H. Shiraishi, P. A. M. Farias, N.

Dontha, D. Luo, Anal. Chim. Acta 1996, 332, 139.[32] M. Vanickova, M. Buckova, J. Labuda, Chem. Anal. (War-

saw) 2000, 45, 125.[33] A. M. Oliveria Brett, S. H. P. Serrano, T. A. Macedo, D.

Raimundo, M. H. Marques, M. A. La-Scalea, Electroanalysis1996, 8, 992.

[34] C. Teijeiro, E. Red, D. Marin, Electroanalysis 2000, 12, 963.[35] D. Marin, P. Perez, C. Teijeiro, E. Palecek, Anal. Chim. Acta

1998, 358, 45.[36] J. Xu, J.-J. Zhu, Y. Zhu, K. Gu, H.-Y. Chen, Anal. Lett. 2001,

34, 503.

[37] J. Labuda, M. Buckova, S. Jantova, I. Stepanek, I. Surugiu, B.Danielsson, M. Mascini, Fresenius J. Anal. Chem. 2000, 367,364.

[38] C. Xia, S. Guoli, J. Jianhui, Y. Ruqin, Anal. Lett. 1999, 32,717.

[39] K. Hashimato, K. Ito, Y. Ishimori, Anal. Chim. Acta 1994,286, 219.

[40] A. Erdem, M. Ozsoz, Anal. Chim. Acta 2001, 437, 107.[41] S. O. Kelley, N. M. Jackson, M. G. Hill, J. K. Barton, Angew.

Chem. Int. Ed. 1999, 38, 941.[42] S. O. Kelley, E. M. Boon, J. K. Barton, N. M. Jackson, M. G.

Hill, Nuc. Acids Res. 1999, 7, 4830.[43] G. Marrazza, G. Chiti, M. Mascini, M. Anichini, Clin. Chem.

2000, 46, 31.[44] F. Jelen, A. Erdem, E. Palecek, Bioelectrochem. 2002, 55,

165.[45] A. Erdem, K. Kerman, D. Ozkan, O. Kucukoglu, E. Erciyas,

M. Ozsoz, Electrochem. Soc. P. V. (the 200th Meeting of theElectrochemical Society and the 52 th meeting of the Interna-tional Society of Electrochemistry, 2 ± 7 September 2001, SanFrancisco, CA, USA), (Eds: M. Butler, P. Vanysek, N.Yamazoe), 2001, 18, 563.

[46] J. R. Bertino, Textbook of Pharmacology, Vol 26, Antineo-plastic Drugs (Eds: N. Cedric, A. M. Reynard), W. B. Sa-unders, Philadelphia 1992, p. 941 ± 963.

[47] H. Kamei, T. Koide, Y. Hashimoto, T. Kojima, M. Hasegawa,Cancer Biother. Radiopharm., 1997, 1, 51.

[48] J. R. Dimmock, E. Erciyas, P. Kumar, M. Hetherington, J. W.Quail, U. Pugazhenthi, S. A. Arpin, S. J. Hayes, T. M. Allen,S. Halleran, E. DeClercq, J. Balzarini, J. P. Stables, Eur. J.Med. Chem. 1997, 7, 583.

[49] P. M. Dean, E. D. Zanders, D. S. Bailey, Trends Biotechnol.2001, 19, 288.

[50] W. G. Kuhr, Nat. Biotech. 2000, 18, 1042.[51] C. Debouck, P. N. Goodfellow, Nature Genetics Suppl. 1999,

21, 48.[52] M. M. Shi, D. Mehrens, K. Dacus, Modern Drug Discov.

2001, July, 27.



electrochemical dna biosensors based on dna-drug interactions

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