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  1. 1. INSTITUTE OF PHYSICS PUBLISHING MEASUREMENT SCIENCE AND TECHNOLOGYMeas. Sci. Technol. 15 (2004) R1–R11 PII: S0957-0233(04)56971-0REVIEW ARTICLERecent trends in electrochemical DNAbiosensor technologyKagan Kerman, Masaaki Kobayashi and Eiichi Tamiya1School of Materials Science, Japan Advanced Institute of Science and Technology,1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292, JapanE-mail: tamiya@jaist.ac.jpReceived 2 October 2003, accepted for publication 7 November 2003Published 11 December 2003Online at stacks.iop.org/MST/15/R1 (DOI: 10.1088/0957-0233/15/2/R01)AbstractRecent trends and challenges in the electrochemical methods for thedetection of DNA hybridization are reviewed. Electrochemistry has superiorproperties over the other existing measurement systems, becauseelectrochemical biosensors can provide rapid, simple and low-cost on-fielddetection. Electrochemical measurement protocols are also suitable formass fabrication of miniaturized devices. Electrochemical detection ofhybridization is mainly based on the differences in the electrochemicalbehaviour of the labels towards the hybridization reaction on the electrodesurface or in the solution. Basic criteria for electrochemical DNA biosensortechnology, and already commercialized products, are also introduced.Future prospects towards PCR-free DNA chips are discussed.Keywords: electrochemical DNA hybridization biosensor, transduction ofDNA hybridization, DNA recognition interface, redox-labels, label-freedetection, metal nanoparticles, electrical detectionIntroduction Electrochemical detection of hybridization is mainly based on the differences in the electrochemical behaviour ofSince the new concept of ‘the electrochemical DNA the labels with or without double-stranded DNA (dsDNA) orhybridization biosensor’ was first introduced by Millan and single-stranded DNA (ssDNA). The labels for hybridizationMikkelsen back in 1993 [1], this exciting research area has detection can be anticancer agents, organic dyes, metalreceived intense attention from several groups around the complexes, enzymes or metal nanoparticles. There areworld. DNA biosensors convert the Watson–Crick base pair basically four different pathways for electrochemical detectionrecognition event into a readable analytical signal. A basic of DNA hybridization:DNA biosensor is designed by the immobilization of a single- (1) A decrease/increase in the oxidation/reduction peakstranded (ss) oligonucleotide (probe) on a transducer surface current of the label, which selectively binds withto recognize its complementary (target) DNA sequence via dsDNA/ssDNA, is monitored.hybridization. The DNA duplex formed on the electrode (2) A decrease/increase in the oxidation/reduction peaksurface is known as a hybrid. This event is then converted current of electroactive DNA bases such as guanine orinto an analytical signal by the transducer, which can adenine is monitored.be an electrochemical [2], optical [3], gravimetric [4], (3) The electrochemical signal of the substrate aftersurface plasmon resonance-based [5] or electrical [6] device. hybridization with an enzyme-tagged probe is monitored.Electrochemistry has superior properties over the other existing (4) The electrochemical signal of a metal nanoparticle probemeasurement systems, because electrochemical biosensors attached after hybridization with the target is monitored.enable fast, simple and low-cost detection. Recently, various reviews of electrochemical DNA1 Author to whom any correspondence should be addressed. biosensors have been reported [7–9]. The present review0957-0233/04/020001+11$30.00 © 2004 IOP Publishing Ltd Printed in the UK R1
  2. 2. Review Article A DNA biosensor for the detection of hepatitis B virus (HBV) was developed by covalently immobilizing ss HBV DNA fragments to a gold (Au) electrode surface via a carboxylate ester to link the 3 -hydroxy end of the DNA to the carboxyl of the thioglycolic acid monolayer [24]. A 181 bp HBV DNA fragment of known sequence was obtained and amplified by the polymerase chain reaction (PCR). The surface hybridization of the immobilized HBV probe with its target DNA fragment was detected by using the electrochemical signal of osmium bipyridine. The formation of the hybrid on the Au electrode resulted in a great increase in the peak current of osmium bipyridine in comparison with those obtained at a bare or probe-modified electrode. The difference between the responses of ferrocenium hexafluorophosphate at the probe- and hybrid-modified Au electrodes suggested that an electrochemical hybridization biosensor could be used to monitor DNA hybridization [25]. An electrochemical hybridization assay was designed by Yamashita et al [26] for the rapid analysis of a heterozygousScheme 1. Label-based electrochemical detection of DNA deficiency related to the human lipoprotein lipase (LPL) gene.hybridization. An intercalator, which can selectively bind to the PCR-amplified samples containing the wild-type sequence, ahybrid on the electrode surface, causes an increase in theelectrochemical signal. At the probe-modified electrode, a lower mutant or a one-base deletion of the LPL gene were subjectedsignal than the one obtained from the hybrid modified electrode is to hybridization with a 13–15 mer DNA probe that representedmonitored, because the intercalator cannot accumulate on the either the wild-type or the mutated sequence immobilized onsurface. an Au electrode. The differential pulse voltammetry of the electrode before and after hybridization was determined in thewill focus on the most recent developments in the technology presence of a bis-intercalator, namely ferrocenylnaphthaleneof electrochemical DNA biosensors, while providing basic diimide (FND), at 460 mV.insight for the detection methods. An overview of the current Electrochemical detection of several types of one-basestatus of DNA biosensor development will include information mismatch in a 20 mer hybrid anchored on a gold electrodeon applications and future prospects for molecular diagnostics. was also performed by Yamashita et al [27] in connection with FND as the label. A probe-modified Au electrode was incubated in 2,6-1. Label-based electrochemical detection of DNA disulfonic acid anthraquinone (AQDS) intercalator solution,hybridization rinsed and placed in an AQDS-free buffer solution, whereupon1.1. Redox-active molecules as labels for hybridization voltammetric experiments were performed [28]. Nodetection voltammetric peaks were observed for probe-modified Au electrodes. Upon DNA hybridization and incubation in AQDS,The electrochemical detection of DNA hybridization based on clear voltammetric peaks were observed. The absence ofa redox-active label is illustrated in scheme 1. Basically, the an AQDS signal for probe-modified surfaces clearly showedhybrid modified electrode is immersed in a solution which that the electrochemistry is due to long-range electron transfercontains a redox-active and DNA-binding molecule. After through the DNA duplex.a period of time for the interaction between the DNA and Since the redox-active molecule-based detection ofthe molecule, an electrochemical technique is applied to the hybridization is well established, two commercialized DNAelectrode to measure the surface species. If the redox-active chips are now being introduced onto the molecular diagnosismolecule is an intercalator such as daunomycin [10], it would market. The first example of such a DNA chip, called thebe inserted between the double-helix structure of the dsDNA eSensorTM , was produced by Motorola Life Sciences Inc. [29].with the help of its planar aromatic ring, and an enhancement Electrochemical detection by using the eSensorTM ,in the redox signal would be observed. On the contrary, if the which is illustrated in scheme 2, proceeds via a sandwichmolecule had an affinity towards ssDNA, such as methylene hybridization assay, in which three critical componentsblue [11–13], then a high signal would be observed from the (capture probe, target and signalling probe) are each presentprobe-modified electrode. These changes in the peak potential in the device. The signalling probe, tagged with a ferrocene,current of the labels for the probe and hybrid molecules provide serves to label the target upon hybridization. Electrons flowthe basis for detection of the label-based hybridization. to the electrode surface only when the target is present, Several metal complexes such as cobalt phenanthro- and are specifically hybridized to both the signalling andline [14, 15], cobalt bipyridine [1, 2] and ruthenium bipyri- capture probes [30]. eSensorTM bioelectric chips alsodine [16, 17], anticancer agents such as echinomycin [18, 19] successfully detected 86% of the HPV types contained inand epirubicin [20], and organic dyes such as methylene clinical samples [31].blue [21–23] were used as labels for the detection of hybridiza- Toshiba’s electrochemical DNA hybridization detectiontion. system is called the GenelyzerTM [32]. It contains anR2
  3. 3. Review ArticleScheme 2. Electrochemical detection protocol of eSensorTM DNA chips, produced by Motorola Life Sciences Inc. The capture probe,which is immobilized on the Au electrode, hybridizes with the target DNA. Then, the ferrocene-tagged signalling probe is exposed to thehybrid on the electrode surface. The redox signal of the tag is measured after the sandwich hybridization between the signalling probe andtarget DNA (Fc: ferrocene tag).electrochemical DNA chip that is able to analyse and typesingle-nucleotide polymorphisms (SNPs) and common DNAsequence variations by using the redox-active dye Hoechst33258 [33–35]. This hand-held device is only 45 cm wide,50 cm deep and 23 cm in height. A typical analysis ofSNPs in a sample takes only about 1 hour. A disposablepeptide nucleic acid (PNA) array with a 20-channel electrodewas microfabricated by Hashimoto and Ishimori [36] for thedetection of the cancer gene c-Ki-ras from PCR-amplified Scheme 3. Enzyme-tagged electrochemical detection of DNAsamples by using Hoechst 33258. hybridization. Specific hybridization between the surface-anchored A new electrochemical hybridization detection method target and the enzyme-tagged signalling probe enables monitoringbased on Hoechst 33258 has recently been reported by of the amplified voltammetric or amperometric signal from theKobayashi et al [37]. Linear sweep and cyclic voltammetry product as the substrate is introduced to the electrode (E: enzyme).methods were employed on a microchip composed of 32 Auelectrodes, for the detection of target DNA related to the human labels is illustrated in scheme 3. When the substrate is intro-immunodeficiency virus (HIV) and hepatitis C virus (HCV). duced to the enzyme-modified electrode surface, the electro-Moreover, a novel electrochemical method for detecting the chemical activity of the product greatly simplifies the detectionhepatitis B virus (HBV) has been reported [38]. Current of DNA hybridization.methods need a compulsory step, i.e. immobilization of the A 38-base DNA sequence has been detected at aprobe DNA on the electrodes, which adds to the complexity concentration of 20 pmol l−1 in 15–35 µl droplets by means ofof making a DNA chip. However, this novel method, based an electrochemical enzyme-amplified sandwich-type assay onon DNA aggregation by an electroactive indicator, can detect a mass-manufacturable screen-printed carbon electrode [43].DNA quantification without applying a compulsory step: the The formation of the sandwich brought the horseradishimmobilization of probe DNA. Furthermore, our new method peroxidase (HRP) label of the detection sequence intousing Hoechst 33258 can detect DNA amplification without electrical contact with a pre-electrodeposited redox polymer,the DNA purification step. This novel method has enabled us making the sandwich an electrocatalyst for the reduction ofto detect HBV DNA from human blood in less than 2 h. hydrogen peroxide to water at +0.2 V (Ag/AgCl). Recently, DNA damage was detected by using catalytic oxidation Zhang et al [44] reported a major advance in the horseradishwith ruthenium bipyridine and by monitoring the binding of peroxidase-amplified amperometric detection of DNA hybri-cobalt bipyridine to DNA [39]. Damaged DNA reacted more dization. They achieved the detection of DNA as low as 3000rapidly than intact dsDNA with ruthenium bipyridine, giving copies in a 10 µl droplet at a concentration of 0.5 fM.electrochemical peaks at approximately 1 V that grew larger Pividori et al [45] implemented a classical dot–blot formatwith reaction time. Cobalt bipyridine bound more strongly to for the enzyme-based amperometric detection of hybridization.intact dsDNA, and its signals at 0.04 V, decreased as DNA was The analytical procedure consisted of five steps: DNAdamaged. target immobilization by adsorption onto a nylon membrane; hybridization between a DNA target and biotin–DNA probe; complexation reaction between a biotin–DNA probe and HRP-1.2. Enzyme labelling for detection of DNA hybridization streptavidin conjugate; integration of the modified membraneLabelling of probes with enzymes has also been effectively onto an electrochemical transducer; and, finally, amperometricused for the electrochemical detection of DNA hybridiza- detection using a suitable substrate for the enzyme-labelledtion [40–42]. The basic detection scheme of using enzymes as duplex. R3
  4. 4. Review ArticleScheme 4. Metal-nanoparticle-based electrochemical detection of DNA hybridization. (A) After the specific hybridization between thesurface-anchored target and the metal-nanoparticle-tagged signalling probe, the intrinsic electrochemical signal of the metal nanoparticlecan be observed on the same electrode. (B) Dissolution of the metal tag with acid treatment enables the detection of hybridization on aseparate bare electrode. (C) Au nanoparticles can be coated with an Ag layer and the electrochemical signal of Ag can be detected with orwithout dissolving the Ag layer (Me: metal nanoparticle). Hairpin-forming probes in connection with HRP were Cholinesterase- and peroxidase-immobilized screen-used for the electrochemical detection of factor V Leiden printed electrodes were also used to detect the DNA–antibodymutations from human blood specimens [46]. After adducts [50].hybridization between the ss target DNA and the fluorescein- Glucose oxidase was also utilized for the detection of virallabelled detector probe, the hybrids were immobilized on a genes on an Au electrode [51]. The generation of a redox-carbon paste electrode (CPE). HRP-linked anti-fluorescein active replica was accomplished by using ferrocene conjugatedantibody–enzyme conjugates were then loaded onto each uracil bases in combination with polymerase. The resultinghybrid. Following the addition of an HRP substrate replica on the electrode surface acted as an electron-transferand mediators such as tetramethylbenzidine, amperometic relay for the bioelectrocatalysis of glucose.signals provided quantitative information on the number of A major breakthrough in DNA biosensors was achievedimmobilized hybrids on the sensor surface. by eliminating the PCR step from DNA diagnosis. This PCR- Magnetic beads have provided easy removal of non- free biosensor based on enzyme amplification was reportedspecifically bound DNA from electrode surfaces [47–49]. An by Patolsky et al [52]. In the presence of polymerase,enzyme-linked sandwich hybridization assay was taken with a a biotinylated nucleotide, complementary to the mutationmagnetic-particle-labelled probe hybridizing to a biotinylated site, was coupled to the ds hybrid on the electrode surface.DNA target that captured a streptavidin-alkaline phosphatase Subsequent binding of avidin-alkaline phosphatase to the assembly, and the biocatalysed precipitation of an insoluble(AP). The 1-naphthol product of the enzymatic reaction was product on the surface, provided the lower limit of sensitivityquantified through its low-potential (+0.1 V versus Ag/AgCl) of 1 × 10−14 mol ml−1 for Tay–Sachs genetic disorder with nodifferential pulse voltammetric peak at the disposable screen- PCR preamplification.printed electrode [48]. Palecek et al [49] developed a new technology inwhich DNA hybridization was performed on commercially 1.3. DNA biosensors meet nanotechnologyavailable magnetic beads and detected on solid electrodes. 1.3.1. Metal nanoparticles as labels for detection ofTarget DNA was modified with osmium tetroxide, 2, 2 - DNA hybridization. The basic protocol for the detectionbipyridine (Os, bipy) and the immunogenic DNA–Os, bipy of metal-nanoparticle-based DNA hybridization is shownadduct was determined by the AP-linked immunoassay in scheme 4. There are currently three strategies forwith electrochemical detection. Electro-inactive 1-naphthyl detection: the intrinsic electrochemical signal of the metalphosphate was used as a substrate, and the electroactive product nanoparticle can be observed with (scheme 4(B)) [53] or(1-naphthol) was measured on the carbon electrodes. without (scheme 4(A)) [54] dissolving it with acid treatmentR4
  5. 5. Review Articleand can be detected on a bare electrode, or the Au nanoparticles internally with ferrocenecarboxyaldehyde marker moleculescan be coated with a silver (Ag) layer to enhance the for amplified electrochemical DNA sensing. A DNA arrayelectrochemical signal of Ag (scheme 4(C)) [55]. microchip utilizing magnetic beads as labels to detect DNA Ozsoz et al [54] reported a DNA biosensor based on a hybridization has recently been reported by Miller et al [64].pencil graphite electrode and modified with the target DNA. Polyvinyl-alcohol-based magnetic beads for rapid and efficientWhen the target DNA hybridized with a complementary probe separation, purification and detection of DNA were reportedconjugated to an Au nanoparticle, the Au oxide wave was by Oster et al [65].monitored to detect the hybridization. The detection of DNA hybridization using cadmium 1.3.2. Carbon nanotubes for electrochemical detection of DNAsulfide nanocluster-based electrochemical stripping was hybridization. A nanoelectrode array based on verticallyreported by Zhu et al [56]. Their protocol consisted of the aligned multi-walled carbon nanotubes (MWNTs) embeddedhybridization of the target DNA with the CdS nanocluster in SiO2 was reported by Li et al [66]. Oligonucleotideoligonucleotide DNA probe, followed by dissolution of the probes were selectively functionalized to the open ends ofCdS nanoclusters anchored on the hybrids and the indirect the MWNTs. The hybridization of subattomole DNA targetsdetermination of the dissolved cadmium ions by sensitive could be detected by combining the nanoelectrode array withanodic stripping voltammetry (ASV) at a mercury-coated ruthenium bipyridine mediated guanine oxidation.glassy-carbon electrode. Carbon-nanotube-based electrochemical assay for hy- Another assay relied on the hybridization of the target bridization detection provided enhanced daunomycin signalsDNA with a silver nanoparticle oligonucleotide DNA probe, due to the large surface area and good charge-transport char-followed by the release of the silver metal atoms anchored acteristics of the MWNTs [67].on the hybrid by oxidative metal dissolution and the indirectdetermination of the solubilized Ag(I) ions by ASV at a carbon 1.3.3. Encoding technology with metal nanoparticles andfibre ultramicroelectrode [57]. enzymes. The labelling of probes bearing different DNA Wang et al [58] reported the hybridization of a target sequences with different metal nanoparticles or enzymesoligonucleotide to magnetic bead-linked oligonucleotide enables the simultaneous detection of more than one targetprobes followed by binding of the streptavidin-coated metal in a sample as shown in scheme 5. The key point in thisnanoparticles to the captured DNA. After the dissolution of protocol is that the signals of labels should not overlap withthe Au tag, potentiometric stripping measurements of the each other, otherwise simultaneous monitoring of signals indissolved Au were performed at single-use thick-film carbon one scan cannot be performed.electrodes. Three encoding nanoparticles (zinc sulfide, cadmium An electrochemical DNA detection method was devel- sulfide and lead sulfide) were used to differentiate the signals ofoped for the sensitive quantification of an amplified 406- three DNA targets in connection with stripping-voltammetricbase pair human cytomegalovirus DNA sequence (HCMV measurements of the heavy metal dissolution products [68].DNA) [59]. The assay relied on These products yielded well-defined and resolved stripping peaks at −1.12 V (Zn), −0.68 V (Cd) and −0.53 V (Pb) at the(a) the hybridization of the target HCMV DNA with Au mercury-coated glassy-carbon electrode (versus the Ag/AgCl nanoparticle-modified oligonucleotide, reference). The position and size of these peaks reflected the(b) followed by the release of Au by acid treatment, and identity and level of the corresponding DNA target.(c) the indirect determination of the solubilized AuIII ions Two encoding enzymes, alkaline phosphatase and β- by ASV at a sandwich-type screen-printed microband galactosidase, were used to differentiate the signals of electrode. two DNA targets from the signals of their electroactive products [69]. These products provided well-defined and Sato et al [60] reported a novel aggregation phenomenon resolved peaks at +0.31 V (α-naphthol) and +0.63 V (phenol)of DNA-modified Au nanoparticles induced by hybridization at the graphite working electrode. The position and size ofof target DNA, which did not crosslink the nanoparticles. Ag- these peaks reflected the identity and level of the correspondinggregation of DNA-functionalized poly(isopropylacrylamide) target.(PNIPAAm) nanoparticles without the crosslinking mech- Recently, encoded redox beads, based on the encapsula-anism was reported by Mori and Maeda [61]. Probe tion of different quantum dots (QD) within polystyrene mi-oligonucleotide was grafted onto PNIPAAm, which produced crospheres, and encoded redox rods, prepared by sequentialnanoparticles above 40 ◦ C. When the target DNA was perfectly plating of different metal tracers into the pores of a host mem-complementary to the probe, the nanoparticles aggregated to- brane, have also been designed for electrochemical authenticitygether at high salt concentration. testing of commercial products [70]. Wang et al [62] recently reported two particle-basedprocedures for monitoring DNA hybridization. The firstprotocol involved detecting the iron content of magnetic 2. Label-free electrochemical detection of DNAbeads, while the second route relied on probes labelled with hybridizationAu-coated iron core-shell nanoparticles. In both protocols, 2.1. Electrical charge flow through DNA for detection ofiron-containing particles were dissolved, and the released hybridizationiron was quantified by cathodic-stripping voltammetry in thepresence of the 1-nitroso-2-naphtol ligand and a bromate Intense debate has occurred on the question of whether orcatalyst. Wang et al [63] loaded microsphere tags not DNA is able to conduct electrical charges. Fluorescence R5
  6. 6. Review ArticleScheme 5. Encoding technology enables simultaneous detection of several DNA targets in one measurement. Different metal nanoparticlesor enzymes can be used as the signalling probes for coding several targets simultaneously (E: enzyme, M: metal nanoparticle, S: substrate,P: product; the subscript numbers represent three different types).quenching experiments in connection with intercalated donor Silver deposition facilitated by the nanoparticles bridged theand acceptor molecules on DNA have generated contradictory microgap and led to a change in conductivity.discussions. The conductivity of DNA has been assessed from The flow of electrical charges through DNA provided theelectron transfer as a function of the distance between the donor basis for the detection of DNA hybridization. One of theand acceptor molecules [71]. leading DNA chip venture companies, GeneOhm Sciences The first electrical transport measurements on DNA Inc., and its technology for detecting single-nucleotidemolecules at least 600 nm long were reported by Fink and polymorphisms (SNPs), uses DNA’s ability to carry electricalSch¨ nenberger [72]. Direct measurements of electrical current o current [77].as a function of the potential applied across DNA indicated A probe, which has a DNA sequence complementaryefficient conduction through the DNA ‘ropes’. The resistivity to a portion (15–30 bases) of a gene with an SNP, isvalues derived from these measurements were comparable to immobilized onto an Au electrode surface as a self-assembledthose of conducting polymers; thus DNA was assumed to monolayer (SAM). After hybridization with the target DNA,transport electrical current as efficiently as a semiconductor. the electrode is exposed to an intercalator solution. As theLarge numbers of DNA molecules aligned on films also intercalator accumulates between the base pairs of the hybrid,showed anisotropic conductivity [73]. In contrast, electrical a current is applied to the hybrid from the electrode. If the hybrid is perfectly matched, then the current flow reaches thetransport through individual poly(G)–poly(C) DNA molecules intercalator and a high charge signal can be obtained. If there10.4 nm long connected to two metal nanoelectrodes indicated is an SNP in the hybrid, the flow of current is blocked andlarge-bandgap semiconducting behaviour [74]. Non-linear no charge signal can be monitored from the DNA SAM. Acurrent–voltage curves that exhibited a voltage gap at low schematic representation of the core technology of GeneOhmapplied bias were obtained. A peak structure, which exhibited Sciences Inc. is shown in scheme 6.the voltage dependence of the differential conductance, A comparison of electron transfer rates of ferrocenoyl-suggested that charge carrier transport was mediated by the linked DNA molecules has recently been reported by Longmolecular energy bands of DNA [74]. et al [78]. Boon et al [79] reported the development of an Braun et al [75] hybridized target DNA with surface- electrochemical assay for protein binding to DNA-modifiedbound oligonucletides on two Au electrodes, thus stretching electrodes based on the detection of associated perturbations inthe DNA molecule in the microgap between these electrodes. DNA base stacking. Au electrode surfaces that were modifiedDNA was then used as the template for the vectoral growth of with loosely packed DNA duplexes, covalently crosslinked toa conductive silver wire. a redox-active intercalator and containing the binding site of Array-based electrical detection of DNA with Au- the test protein, were constructed. Charge transport throughnanoparticle-tagged probes was reported by Park et al [76]. DNA as a function of protein binding was then assayed.Probe oligonucleotides were immobilized into a 20 µm gap Marques et al [80] used electrochemical techniquesbetween Au and Ti microelectrodes. Hybridization between to detect DNA polymorphisms in human genes by usingthe probes and the Au-nanoparticle-tagged complementary cytochrome P450 3A4 (CYP3A4) as a model gene. TheirDNA localized the Au nanoparticles in the microgap. detection protocol was based on dsDNA’s ability to transportR6
  7. 7. Review ArticleScheme 6. Electrochemical detection protocol of electrical DNA chips, produced by GeneOhm Sciences Inc. After hybridization of theprobe with the target DNA, the hybrid-modified Au electrode is exposed to an intercalator solution. Then a current is applied to the hybridfrom the electrode. If the hybrid is perfectly matched, the current flow reaches the intercalator and a high charge signal can be obtained. Amismatch in the hybrid blocks the flow of current, so no charge signal can be monitored from the hybrid.charge along nucleotide stacking. The perturbation of thedouble-helix pi-stack introduced by a mismatched nucleotidereduced electron flow, and could be detected by measuring theattenuation of the charge transfer. CYP3A4*1A homozygotescould be detected by 5 µC charge attenuation. The reagentless transduction of DNA hybridization intoa readily detectable electrochemical signal by means of aconformational change approach was reported by Fan et al[81]. The strategy involved an electroactive, ferrocene-taggedDNA stem-loop structure that self-assembled onto an Auelectrode by means of facile Au-thiol chemistry. Hybridization Scheme 7. Label-free electrochemical detection of DNAinduced a large conformational change in this surface-confined hybridization eliminates the use of redox molecules, and shortensDNA structure, which in turn significantly altered the electron- the assay time. The inosine (I) substituted probe shows no electrochemical signals, since inosine is electro-inactive. Aftertransfer tunnelling distance between the electrode and the hybridization with the target DNA, the appearance of the guanineredoxable label. The resulting change in electron-transfer (G) oxidation signal provides specific detection.efficiency was readily measured by cyclic voltammetry attarget DNA concentrations as low as 10 pM. a new detection method for DNA hybridization as shown in A new label-free electrochemical DNA hybridization scheme 7. This procedure eliminated the use of external labelsdetection method has recently been reported by Gooding et al and shortened the assay time. Inosine-substituted probes for[82]. The change in flexibility of an ss probe before and after DNA hybridization biosensors were applied onto indium tinhybridization, caused an ion-gating effect, where the rigid rod- oxide electrodes by Thorp [89]. Jelen et al [90] showed thatlike structure of the duplex opened up the SAM-modified Auelectrode to the access of ions. subnanomolar concentrations (related to monomer content) of unlabelled DNA could be determined using copper solid amalgam electrodes or hanging mercury drop electrodes in the2.2. Intrinsic DNA signals for the detection of hybridization presence of copper ions.Guanine and adenine are the most electroactive bases of Strepavidin and biotin are widely used in affinity-basedDNA, because they can easily be adsorbed and oxidized on separations and diagnostic assays. Recently, strepavidincarbon electrodes. Guanine and adenine oxidation signals on and avidin were electrochemically detected in solution bycarbon electrodes can be observed at around 1.0 and 1.3 V in adsorptive transfer-stripping voltammetry at a CPE [91].0.50 M acetate buffer solution (pH 4.80, ABS), respectively, Especially, an avidin-modified CPE could successfully bindas reported by Jelen et al [83]. Monitoring the changes in biotin-modified oligonucleotides onto the electrode surface.these signals upon duplex formation enabled the detection of This avidin–biotin binding technology was applied for thehybridization [84–88]. The electrochemical signals obtained detection of transgenic avidin maize.from free adenine and guanine bases decreased on binding Toxicity controls of wastewater samples were alsoto their complementary thymine and cytosine bases after conducted by using DNA biosensors [92]. The DNA biosensorhybridization. was assembled by immobilizing dsDNA on the surface of The use of inosine-substituted probes and the appearance a disposable, carbon screen-printed electrode (SPE). Theof a guanine signal upon hybridization with the target enabled oxidation signal of guanine was used as the analytical signal. R7
  8. 8. Review ArticleThe presence of compounds with an affinity for DNA was oligonucleotides onto silica surfaces and their subsequent hy-measured by their effect on the guanine oxidation. A bridization with complementary strands [99]. The impedancecomparison of the results with a toxicity test based on measurements, which provided a direct means of detectingbioluminescent bacteria confirmed the applicability of the variations in electrical charge accumulation across the semi-method to real samples. conductor/oxide/electrolyte structure when the oxide surface The interaction of small molecules with DNA was was chemically modified, showed that the semiconductor’sdetected by monitoring the changes in the adenine and flat band potential underwent reproducible shifts of −150 andguanine signals. The interaction of an alkylating agent, 4, 4 - −100 mV following the immobilization and the hybridizationdihydroxy chalcone, with dsDNA and ssDNA was studied steps, respectively.electrochemically based on the oxidation signals of guanine An electrochemical method to detect DNA hybridizationand adenine by using differential pulse voltammetry (DPV) at directly was developed on the basis of a new conductivea CPE [93]. polymer, which was polymerized on a glassy-carbon electrode The interaction of arsenic trioxide (As2 O3 ) with dsDNA, with a terthiophene monomer having the carboxyl groupssDNA and also 17 mer short oligonucleotide was studied (3 -carboxyl-5, 2 , 5 , 2 -terthiophene) [100]. A difference inelectrochemically by using DPV with a CPE at the surface admittance was observed before and after hybridization as aand also in solution [94]. Potentiometric stripping analysis result of the reduction of the resistance after hybridization.(PSA) was employed to monitor the interaction of As2 O3 with The highest difference in admittance was observed arounddsDNA in solution phase by using a renewable pencil graphite 1 kHz before and after hybridization. Hybridization amountselectrode. of end two-base and centre one-base mismatched sequences An investigation of the niclosamide–DNA interaction were obtained only in a 14.3% response when compared tousing an electrochemical DNA biosensor showed for the first that for the complementary matched sequence.time clear evidence of interaction with DNA and suggested The use of electrochemical impedance spectroscopy (EIS)that niclosamide toxicity can be caused by this interaction, and the conducting polymer, poly(pyrrole), as an integratedafter reductive activation [95]. recognition and transduction system for reagentless biosensor The interaction of rhodium dimers, including the carboxy- systems was demonstrated for two different systems by Faracelates (acetate, propionate, butyrate, trifluoroacetate, citrate and et al [101]. A construct for discriminating DNA hybridizationgluconate), amidates (acetamidate and trifluoroacetamidate) and able to differentiate ssDNA and dsDNA based on theand carboxamidate (Doyle catalyst S), with DNA was investi- interaction of the DNA with poly(pyrrole) was achieved.gated by electrochemical methods [96]. DPV measurementsshowed that most of the rhodium carboxylates had a higher 2.4. Field-effect sensors for the detection of DNAaffinity for adenine than guanine residues. hybridization Microfabricated silicon field-effect sensors were used to2.3. Electrochemical impedance for the detection of DNA directly monitor the increase in surface charge when DNAhybridization was hybridized on the sensor surface [102]. The electrostaticHybridization of two complementary DNA strands on the immobilization of probe DNA on a positively charged poly-l-surface of the structure induced a variation in the flat band lysine layer allowed hybridization at low ionic strength, wherepotential of the semiconductor leading to a shift of impedance field-effect sensing was the most sensitive case. Nanomolarcurves along the potential axis. This means that it is possible to DNA concentrations could be detected within minutes, and adetect DNA hybridization directly without the use of labelled single-base mismatch within 12 mer oligonucleotides could beprobes by monitoring impedance [97]. distinguished by using a differential detection technique with Alkaline phosphatase oxidative hydrolysis of the soluble two sensors in parallel.5-bromo-4-chloro-3-indoyl phosphate to the insoluble indigoproduct was utilized by Patolsky et al [98] in two different 3. Future prospectssensing configurations. The accumulation of the insolubleproduct on Au electrodes or Au/quartz crystals changed the As the three major electrochemical DNA chips producedelectron-transfer resistance at the electrode surface or the mass by GeneOhm Sciences Inc., Toshiba Corp. and Motorolaassociated with the piezoelectric crystal, thus enabling the Life Sciences Inc. enter the molecular diagnosis market inquantitative transduction of DNA hybridization by Faraday the near future, it is likely that the competition and interestimpedance spectroscopy or microgravimetric quartz-crystal in the electrochemical detection of DNA hybridization willmicrobalance measurements, respectively [98]. intensify. Although the main target in all the DNA chip The capacitance measurement, electrochemical impedance technologies has been to eliminate the role of the polymerasespectroscopy and constant current chronopotentiometry have chain reaction (PCR) from their protocols, this goal hasbeen used for the electrochemical study of echinomycin and not yet been reached by commercialized electrochemicalits interaction with ssDNA and dsDNA at the hanging mercury technologies. Some efforts are now being made towardsdrop electrode (HMDE) [18]. Echinomycin was found to be decreasing the time needed for PCR. The only promisinga promising redox indicator for hybridization detection due to example for the achievement of a PCR-free DNA biosensorits strong binding ability with dsDNA. based on the enzymatic amplification of electrochemical Radiolabelling and electrochemical impedance measure- signals was reported by Patolsky et al [52]. With the rapidments were used to characterize the immobilization of ss homo- progress in the electrochemical biosensing world, it can beR8
  9. 9. Review Articleenvisaged that DNA biosensors without PCR amplification [15] Erdem A, Meric B, Kerman K, Dalbasti T and Ozsoz M 1999will soon be on the market. Therefore, electrochemical Detection of interaction between metal complex indicatorDNA biosensors with their cost-effectiveness and suitability and DNA by using electrochemical biosensor Electroanalysis 11 1372–6for microfabrication can be expected to become increasingly [16] Napier M E, Loomis C R, Sistare M F, Kim J,popular in the near future. Eckhardt A E and Thorp H H 1997 Probing biomolecule recognition with electron transfer: electrochemical sensors for DNA hybridization Bioconjug. Chem. 8 906–13Acknowledgments [17] Yang I V, Ropp P A and Thorp H H 2002 Toward electrochemical resolution of two genes on one electrode:KK acknowledges the Monbukagakusho scholarship for using 7-deaza analogues of guanine and adenine to prepareresearch students from the Japan Ministry of Education, PCR products with differential redox activity Anal. Chem.Culture, Sports, Science and Technology (MEXT). The authors 74 347–54express their gratitude to Dr Yasutaka Morita for a critical [18] Hason S, Dvorak J, Jelen F and Vetterl V 2002 Interaction ofreading of the manuscript. DNA with echinomycin at the mercury electrode surface as detected by impedance and chronopotentiometric measurements Talanta 56 905–13References [19] Jelen F, Erdem A and Palecek E 2002 Cyclic voltammetry of echinomycin and its interaction with double-stranded and [1] Millan K M and Mikkelsen S R 1993 Sequence-selective single-stranded DNA adsorbed at the electrode biosensor for DNA based on electroactive hybridization Bioelectrochemistry 55 165–7 indicators Anal. Chem. 65 2317–23 [20] Erdem A and Ozsoz M 2001 Interaction of the anticancer [2] Millan K M, Saraullo S and Mikkelsen S R 1994 drug epirubicin with DNA Anal. Chim. Acta Voltammetric DNA biosensor for cystic fibrosis based on a 437 107–14 modified carbon paste electrode Anal. Chem. 66 2943–8 [21] Ozkan D, Kara P, Kerman K, Meric B, Erdem A, Jelen F, [3] Piunno P A E, Krull U J, Hudson R H E, Damha M J and Nielsen P E and Ozsoz M 2002 DNA and PNA sensing on Cohen H 1994 Fiber optic biosensor for fluorimetric mercury and carbon electrodes by using methylene detection of DNA hybridization Anal. Chim. Acta 288 blue as an electrochemical label Bioelectrochemistry 58 205–14 119–26 [4] Minunni M, Tombelli S, Scielzi R, Mannelli I, Mascini M and [22] Ozkan D, Erdem A, Kara P, Kerman K, Gooding J J, Gaudiano C 2003 Detection of β-thalassemia by a DNA Nielsen P E and Ozsoz M 2002 Electrochemical detection piezoelectric biosensor coupled with polymerase chain of hybridization using peptide nucleic acids and methylene reaction Anal. Chim. Acta 481 55–64 blue on self-assembled alkanethiol monolayer modified [5] Sawata S, Kai E, Ikebukuro K, Iida T, Honda T and Au electrodes Electrochem. Commun. 4 796–802 Karube I 1999 Application of peptide nucleic acid to the [23] Kara P, Kerman K, Ozkan D, Meric B, Erdem A, direct detection of deoxyribonucleic acid amplified by Ozkan Z and Ozsoz M 2002 Electrochemical genosensor polymerase chain reaction Biosens. Bioelectron. 14 for the detection of interaction between methylene blue 397–404 and DNA Electrochem. Commun. 4 705–9 [6] Kelley S O, Boon E M, Barton J K, Jackson N M and [24] Ju H X, Ye Y K, Zhao J H and Zhu Y L 2003 Hybridization Hill M G 1999 Single-base mismatch detection based on biosensor using di(2, 2( )-bipyridine)osmium (III) as charge transduction through DNA Nucleic Acids Res. 27 electrochemical indicator for detection of polymerase 4830–7 chain reaction product of hepatitis B virus DNA Anal. [7] Drummond T G, Hill M G and Barton J K 2003 Biochem. 313 255–61 Electrochemical DNA biosensors Nat. Biotechnol. 21 [25] Ye Y K, Zhao J H, Yan F, Zhu Y L and Ju H X 2003 1192–9 Electrochemical behaviour and detection of hepatitis B [8] Wang J 2002 Electrochemical nucleic acid biosensors Anal. virus DNA PCR production at Au electrode Biosens. Chim. Acta 288 205–14 Bioelectron. 18 1501–8 [9] Gooding J J 2002 Electrochemical DNA hybridization [26] Yamashita K, Takagi M, Kondo H and Takenaka S 2002 biosensors Electroanalysis 14 1149–56 Electrochemical detection of nucleic base mismatches [10] Wang J, Ozsoz M, Cai X, Rivas G, Shiraishi H, Grant D H, with ferrocenyl naphthalene diimide Anal. Biochem. 306 Chicharro M, Fernandes J and Palecek E 1998 Interactions 188–96 of antitumor drug daunomycin with DNA in solution and [27] Yamashita K, Takagi A, Takagi M, Kondo H, Ikeda Y and at the surface Bioelectrochem. Bioenerg. 45 33–40 Takenaka S 2002 Ferrocenylnaphthalene diimide-based [11] Erdem A, Kerman K, Meric B, Akarca U S and electrochemical hybridization assay for heterozygote Ozsoz M 2000 Novel hybridization indicator methylene deficiency of the lipoprotein lipase gene Bioconjug. Chem. blue for the electrochemical detection of short DNA 13 1193–9 sequences related to the hepatitis B virus Anal. Chim. Acta [28] Wong E L S and Gooding J J 2003 Electronic detection of 422 139–49 target nucleic acids by a 2,6-disulfonic acid anthraquinone [12] Meric B, Kerman K, Ozkan D, Kara P, Erensoy S, intercalator Anal. Chem. 75 3845–52 Akarca U S, Mascini M and Ozsoz M 2002 [29] Motorola Life Sciences Inc. Electrochemical DNA biosensor for the detection of TT http://www.motorola.com/lifesciences/ and hepatitis B virus from PCR amplified real samples by [30] Yu C J, Wan Y, Yowanto H, Li J, Tao C, James M D, using methylene blue Talanta 56 837–46 Tan C L, Blackburn G F and Meade T J 2001 Electronic [13] Kerman K, Ozkan D, Kara P, Meric B, Gooding J J and detection of single-base mismatches in DNA with Ozsoz M 2002 Voltammetric determination of DNA ferrocene-modified probes J. Am. Chem. Soc. 123 hybridization using methylene blue and self-assembled 11155–61 alkanethiol monolayer on Au electrodes Anal. Chim. Acta [31] Vernon S D, Farkas D H, Unger E R, Chan V, Miller D L, 462 39–47 Chen Y P, Blackburn G F and Reeves W C 2003 [14] Erdem A, Kerman K, Meric B, Akarca U S and Bioelectronic DNA detection of human papillomaviruses Ozsoz M 1999 Electrochemical biosensor for the detection using eSensorTM: a model system for detection of of short DNA sequences related to the hepatitis B virus multiple pathogens BMC Infect. Dis. 3 12 Electroanalysis 11 586–7 [32] Toshiba Corporation http://dna-chip.toshiba.co.jp/eng/ R9
  10. 10. Review Article [33] Hashimoto K, Ito K and Ishimori Y 1994 Sequence-specific [53] Wang J, Xu D, Kawde A-N and Polsky R 2001 Metal gene detection with a gold electrode modified with DNA nanoparticle-based electrochemical stripping probes and an electrochemically active dye Anal. Chem. potentiometric detection of DNA hybridization Anal. 66 3830–3 Chem. 73 5576–81 [34] Hashimoto K, Ito K and Ishimori Y 1994 Novel DNA sensor [54] Ozsoz M, Erdem A, Kerman K, Ozkan D, Tugrul B, for electrochemical gene detection Anal. Chim. Acta 286 Topcuoglu N, Ekren H and Taylan M 2003 219–24 Electrochemical genosensor based on colloidal Au [35] Hashimoto K, Ito K and Ishimori Y 1998 Microfabricated nanoparticles for the detection of factor V Leiden mutation disposable DNA sensor for detection of hepatitis B virus using disposable pencil graphite electrodes Anal. Chem. DNA Sensors Actuators B 46 220–5 75 2181–7 [36] Hashimoto K and Ishimori Y 2001 Preliminary evaluation of [55] Wang J, Polsky R and Xu D 2001 Silver-enhanced colloidal electrochemical PNA array for detection of single base Au electrochemical stripping detection of DNA mismatch mutations Lab. Chip 1 61–3 hybridization Langmuir 17 5739–41 [37] Kobayashi M, Mizukami T, Morita Y, Murakami Y, [56] Zhu N, Zhang A, He P and Fang Y 2003 Cadmium sulfide Yokoyama K and Tamiya E 2001 Electrochemical gene nanocluster-based electrochemical stripping detection of detection using microelectrode array on a DNA chip DNA hybridization Analyst 128 260–4 Electrochemistry 69 1013–6 [57] Cai H, Xu Y, Zhu N, He P and Fang Y 2002 An [38] Kobayashi M, Mizukami T, Morita Y, Murakami Y, electrochemical DNA hybridization detection assay based Yokoyama K and Tamiya E 2001 Electrochemical gene on a silver nanoparticle label Analyst 127 803–8 detection using microelectrode array and PCR New Tech. [58] Wang J, Xu D and Polsky R 2002 Magnetically induced Japan 29 11–7 solid-state electrochemical detection of DNA [39] Zhou L, Yang J, Estavillo C, Stuart J D, Schenkman J B and hybridization J. Am. Chem. Soc. 124 4208–9 Rusling J F 2003 Toxicity screening by electrochemical [59] Authier L, Grossiord C, Brossier P and Limoges B 2001 Au detection of DNA damage by metabolites generated in situ nanoparticle-based quantitative electrochemical detection in ultrathin DNA-enzyme films J. Am. Chem. Soc. 125 of amplified human cytomegalovirus DNA using 1431–6 disposable microband electrodes Anal. Chem. 73 [40] de Lumley-Woodyear T, Campbell C N, Freeman E, 4450–6 Freeman A, Georgiou G and Heller A 1999 Rapid [60] Sato K, Hosokawa K and Maeda M 2003 Rapid aggregation amperometric verification of PCR amplification of DNA of Au nanoparticles induced by non-cross-linking DNA Anal. Chem. 71 535–8 hybridization J. Am. Chem. Soc. 125 8102–3 [41] Campbell C N, Gal D, Cristler N, Banditrat C and [61] Mori T and Maeda T 2002 Stability change of DNA-carrying Heller A 2002 Enzyme-amplified amperometric sandwich colloidal particle induced by hybridization with target test for RNA and DNA Anal. Chem. 74 158–62 DNA Polym. J. 34 624–8 [42] Dequaire M and Heller A 2002 Screen printing of nucleic [62] Wang J, Liu G and Merko¸ i A 2003 Particle-based detection c acid detecting carbon electrodes Anal. Chem. 74 4370–7 of DNA hybridization using electrochemical stripping [43] Zhang Y, Kim H-H, Mano N, Dequaire M and Heller A 2002 measurements of an iron tracer Anal. Chim. Acta 482 Simple enzyme-amplified amperometric detection of a 149–55 38-base oligonucleotide at 20 pmol l−1 concentration in a 30 µl droplet Anal. Bioanal. Chem. 374 1050–5 [63] Wang J, Polsky R, Merkoci A and Turner K L 2003 [44] Zhang Y, Kim H-H and Heller A 2003 Enzyme-amplified ‘Electroactive beads’ for ultrasensitive DNA detection amperometric detection of 3000 copies of DNA in a Langmuir 19 989–91 10 µl droplet at 0.5 fM concentration Anal. Chem. 75 [64] Miller M M, Sheehan P E, Edelstein R L, Tamanaha C R, 3267–9 Zhong L, Bounnak S, Whitman L J and Colton R J 2001 [45] Pividori M I, Merko¸ i A and Alegret S 2001 Classical c A DNA array sensor utilizing magnetic microbeads and dot–blot format implemented as an amperometric magnetoelectronic detection J. Magn. Magn. Mater. 225 hybridisation genosensor Biosens. Bioelectron. 16 138–44 1133–42 [65] Oster J, Parker J and Brassard L 2001 [46] Huang T J, Liu M, Knight L D, Grody W W, Miller J F and Polyvinyl-alcohol-based magnetic beads for rapid and Ho C-M 2002 An electrochemical detection scheme for efficient separation of specific or unspecific nucleic acid identification of single nucleotide polymorphisms using sequences J. Magn. Magn. Mater. 225 145–50 hairpin-forming probes Nucleic Acids Res. 30 e55 [66] Li J, Ng H T, Cassell A, Fan W, Chen H, Ye Q, Koehne J, [47] Patolsky F, Weizmann Y, Katz E and Willner I 2003 Han J and Meyyappan M 2003 Carbon nanotube Magnetically amplified DNA assays (MADA): sensing of nanoelectrode array for ultrasensitive DNA detection Nano viral DNA and single-base mismatches by using nucleic Lett. 3 597–602 acid modified magnetic particles Angew. Chem. Int. Edn. [67] Cai H, Cao X, Jiang Y, He P and Fang Y 2003 Carbon Engl. 42 2372–6 nanotube-enhanced electrochemical DNA biosensor for [48] Wang J, Xu D, Erdem A, Polsky R and Salazar M A 2002 DNA hybridization detection Anal. Bioanal. Chem. 375 Genomagnetic electrochemical assays of DNA 287–93 hybridization Talanta 56 931–8 [68] Wang J, Liu G and Merkoci A 2003 Electrochemical coding [49] Palecek E, Kizek R, Havran L, Billova S and Fojta M 2002 technology for simultaneous detection of multiple DNA Electrochemical enzyme-linked immunoassay in a DNA targets J. Am. Chem. Soc. 125 3214–5 hybridization sensor Anal. Chim. Acta 469 73–83 [69] Wang J, Kawde A-N, Musameh M and Rivas G 2002 Dual [50] Evtugyn G, Mingaleva A, Budnikov H, Stoikova E, enzyme electrochemical coding for detecting DNA Vinter V and Eremin S 2003 Affinity biosensors based on hybridization Analyst 127 1279–82 disposable screen-printed electrodes modified with DNA [70] Wang J, Liu G and Rivas G 2003 Encoded beads for Anal. Chim. Acta 479 125–34 electrochemical identification Anal. Chem. 75 4667–71 [51] Patolsky F, Weizmann Y and Willner I 2002 Redox-active [71] Lewis F D, Wu T, Zhang Y, Letsinger R L, nucleic-acid replica for the amplified bioelectrocatalytic Greenfield S R and Wasielewski M R 1997 detection of viral DNA J. Am. Chem. Soc. 124 770–2 Distance-dependent electron transfer in DNA hairpins [52] Patolsky F, Lichtenstein A and Willner I 2001 Detection of Science 277 673–6 single-base DNA mutations by enzyme-amplified [72] Fink H-W and Sch¨ nenberger C 1999 Electrical conduction o electronic transduction Nat. Biotechnol. 19 253–7 through DNA molecules Nature 398 407–10R10
  11. 11. Review Article[73] Okahata Y, Kobayashi T, Tanaka K and Shimomura M 1998 [89] Thorp H H 1998 Cutting out the middleman: DNA biosensors Anisotropic electric conductivity in an aligned DNA cast based on electrochemical oxidation TIBTECH 16 117–21 film J. Am. Chem. Soc. 120 6165–6 [90] Jelen F, Yosypchuk B, Kourilova A, Novotny L and[74] Porath D, Bezryadin A, de Vries S and Dekker C 2000 Direct Palecek E 2002 Label-free determination of picogram measurement of electrical transport through DNA quantities of DNA by stripping voltammetry with solid molecules Nature 403 635–8 copper amalgam or mercury electrodes in the presence of[75] Braun E, Eichen Y, Sivan U and Ben-Yoseph G 1998 copper Anal. Chem. 74 4788–93 DNA-templated assembly and electrode attachment of a [91] Masarik M, Kizek R, Kramer K J, Billova S, Brazdova M, conducting silver wire Nature 391 775–8 Vacek J, Bailey M, Jelen F and Howard J A 2003[76] Park S-J, Taton A and Mirkin C A 2002 Array-based Application of avidin–biotin technology and adsorptive electrical detection of DNA with nanoparticle probes transfer stripping square-wave voltammetry for detection Science 295 1503–6 of DNA hybridization and avidin in transgenic avidin[77] GeneOhm Sciences, Inc. http://www.geneohm.com/ maize Anal. Chem. 75 2663–9[78] Long Y-T, Li C-Z, Sutherland T C, Chahma M, Lee J S and [92] Lucarelli F, Kicela A, Palchetti I, Marrazza G and Kraatz H-B 2003 A comparison of electron-transfer rates Mascini M 2002 Electrochemical DNA biosensor for of ferrocenoyl-linked DNA J. Am. Chem. Soc. analysis of wastewater samples Bioelectrochemistry 58 125 8724–5 113–8[79] Boon E M, Salas J E and Barton J K 2002 An electrical probe [93] Meric B, Kerman K, Ozkan D, Kara P, Erdem A, of protein–DNA interactions on DNA-modified surfaces Kucukoglu O, Erciyas E and Ozsoz M Electrochemical Nat. Biotechnol. 20 282–6 biosensor for the interaction of DNA with the alkylating[80] Marques L P, Cavaco I, Pinheiro J P, Ribeiro V and agent 4,4 -dihydroxy chalcone based on guanine and Ferreira G N 2003 Electrochemical DNA sensor for adenine signals J. Pharm. Biomed. Anal. 30 1339–46 detection of single nucleotide polymorphisms Clin. Chem. [94] Ozsoz M, Erdem A, Kara P, Kerman K and Ozkan D 2003 Lab. Med. 41 475–81 Electrochemical biosensor for the detection of interaction[81] Fan C, Plaxco K W and Heeger A J 2003 Electrochemical between arsenic trioxide and DNA based on guanine interrogation of conformational changes as a reagentless signal Electroanalysis 15 613–9 method for the sequence-specific detection of DNA Proc. [95] Abreu F C, Goulart M O and Brett A M 2002 Detection of Natl Acad. Sci. USA 100 9134–7 the damage caused to DNA by niclosamide using an[82] Gooding J J, Chou A, Mearns F J, Wong E (L-S) and electrochemical DNA-biosensor Biosens. Bioelectron. 17 Jericho K L 2003 The ion-gating effect: using a change in 913–9 flexibility to allow label free electrochemical detection of [96] Gil Ede S, Serrano S H, Ferreira E I and Kubota L T 2002 DNA hybridization Chem. Commun. 1938–9 Electrochemical evaluation of rhodium dimer-DNA[83] Jelen F, Fojta M and Palecek E 1997 Voltammetry of native interactions J. Pharm. Biomed. Anal. 29 579–84 double-stranded, denatured and degraded DNAs [97] Katz E and Willner I 2003 Probing biomolecular interactions J. Electroanal. Chem. 427 49–56 at conductive and semiconductive surfaces by impedance[84] Meric B, Kerman K, Ozkan D, Kara P and Ozsoz M 2002 spectroscopy: routes to impedimetric immunosensors, Indicator-free electrochemical DNA biosensor based on DNA-sensors, and enzyme biosensors Electroanalysis 15 adenine and guanine signals Electroanalysis 14 1245–50 913–47[85] Kara P, Kerman K, Ozkan D, Meric B, Erdem A, [98] Patolsky F, Lichtenstein A and Willner I 2003 Highly Nielsen P E and Ozsoz M 2002 Label-free and label based sensitive amplified electronic detection of DNA by electrochemical detection of hybridization by using biocatalyzed precipitation of an insoluble product onto methylene blue and peptide nucleic acid probes at chitosan electrodes Chem. Eur. J. 9 1137–45 modified carbon paste electrodes Electroanalysis 14 [99] Cloarec J P, Deligianis N, Martin J R, Lawrence I, 1685–90 Souteyrand E, Polychronakos C and Lawrence M F 2002[86] Kerman K, Ozkan D, Kara P, Erdem A, Meric B, Immobilization of homooligonucleotide probe layers onto Nielsen P E and Ozsoz M 2003 Label-free bioelectronic Si/SiO(2) substrates: characterization by electrochemical detection of point mutation by using peptide nucleic acid impedance measurements and radiolabelling Biosens. probes Electroanalysis 15 667–70 Bioelectron. 17 405–12[87] Ozkan D, Erdem A, Kara P, Kerman K, Meric B, [100] Lee T Y and Shim Y B 2001 Direct DNA hybridization Hassmann J and Ozsoz M 2002 Allele-specific genotype detection based on the oligonucleotide functionalized detection of Factor V Leiden mutation from polymerase conductive polymer Anal. Chem. 73 5629–32 chain reaction amplicons based on label-free [101] Farace G, Lillie G, Hianik T, Payne P and Vadgama P 2002 electrochemical genosensor Anal. Chem. 74 5931–6 Reagentless biosensing using electrochemical impedance[88] Lucarelli F, Marrazza G, Palchetti I, Cesaretti S and spectroscopy Bioelectrochemistry 55 1–3 Mascini M 2002 Coupling of an indicator-free [102] Fritz J, Cooper E B, Gaudet S, Sorger P K and electrochemical DNA biosensor with polymerase chain Manalis S R 2002 Electronic detection of DNA by its reaction for the detection of DNA sequences related to the intrinsic molecular charge Proc. Natl Acad. Sci. USA 99 apolipoprotein E Anal. Chim. Acta 469 93–9 14142–6 R11