Electrochemical Detection of Single-Nucleotide Mismatches: Application of M-DNA

Electrochemical Detection of Single-NucleotideMismatches: Application of M-DNA

Yi-Tao Long,†,‡ Chen-Zhong Li,†,‡ Todd C. Sutherland,†,‡ Heinz-Bernhard Kraatz,*,† andJeremy S. Lee*,‡

Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, S7N 5C9 Saskatchewan, Canada,and Department of Biochemistry, University of Saskatchewan, 107 Wiggins Road,Saskatoon, S7N 5E5 Saskatchewan, Canada

The detection of a single-nucleotide mismatch in un-labeled duplex DNA by electrochemical methods is pre-sented. Impedance spectroscopy is used to characterizea perfect duplex monolayer and three DNA monolayersdiffering in the position of the mismatch. The monolayerswere studied as B-DNA (normal duplex DNA) and afterconversion to M-DNA (a metalated duplex). Modeling ofthe impedance data to an equivalent circuit providesparameters that are useful in discriminating the fourmonolayer configurations. The resistance to charge trans-fer, RCT, was lower for all duplexes after conversion toM-DNA. Contrary to expectations, RCT was also found todecrease for duplexes containing a mismatch. However,RCT was found to be diagnostic for mismatch detection.In particular, the difference in RCT between B- and M-DNA(∆RCT) decreased from 190(22) Ω‚cm2 for a perfectlymatched duplex to 95(20), 30(20), and 85(20) Ω‚cm2

for a mismatch at the top (distal), middle, and bottom(proximal) positions of the monolayer with respect to thegold surface. Further, a method to form loosely packedsingle-stranded (ss)-DNA monolayers by duplex dehy-bridization that is able to rehybridize to target strands ispresented. Rehybridization efficiencies were in the rangeof 40-70%. Under incomplete hybridization conditions,the RCT was the same for matched and mismatchedduplexes under B-DNA conditions. However, ∆RCT be-tween B- and M-DNA, under incomplete hybridization,still provided a distinction. The ∆RCT for a perfect duplexwas 76(12) Ω‚cm2, whereas a mismatch in the middle ofthe sequence yielded a ∆RCT value of 30(15) Ω‚cm2. Thedetection limit was measured and the impedance meth-odology reliably detected single DNA base pair mis-matches at concentrations as low as 100 pM.

DNA biosensors provide a powerful means of recognizingspecific DNA sequences. The design of sequence-selective DNAbiosensors, via hybridization, has received much attention inrecent years.1-9 In particular, methods for the rapid identification

of base mutations or single nucleotide polymorphisms (SNPs)would prove useful for the diagnosis of many genetic diseasesand in clinical pharmacology.10-15 Two classes of DNA mutationbiosensors are commonly employed. The first requires covalentmodification of the target DNA strands with groups includingredox probes,16-19 fluorescent dyes, or radioactive markers.2,20,21

The second class employs reporter molecules that are notcovalently bound to the DNA, such as biomolecular beacons22-24

and electrochemically active intercalators.25-29 The latter methods

* Authors to whom correspondence should be addressed. E-mail: [email protected] (H.-B. Kraatz); [email protected] (J. S. Lee).

† Department of Chemistry.‡ Department of Biochemistry.

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10.1021/ac049482d CCC: $27.50 © 2004 American Chemical Society Analytical Chemistry, Vol. 76, No. 14, July 15, 2004 4059Published on Web 05/28/2004

have the potential advantage that the target DNA does not requireany post-isolational modification, thereby reducing the numberof manipulations. Electrochemical methods for SNP detection areattractive because they allow a direct electrical readout, whichagain reduces the complexity of the assay.13,29-31 For example,Barton and colleagues have developed a chronoamperometrictechnique that is dependent on the intercalator Methylene Blue(MB+), which acts as a redox mediator between the DNA duplexattached to a gold electrode and the redox probe [Fe(CN)6]3-/4-

in solution. Duplex DNA containing a single mismatch has a lowerrate of charge transport and, thus, can be distinguished from aperfect duplex.32,33 Heller and co-workers have used cyclic voltam-metry (CV) to detect DNA mismatches at Au electrodes using aredox-active polymer adjacent to the Au surface and the covalentattachment of an enzyme to the target DNA sequence.34,35 TheCV provides a readout of the enzyme-amplified signal and is ableto discriminate a DNA mismatch from a match. Along the samelines but using impedance spectroscopy, Willner and co-workerswere able to detect mismatches in B-DNA (normal double-stranded-(ds)-DNA) by enzymatic amplification.36,37 Again, thisuniquely sensitive technique relies on the covalent modificationof the DNA. The weakness of such techniques is that they arevery dependent on hybridization efficiencies. Recently, anothermethod was proposed38,39 to detect DNA hybridization andpotentially DNA-mismatch detection. One end of a ferrocene-(Fc)-DNA construct containing a self-complementary region wasattached to an Au surface. Upon monolayer formation, the single-stranded-(ss)-DNA induces a hairpin, forcing the Fc group to bepositioned close to the electrode surface. Hybridization with acomplementary strand released the hairpin structure to formB-DNA, resulting in the Fc moiety increasing the distance to theelectrode surface. Complementary sequences result in a changein the redox potential and kinetics of electron transfer of the Fcgroup, thus enabling an effective electrochemical hybridizationsensor. The method was recently tested38 and proved very effectiveas a hydridization sensor. Furthermore, a recent study19 hassuccessfully used a similar ss-DNA hairpin system to determinesingle base pair mismatches but again the hybridization efficiency

can lead to erroneous conclusions. In summary, it is highlydesirable to develop an approach to single-nucleotide mismatchdetection that does not require DNA labeling and does not dependon hybridization efficiency.

Previously, we studied, by electrochemical impedance spec-troscopy (EIS)40 and cyclic voltammetry,41 the electrochemicalbehavior of DNA duplexes attached to gold electrodes under B-and M-DNA conditions. M-DNA is a metalated form of DNA whichforms at pH 8.5 with Zn2+;42-44 see refs 45-47 for recent reviews.In EIS, the impedance of an electrode undergoing heterogeneouselectron transfer through a self-assembled monolayer can bedescribed using an equivalent electrical circuit consisting ofresistance and capacitance elements, such as RS (the solutionresistance), RCT (the charge-transfer resistance), CPE (the constant-phase element), and a mass transfer element W (Warburgimpedance). M-DNA formation causes significant changes in theelectronic properties of the DNA that are readily detected by EIS.Nyquist plots show large differences between B- and M-DNA,which when analyzed by a modified Randle’s circuit demonstratethat the charge-transfer resistance through the DNA is decreasedfor M-DNA. The charge-transfer resistance increases with increas-ing duplex length for M-DNA, but in each case, the resistancefor M-DNA is lower than its corresponding B-DNA construct. Inaddition, electron transfer is faster in M-DNA as compared to thatin B-DNA.40,41

We hypothesized that any disruption of the electron transferpathway in DNA, caused by bulges and kinks due to a mismatchedsequence, would have an effect on the electronic and electrontransfer properties of the DNA construct. As was previouslyreported, mismatches result in a larger motion of the DNA helixwhich will influence the electronic coupling between the base pairsand lead to a decrease in electron transfer rate.29 Single-nucleotidemismatches in B-DNA will affect the local environment of adjacentbases. This effect is dependent on the identity of the base andthe position of the mismatch in the helix.48,49

EIS was deemed to be the most suitable technique to evaluatethe effects of mismatches on the electronic and electron-transferproperties of DNA and has been used before in enzyme-amplifiedmethods for mismatch detection.36 In this contribution, wedemonstrate that the presence and position of a single-nucleotidemismatch in unlabeled M- and B-DNA monolayers give rise tocharacteristic changes in the impedance spectrum, which can beexploited for the detection of mismatches even under conditionsof incomplete hybridization with a detection limit of 100 pM. This

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C. N.; Heller, A. J. Am. Chem. Soc. 1999, 121, 10803-10812.(35) Caruana, D. J.; Heller, A. J. Am. Chem. Soc. 1999, 121, 769-774.(36) Patolsky, F.; Lichtenstein, A.; Willner, I. Nat. Biotechnol. 2001, 19, 253-

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100, 9134-9137.(39) Hu, X. Ph.D. Dissertation, Duke University: Durham, NC, 2001.

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4060 Analytical Chemistry, Vol. 76, No. 14, July 15, 2004

represents a significant improvement over other methods anddemonstrates the utility of M-DNA.

EXPERIMENTAL SECTIONMaterials. The 5′-disulfide-labeled and unlabeled oligonucle-

otide strands were synthesized by standard phosphoamidate solid-phase DNA synthesis using a fully automated DNA synthesizer,purified by reversed-phase HPLC and then characterized byelectrospray ionization mass spectrometry (see Supporting Infor-mation).40 The DNA sequences and position of the mismatchesare shown in Chart 1.

Monolayer Preparation. The freshly cleaned gold electrodes(BAS, 1.6-mm diameter) were incubated in 0.05-mM ss-DNA ords-DNA B-DNA, 20-mM Tris-ClO4 buffer solution (pH 8.6) for 5days. Then the electrodes were washed with Tris-ClO4 bufferand mounted into an electrochemical cell. Dehybridization andregeneration of the single-stranded probe electrode was achievedby denaturing the duplex DNA by soaking the electrode in aheated (60 °C) water/EtOH (60:40) bath for 10 min and thenrinsing in room temperature 20-mM Tris-ClO4 buffer. Reproduc-ible behavior was found for repeated measurements on differentelectrodes. Rehybridization was performed by exposing the ss-DNA self-assembled monolayer (SAM) to SSC buffer (300-mMNaCl, 30-mM sodium citrate, pH 7) heated to 37 °C in the presenceof target DNA for 10 min and then was allowed to cool to roomtemperature for an additional 3 h. B-DNA was converted toM-DNA by the addition of 0.4-mM Zn(ClO4)2‚6H2O for 2 h at pH8.6.40,41

The formation of the monolayer was assessed by standardblocking studies with [Fe(CN)6]3-/4-, X-ray photoelectron spec-troscopy (XPS), and EIS, as described previously.40 The blockingstudies showed a decrease in peak current attributed to thereduced diffusion of the redox probe to the Au surface. The XPSdata show the presence of a an Au-thiolate bond and a thicknessof 44 Å for a 1:2 monolayer.40

Electrochemical Measurements. A conventional three-electrode cell was used. All experiments were conducted at roomtemperature (22 °C). The cell was enclosed in a grounded Faradaycage. The reference electrode was constructed by sealing a Ag/AgCl wire into a glass tube with a solution of 3 m KCl that wascapped with a Vycor tip. The counter electrode was a platinumwire. Impedance spectra were measured using an EG&G 1025

frequency response analyzer interfaced to an EG&G 283 poten-tiostat/galvanostat. The ac voltage amplitude was 5 mV and thevoltage frequencies used for EIS measurements ranged from 100kHz to 100 mHz. The applied potential was 250 mV vs Ag/AgCl(formal potential, E°′, of the redox probe [Fe(CN)6]3-/4-. Allmeasurements were repeated a minimum of five times withseparate electrodes to obtain statistically meaningful results.

RESULTS AND DISCUSSIONMonolayers of fully matched B-DNA on gold were prepared

from the oligonucleotide 1 and its fully matched complementarystrand 2. The properties of the resulting 1:2 B-DNA surfacecompares well with those described before.40,41 To evaluate theeffect of mismatches by EIS, we prepared three types of mis-matched monolayers, each containing a single pyrimidine‚pyrimidine mismatch in the complementary strand. Complemen-tary mismatched strand 3 contains a mismatch in the second topbase pair, resulting in a mismatch distal to the electrode surface.Complementary mismatched strand 4 contains a T instead of aG in position 11, giving a monolayer with the mismatch in themiddle of the duplex. Complementary mismatched strand 5possesses a C instead of an A in position 19, resulting in amismatch proximal to the electrode surface. Mismatched B-DNAmonolayers of 1:3, 1:4, and 1:5 were prepared in an analogousmanner (see Figure 1). Impedance measurements were carriedout on all monolayers in 20-mM Tris-ClO4 (pH 8.6) in the presenceof a 4 mM [Fe(CN)6]3-/4- (1:1) mixture, as the solution-basedredox probe. The B-DNA monolayers were then converted toM-DNA monolayers by the addition of 0.4 mM Zn2+ at pH 8.6 asdescribed before.40,41 The impedance measurements were repeatedunder M-DNA conditions for all four monolayers. Control experi-ments were performed with longer incubation times with Zn2+

and repeated EIS measurements. Neither procedure producedchanges in the impedance spectra, demonstrating that the DNAwas not significantly damaged, for example, by oxidation due toelectron transfer.25,31 Typical impedance spectra, in the form ofNyquist plots, for B-DNA and M-DNA monolayers of a perfectlymatched duplex (1:2) and a duplex containing a mismatch in themiddle of the helix (1:4) are shown in Figure 2. Each pointrepresents a value of Zim and Zre measured at a particular acfrequency. The spectra show a lower impedance for M-DNA thanfor B-DNA, as would be expected from previous observations.40-44

Chart 1. List of DNA Sequences Used for Monolayer Film Preparationa

a Mismatched base pairs are indicated by the blocked characters in the sequence.

Analytical Chemistry, Vol. 76, No. 14, July 15, 2004 4061

More importantly, the presence of a mismatch in the DNA duplexdecreases the impedance of B-DNA while increasing the imped-ance of M-DNA. To provide a rationale for this behavior, theimpedance spectra of all DNA films were analyzed with a modifiedRandles equivalent circuit.40 The circuit drawing is shown in Figure2. The same model was used to fit all monolayers described here.The fit of the equivalence circuit to the experimental values isgiven as a solid line. This treatment allows the interpretation ofthe impedance data in terms of electronic circuit components,

which are listed for all monolayers in Table 1. The equivalentcircuit contains five elements that are described below.

A solution resistance term, RS, remains constant at 5-6 Ω‚cm2, as would be expected for measurements under identicalconditions of supporting electrolyte concentration and tempera-ture. The circuit contains a constant phase element (CPE) actingas a nonideal capacitor, which is commonly used instead of acapacitor to account for inhomogeneity on the electrode surface.51

Here, the CPE will be interpreted as a capacitor as describedelsewhere in situations where the exponential modifier is greaterthan 0.9.50 This is the case for all monolayers presented in thispaper. Monolayer composition and thickness are contributingfactors to the CPE. The magnitude of the CPE for films of thematched duplex 1:2 and the two top and bottom mismatchedduplexes 1:3 and 1:5 are in the range of 10-25 µF‚cm-2.However, for films of 1:4, B- and M-DNA containing the middlemismatch, a significantly higher capacitance of about 40(2)µF‚cm-2 was observed. A possible interpretation is a change inthe monolayer thickness for films of 1:4 due to kinking of thehelix, which is caused by the mismatch in the center of the DNAsequence. This kink will cause the duplex to bend toward thesurface, resulting in a decrease in the film thickness. The top andbottom mismatch containing films, 1:3 and 1:5, respectively, yieldCPE values that also indicate slight variations in the thickness ofthe monolayer due to fraying of the strands for both films.However, the effects of fraying for the top and bottom mismatchfilms are opposite. Fraying in the top of the helix will result in abetter interaction between individual nonpaired base pairs andthe solvent, thus leading to a thicker monolayer. In the case ofthe mismatch being proximal to the electrode surface, as for film1:5, fraying is expected to enhance flexibility of the DNA-linkerregion, which may result in a lower film thickness by compaction.Effective monolayer thickness can be calculated assuming the CPEis an ideal capacitor and the permittivity of the DNA monolayeris 1 F‚m-1. Under these assumptions, the effective thicknessesfor B-DNA monolayers of 1:2, 1:3, 1:4, and 1:5 are 120, 140, 40,and 70 Å, respectively.

The RX component of the equivalence circuit can be attributedto pinholes in the monolayer structure. The value of RX is similarfor each of the B-DNA monolayers; indicating the number andsize of the pinholes does not change between monolayers.However, RX tends to decrease upon conversion to M-DNA. Thisbehavior can be attributed to M-DNA having a slightly morecompact structure than B-DNA due to the divalent metal ionsreducing the repulsion between the phosphate backbone residuesof adjacent helices.40-43 The Warburg impedance element, W, isdependent on the rate of diffusion of the [Fe(CN)6]3-/4- redoxprobe. The Warburg impedance is smallest for the perfect duplexin the B-DNA conformation, suggesting that this is the mostordered monolayer, which offers the least access of the solutionelectrophore through the DNA monolayer.

The charge-transfer resistance term, RCT, comprises resistanceterms resulting from (a) transfer of the electron from the[Fe(CN)6]3-/4- redox probe to the DNA monolayer, (b) theresistance to charge transfer between the base pairs of the DNAhelix, and (c) from the helix to the surface of the gold electrode.

(50) Creager, S. E.; Wooster, T. T. Anal. Chem. 1998, 70, 4257-4263.(51) Dijksma, M.; Boukamp, B. A.; Kamp, B.; van Bennekom, W. P. Langmuir

2002, 18, 3105-3112.

Figure 1. Proposed structures of the monolayers prepared in thisstudy. (a) 1:2, the complementary ds-DNA monolayer. (b) 1:3,mismatch distal to the Au surface of the ds-DNA monolayer. (c) 1:4,mismatch in the middle of the ds-DNA monolayer. (d) 1:5, mismatchproximal to the Au surface of the ds-DNA monolayer. The arrowindicates the position of the mismatched base pair.

Figure 2. Nyquist plots (-Zim vs Zre) of the 20 base pair comple-mentary B-DNA (O), middle mismatch B-DNA (0), complementaryM-DNA (b), and middle mismatch M-DNA (9) assembled on gold ina 4 mM [Fe(CN)6]3-/4- (1:1) mixture as the redox probe in 20 mMTris-ClO4 and 20 mM NaClO4 solution. Applied potential of 250 mVversus Ag/AgCl. [Zn2+] ) 0.4 mM; pH 8.6. In all cases the measureddata points are shown as symbols with the calculated fit to theequivalent circuit as solid lines. Inset: The experimental data werefit to the equivalent circuit. RS, solution resistance; RX, monolayerpinhole/defect resistance; RCT, charge transfer resistance; CPE,constant phase element; W, Warburg impedance.


As expected, for all monolayers, RCT is lower for M-DNA than forB-DNA. More importantly, RCT allows the discrimination betweena single nucleotide mismatch and a perfectly matched DNA film.The presence of a mismatch causes a decrease in RCT for all filmscontaining mismatches. This is contrary to expectations since theelectron transfer (ET) kinetics through an unstacked region isretarded.25,32,33 Therefore, thickness and/or disorder in the mono-layer, as signified by the changes in the CPE term, must dominaterates of ET rather than simple RCT. As far as mismatch detectionis concerned, the evaluation of the difference in charge-transferresistance, ∆RCT, between B- and M-DNA for a given film givesexcellent discrimination between a perfect duplex and onecontaining a single mismatch at either the top or the middlepositions of the duplex. Table 1 lists the ∆RCT for all films. ∆RCT

for the perfectly matched duplex film 1:2 is 190 (22) Ω‚cm2

whereas for the mismatched films, ∆RCT is significantly smaller.Interestingly, ∆RCT for the top mismatch containing the film of1:3 and the bottom mismatch (1:5) are similar (95(19) Ω‚cm2

for 1:3 and 85(20) Ω‚cm2 for 1:5). ∆RCT for the duplex containingthe middle mismatch is much lower (30(18) Ω‚cm2 for 1:4). Theuse of ∆RCT was attractive from an application perspective becausedifferent electrode morphologies can yield different impedancesbut the comparative impedance measurements between B- andM-DNA are reproducible.

To assess the ability for mismatch determination undernonideal conditions, we investigated the effect of rehybridization.In this format, the DNA probe sequence is washed across a ss-DNA monolayer which may result in differences in hybridization.The direct formation of a ss-DNA monolayer yields a film in whichthe DNA strands are densely packed and may interfere with thebinding of the complementary strand.52,53 Therefore, we decidedto form a ds-DNA film and then dehybridize to a more looselypacked ss-DNA monolayer. In this way, rehybridization efficienciesfor the target DNA in the range of 40-70% can be achieved.53,54

Figure 3a outlines the experimental procedure. Washing of a ds-DNA film with a hot (60 °C) water/EtOH (60:40) bath followedby rinsing in room-temperature Tris-ClO4 buffer results in dehy-bridization and formation of a ss-DNA film consisting of DNAstrand 1. This film is then exposed to solutions of complementarytarget ss-DNA and allowed to hybridize for 3 h. The heating could

have deleterious effects on the monolayer; however, this is ruledout because the RX component has remained the same orincreased, indicating that no new pinholes or defect sites werecreated. We expected from the results of Table 1 that thecombination using the middle mismatch, strand 4, would givethe smallest difference in ∆RCT under these conditions. Uponreformation of the ds-DNA film using 2 or 4 following thedehybridization-rehybridization procedure, the impedance signaldoes not return to the values for a perfect ds-DNA monolayer asshown by Figure 3b, indicating that the resulting film most likelyconsists of ds- and ss-DNA. Importantly, despite incompleterehybridization the presence of a mismatch can still be detectedas shown by the impedance spectra in Figure 4. After rehybrid-ization of the ss-DNA film with a matching complementary strand,2, and one containing a single mismatch in the middle of thestrand, 4, all spectra were fit to the same equivalent circuitdescribed above and the electronic circuit parameters are shown

(52) Yang, M.-S.; Yau, H. C. M.; Chan, H. L. Langmuir 1998, 14, 6121-6129.(53) Peterson, A. W.; Wolf, L. K.; Georgiadis, R. M. J. Am. Chem. Soc. 2002,

124, 14601-14607.(54) Peterlinz, K. A.; Georgiadis., R. M. J. Am. Chem. Soc. 1997, 119, 3401-

3402.

Figure 3. (a) Hybridization-dehybridization procedure. (i) Soakedin a water:EtOH (60:40) bath at 60 °C for 10 min and then rinsedwith room temperature 20 mM Tris-ClO4 buffer. (ii) Add target ss-DNA in SSC buffer and allow duplex formation to occur for 10 min at37 °C followed by 3 h at room temperature. (b) Nyquist plot of fullyhybridized “ideal” monolayer of 1:2 construct (O), ss-DNA monolayerof 1 (0) after dehybridization procedure, and rehybridized ds-DNAfilm of 1:2 (b) following the rehybridization procedure. The impedancespectra of the rehybridized film is different compared to that of the“ideal” 1:2 films, indicating the heterogeneity of the monolayer as aresult of incomplete hybridization.

Table 1. Circuit Element Values for the Complementary DNA Monolayer and the Series of Mismatch DNAMonolayersa

1:2 1:3 1:4 1:5

circuit element B-DNA M-DNA B-DNA M-DNA B-DNA M-DNA B-DNA M-DNA

RS/Ω‚cm2 5.8(0.5) 6.0(0.6) 4.9(0.9) 4.8(0.7) 6.5(0.7) 5.8(0.6) 5.3(0.7) 6.1(0.8)RX/Ω‚cm2 300(21) 245(18) 357(19) 319(16) 351(17) 323(16) 312(15) 255(12)CPEb/µF‚cm-2 15.0(0.4) 15.7(0.5) 12.8(0.3) 10.9(0.2) 42.1(0.9) 38.0(0.6) 25.0(0.3) 23.1(0.2)RCT/Ω‚cm2 390(20) 200(10) 299(15) 204(12) 258(14) 228(11) 317(18) 232(10)∆RCT/Ω‚cm2 190(22) 95(19) 30(18) 85(20)W/10-3 Ω‚s-1/2‚cm2 1.5(0.3) 3.9 (0.4) 3.8(0.6) 3.4(0.2) 7.8(0.4) 8.1(0.5) 3.0(0.3) 3.9(0.2)

a The values in parentheses represent the standard deviations from several electrode measurements (n g 5) not the non-linear curve fittingerrors. b CPE and associated units are interpreted as a capacitor with an exponential modifier >0.9.50


in Table 2. Apparent from Figure 4 and Table 2 is that the B-DNAfilms, which result from the hybridization of a matched andmismatched DNA target, are indistinguishable. However, the filmsclearly show a difference under M-DNA conditions. Again, RCT isused to discriminate between matched and mismatched DNAfilms. The difference in RCT between B- and M-DNA is consistentlylarger (76(12) Ω‚cm2) for a perfect duplex compared to amismatched film in which ∆RCT decreases to 29(15) Ω‚cm2. Theusefulness of M-DNA could be due to the inherent ability of Zn2+

to bind and electronically alter the duplex DNA without changingthe electronic properties of ss-DNA monolayers. Thus, incompletehybridization does not have a drastic effect on mismatch detection;rather a surface that contains more duplex DNA will simply resultin a larger ∆RCT. In support of this hypothesis, a controlexperiment showing the small effect that Zn2+ addition has onss-DNA is included in the Supporting Information.

From a practical perspective, hybridization of a target strandis likely to be performed with a target DNA that is longer thanthe 20 bases of the probe. Therefore, strands 6 (match) and 7(mismatch) were synthesized which contain three additionaladenosines at both the 3′ and 5′ ends, creating overhangs. Afterhybridization of either 6 or 7 to strand 1, EIS was performed

under standard conditions; the resulting Nyquist plot is shown inFigure 5. It is clear that under B-DNA conditions there is littledifference between RCT for 1:6 (match with overhangs ) 510Ω‚cm-2) and 1:7 (mismatch with overhangs ) 470 Ω‚cm-2)duplexes but after conversion to M-DNA this difference isenhanced (∆RCT is 160 and 70 Ω‚cm-2 for match and mismatch,respectively). Thus, as was the case for duplexes withoutoverhangs, the measurement of ∆RCT gives good discriminationfor targets with short overhangs. Control experiments were alsoperformed where the rehybridization was carried out in eitherstandard PCR buffer or in the presence of a 1000-fold excess of anoncomplimentary target sequence. In neither case was there asignificant change in the value of ∆RCT (data not shown).

Finally, to detect the sensitivity of the dehybridization-rehydridization method, a target concentration range from 10-5

to 10-12 M has been employed. Impedance spectra were recordedfor matched and mismatched DNA in both B- and M-DNAconditions and the resulting impedance was fit to the equivalentcircuit shown in Figure 2. As shown in Figure 6, ∆RCT remainsrelatively constant down to concentrations of 100 pM of targetss-DNA. It is important to emphasize that a clear discriminationbetween matched and mismatched DNA is obtained by thedifference in RCT between B- and M-DNA.

CONCLUSIONAlthough this study is restricted to the detection of a pyrimidine‚

pyrimidine mismatch, purine‚purine mismatches should also bedetectable since they cause similar disruptions to the helical stackof a DNA duplex.55 The presence of a mismatch causes a decreasein RCT regardless of the position of the mismatch. The differencein charge-transfer resistance, ∆RCT, between B- and M-DNArepresents a reliable measure of the presence of a singlenucleotide mismatch under ideal conditions. Even under condi-tions in which incomplete hybridization is observed, single

(55) Yamashita, K.; Takagi, M.; Kondo, H.; Takenaka, S. Chem. Lett. 2000, 1038-1039.

Figure 4. Nyquist plots (-Zim vs Zre) of the rehybridized 20 basepair complementary B-DNA (O), middle mismatch B-DNA (0),complementary M-DNA (b), and middle mismatch M-DNA (9) as-sembled on gold with 4 mM Fe(CN)6

3-/4- (1:1) mixture as the redoxprobe in 20 mM Tris-ClO4 and 20 mM NaClO4 solution. Appliedpotential of 250 mV versus Ag/AgCl. [Zn2+] ) 0.4 mM; pH 8.6. In allcases the measured data points are shown as symbols with thecalculated fit to the equivalent circuit as solid lines.

Table 2. Fitted Impedance Values for the RehybridizedComplementary DNA Monolayer and the RehybridizedMiddle Mismatch DNA Monolayera

rehybridized 1:2 rehybridized 1:4

element B-DNA M-DNA B-DNA M-DNA

RS/Ω‚cm2 6.3 (0.6) 6.0 (0.5) 5.8 (0.6) 5.9 (0.8)RX/Ω‚cm2 345 (11) 311 (15) 355 (17) 334 (9)CPEb/µF‚cm-2 26 (1.5) 17 (0.6) 34 (0.9) 20 (1.5)RCT/Ω‚cm2 295 (11) 219 (5) 284 (12) 255(9)∆RCT/Ω‚cm2 76 (12) 29 (15)W/10-3 Ω‚s-1/2‚cm2 3.1 (0.1) 4.9 (0.3) 3.9 (0.4) 5.2 (0.4)

a Values in parentheses represent the standard deviations fromseveral electrode measurements (n g 5) not the nonlinear curve fittingerrors. b See Table 1.

Figure 5. Nyquist plots (-Zim vs Zre) of the rehybridized 1:6 B-DNA(0), 1:7 B-DNA (O), 1:7 M-DNA (b), and 1:6 M-DNA (0) assembledon gold with a 4 mM Fe(CN)6

3-/4- (1:1) mixture as the redox probein 20 mM Tris-ClO4 and 20 mM NaClO4 solution. Applied potential of250 mV versus Ag/AgCl. [Zn2+] ) 0.4 mM; pH 8.6. In all cases themeasured data points are shown as symbols with the calculated fitto the equivalent circuit as solid lines.


nucleotide mismatches can be detected by the formation ofM-DNA. Thus, these results present a significant step forward inthe electrochemical detection of SNPs using an unlabeled DNAhybrid.

A device with a detection limit of 100 pM can potentially beused for such applications as clinical diagnosis of mutations.However, an amplification step to increase DNA concentrations

from biological samples is required before this sensor can beutilized. In the long term, greater sensitivity can be achieved byreducing the size of the electrode. For example, if a macroelec-trode can detect 100 pM of target, then a microelectrode with acorresponding reduction in target volume would have a sensitivityin the femtomolar range at which point direct detection withoutamplification of the target becomes realistic.

ACKNOWLEDGMENTThe authors wish to thank CIHR, NSERC, and UMDI for

financial support, H.-B.K. is the Canada Research Chair inBiomaterials and J.S.L. is supported by a Senior InvestigatorsAward from the Regional Partnership Program of CIHR. Theauthors also thank Don Schwab, the Plant Biotechnology Institute,Canada, for the preparation of DNA samples.

SUPPORTING INFORMATION AVAILABLEHPLC and UV-visible spectra of 1, EIS mass spectrum of 1,

and impedance spectra of ss-DNA monolayer and ss-DNA underM-DNA conditions. This material is available free of charge viathe Internet at http://pubs.acs.org.

Received for review April 2, 2004. Accepted April 23, 2004.

AC049482D

Figure 6. Determination of detection limits by monitoring the changein RCT between B-DNA and M-DNA as a function of target single-stranded DNA concentration. Complementary DNA strands (0) andmiddle mismatch DNA strands (O). Error bars are derived from aminimum of five electrodes.


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Electrochemical Detection of Single-Nucleotide Mismatches: Application of M-DNA