10392 DOI: 10.1021/la100227s Langmuir 2010, 26(12), 10392–10396Published on Web 03/17/2010

pubs.acs.org/Langmuir

© 2010 American Chemical Society

Electrochemical DNA Detection via Exonuclease and Target-Catalyzed

Transformation of Surface-Bound Probes

Kuangwen Hsieh,† Yi Xiao,*,†,‡ and H. Tom Soh*,†,‡

†Department of Mechanical Engineering and ‡Materials Department, University of California, Santa Barbara,California 93106

Received January 16, 2010. Revised Manuscript Received February 18, 2010

We report a single-step, single-reagent, label-free, isothermal electrochemical DNA sensor based on the phenomenonof target recycling. The sensor exploits strand-specific exonuclease activity to achieve the selective enzymatic digestion oftarget/probe duplexes. This results in a permanent change in the probe structure that yields an increased faradaic currentand liberates the intact target molecule to interact with additional detection probes to achieve further signalamplification. Using this architecture, we achieve an improved detection limit in comparison to hybridization-basedsensors without amplification. We also demonstrate a 16-fold signal amplification factor at low target concentrations.Combined with the advantages of electrochemical detection and its ready integration with microelectronics, ourapproach may represent a promising path toward direct DNA detection at the point of care.

Recent years have witnessed the development of DNA sensorscapable of the rapid detection of trace amounts of DNA to addressincreasingly important applications in molecular diagnostics,1,2

pathogen detection,3-6 forensic investigations,7 and environmentalmonitoring.8-11 Strategies based on the isothermal amplification ofsignal produced by hybridization events have demonstrated espe-cially great potential for the direct detection of small amounts ofDNA with impressive limits of detection (LOD). Examples of suchtechniques include target-catalyzed transfer reactions for DNAdetection,12-14 catalytic silver deposition or stripping assays,15-17

gold nanoparticle (AuNP)-based biobar-code assays,18 and enzyme-linked electrochemical assays.19-23 Unfortunately, these methodsgenerally involve multiple steps and require the addition of manyexogenous reagents. For example, the biobar-code assay18 relies onacombination of two-component oligonucleotide-modified AuNPsand single-componentoligonucleotide-modifiedmagneticmicropar-ticles, with subsequent detection of amplified bar-code DNAachieved via a chip-based silver deposition assay. Target detectionwith an enzyme-linked, electrochemical sensor15 entails a five-stepprocess involving an enzyme-conjugated secondary probe, enzy-matic reduction of p-aminophenyl phosphate, concomitant reduc-tive depositionof silver and, finally, anodic stripping voltammetry toquantify the deposited silver. As such, there is a compelling need forsimple, single-step assays for sensitive, specific nucleic acid detectionat the point of care.

Target recycling, wherein signal amplification is achieved byallowing a single DNA target molecule to interact with multiplenucleic acid-based signaling probes, represents an interestingalternative. In this approach, target-probe hybridization catalyzesthe selective enzymatic digestion of the signaling probe, releasingthe intact DNA target to initiate the digestion of other probemolecules, thereby generating multiple signaling events andachieving signal amplification. This approach has previously beendemonstrated using exonucleases,24-28 nicking enzymes,29,30 and

*Corresponding authors. E-mail: yixiao@physics.ucsb.edu; tsoh@engineering.ucsb.edu.(1) Umek, R. M.; Lin, S. W.; Vielmetter, J.; Terbrueggen, R. H.; Irvine, B.; Yu,

C. J.; Kayyem, J. F.; Yowanto, H.; Blackburn, G. F.; Farkas, D. H.; Chen, Y. P.J. Mol. Diagn. 2001, 3, 74–84.(2) Liao, J. C.; Mastali, M.; Gau, V.; Suchard, M. A.; Moller, A. K.; Bruckner,

D. A.; Babbitt, J. T.; Li, Y.; Gornbein, J.; Landaw, E. M.; McCabe, E. R. B.;Churchill, B. M.; Haake, D. A. J. Clin. Microbiol. 2006, 44, 561–570.(3) Lee, J. G.; Cheong, K. H.; Huh, N.; Kim, S.; Choi, J. W.; Ko, C. Lab Chip

2006, 6, 886–895.(4) Yeung, S. W.; Lee, T. M. H.; Cai, H.; Hsing, I. M. Nucleic Acids Res. 2006,

34, e118.(5) Palchetti, I.; Mascini, M. Anal. Bioanal. Chem. 2008, 391, 455–471.(6) Csordas, A. I.; Delwiche, M. J.; Barak, J. D. Sens. Actuators, B 2008, 134,

1–8.(7) Carey, L.; Mitnik, L. Electrophoresis 2002, 23, 1386–1397.(8) Wang, J.; Rivas, G.; Cai, X.; Palecek, E.; Nielsen, P.; Shiraishi, H.; Dontha,

N.; Luo, D.; Parrado, C.; Chicharro, M.; Farias, P. A. M.; Valera, F. S.; Grant,D. H.; Ozsoz, M.; Flair, M. N. Anal. Chim. Acta 1997, 347, 1–8.(9) Gardeniers, J. G. E.; van den Berg, A.Anal. Bioanal. Chem. 2004, 378, 1700–

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2006, 386, 1025–1041.(11) Palchetti, I.; Mascini, M. Analyst 2008, 133, 846–854.(12) Graf, N.; Goritz, M.; Kramer, R. Angew. Chem., Int. Ed. 2006, 45, 4013–

4015.(13) Grossmann, T. N.; Roglin, L.; Seitz, O. Angew. Chem., Int. Ed. 2008, 47,

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Chem. Soc. 2003, 125, 344–345.(15) Wang, J.; Polsky, R.; Xu, D. K. Langmuir 2001, 17, 5739–5741.(16) Park, S. J.; Taton, T. A.; Mirkin, C. A. Science 2002, 295, 1503–1506.(17) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289, 1757–1760.(18) Nam, J. M.; Stoeva, S. I.; Mirkin, C. A. J. Am. Chem. Soc. 2004, 126, 5932–

5933.(19) Caruana, D. J.; Heller, A. J. Am. Chem. Soc. 1999, 121, 769–774.(20) Liu, G.; Wan, Y.; Gau, V.; Zhang, J.; Wang, L. H.; Song, S. P.; Fan, C. H.

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(22) Patolsky, F.; Weizmann, Y.; Willner, I. J. Am. Chem. Soc. 2002, 124, 770–772.

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(24) Duck, P.; Alvarado-Urbina, G.; Burdick, B.; Collier, B.Biotechniques 1990,9, 142–148.

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5100.(29) Kiesling, T.; Cox, K.; Davidson, E. A.; Dretchen, K.; Grater, G.; Hibbard,

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DNAzymes.31,32 However, to date, the sensor response has beenmeasured predominantly through optical methods (e.g., fluore-scence intensity, surface plasmon resonance imaging, or capillaryelectrophoresis). As an alternative, we sought to combine targetrecycling with electrochemical detection,33-36 which offers nu-merous advantages for rapid detection including a relatively lowbackground, simpler instrumentation, and ready integration withmicroelectronics,37,38 making it a promising detection modalityfor use in point-of-care settings.

Here we present a single-step, single-reagent, isothermal,label-free electrochemical DNA detection assay based on theexonuclease/target-catalyzed transformation (ExTCT) of signal-ing probes bound to the surface of a gold electrode. The assay isdesigned such that, in the absence of a target and exonuclease, themethylene blue (MB)-modified signaling probe assumes a stem-loop structure that sequesters the MB tag away from the elec-trode, producing a reduced faradaic current. Hybridization of theDNA target to the signaling probe enables selective exonucleasedigestion of the target/probe duplex, permanently transformingthe probe into a flexible, MB-tagged, single-stranded fragmentthat can efficiently collide with and transfer electrons to theelectrode, giving rise to an increased faradaic current. After thedigestion of the probe, the target DNA is recycled into solution,allowing hybridization with a new, undigested probe. In this way,a single DNA target can catalyze the transformation of multiplesignaling probes, giving rise to an amplified electrochemicalsignal.

Experimental Section

Materials. The DNA signaling probe (1, 50-PHOS-GATT-GAAGTGGATCGGCGTCTCTCCAGGTTT-T(MB)-TTTTT-TTTTTTTGACGCCGT-(CH2)6-HS-30) with 30 C6-linked thiol,an internal MB-label, and 50-phosphate modification was synthe-sized by Biosearch Technologies, Inc. (Novato, CA), purified byC18 dual HPLC, and confirmed by mass spectrometry. Here, theunderlined sequences represent the complementary bases to thematched target. The 29-base perfectly matched target (2, 50-ACC-TGGAGAGACGCCGATCCACTTCAATC-30) and noncog-nate target (3, 50- CTAGTTCTCTCATAATGTAACATGAC-TAA-30) were purchased from Integrated DNA Technologies Inc.(Coralville, IA) and purified by HPLC. Tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) was purchased from Invitrogen(Carlsbad, CA). 6-Mercaptohexanol was purchased from Sigma-Aldrich (St. Louis, MO). Lambda exonuclease (specific activity,100 000 units/mg; concentration, 5000 units/mL; extinction coeff-fiecient, 46660 cm-1M-1) and its 10� reaction buffer were pur-chased from New England Biolabs (Ipswich, MA). All chemicalswere used as receivedwithout further purification. The 1� reactionbuffer for lambda exonuclease contains 67 mM glycine KOH, 2.5mM MgCl2, and 50 μg μL-1 bovine serum albumin (BSA) (pH9.4).Aphosphate buffer (100mMsodiumphosphate, 1.0MNaCl,1 mM MgCl2, pH 7.2) was prepared and used for all electroche-mical measurements.

ExTCT DNA Sensor Preparation. The ExTCT DNAsensor was fabricated by modifying the clean surface of poly-

crystalline gold disk electrodes (1.6 mm diameter; BASi, WestLafayette, IN) with thiolated probe DNA (1) as detailed inprevious work.39 Briefly, the gold electrodes were polished with1.0 μmdiamondpolish and 0.05μmalumina polish (BuehlerLtd.,Lake Bluff, IL), sonicated in ethanol and deionized (DI) water,and electrochemically cleaned in a series of oxidation and reduc-tion steps in 0.5 M NaOH, 0.5 M H2SO4, 0.01 M KCl/0.1 MH2SO4, and 0.05 M H2SO4. Prior to attachment to the goldsurface, thiolated probe DNA (1) was incubated with 100 mMTCEP for 1 h to reduce disulfide bonds and subsequently dilutedto 1.0 μM with phosphate buffer. The clean gold electrodes wereincubated in this reduced probe solution for 1 h at room tem-perature in the dark. The probe-functionalized surface was sub-sequently passivated with 6 mM 6-mercaptohexanol (diluted inphosphate buffer) for 2 h at room temperature. The electrodeswere rinsed with DI water and stored in phosphate buffer forat least 1 h to equilibrate the probe structures prior to electro-chemical measurements.

DNA Target Hybridization, Exonuclease Digestion, and

Electrochemical Measurements. All electrochemical measure-ments were performed on a CHI 730 potentiostat (CH Instru-ments,Austin, TX) in a standard cellwith a platinumwire counterelectrode and an Ag/AgCl reference electrode (saturated with3.0 M NaCl). The measurements were conducted with alter-nating-current voltammetry (ACV) in phosphate buffer using astep potential of 10 mV, an amplitude of 25 mV, and a frequencyof 10Hz. TheExTCT sensorswere first tested in phosphate bufferto establish the baseline of the MB redox current. The enzymaticreaction buffer consists of 1� lambda exonuclease reaction bufferaugmented with MgCl2 (final concentration 10 mM). Reactionswere preparedbyaddingDNAtargets and 50units (0.25U/μL) oflambda exonuclease to the enzymatic reaction buffer (200 μL offinal reaction volume). The ExTCT sensor was then incubated inthe reaction solution to allow surface probeDNA (1) to react withthe target DNA and exonuclease in solution. All reactions wereconducted in a 37 �Cwater bath in the dark for 1 h (except for thetime-course study). After the reaction, the sensor was switched tophosphate buffer and allowed to equilibrate at room temperaturefor at least 20 min prior to measurements. The signal gain in MBredox current was determined as the relative difference betweenthe baseline and postincubation redox currents. The relativesensor response (%) was employed in the interest of reproduci-bility. Of note, the present ExTCT sensor architecture is designedfor single use; therefore, multiple electrodes were used to collectthe various data sets presented in this work.

Time-Course Response Experiments. Measurements weretaken after 10, 30, 60, and 120 min of enzymatic reaction. At eachtime point, the sensor was switched to the phosphate buffer toequilibrate at room temperature for 20 min prior to ACVmeasurements. After measurement, the sensor was returned tothe same reagent-containing reaction solution to continue theenzyme reaction. All other reaction conditions and reagents in thetime-course response experiments were the same as describedabove.

Results and Discussion

Design of the ExTCTSensor.The ExTCTprobe (1) consistsof a 52-nucleotide (nt) DNA strand that has beenmodified with athiol group at its 30 terminus and a phosphate group at its 50

terminus and contains an MB tag at an internal position. In theabsence of the target, the probe self-hybridizes into a stem-loopstructure consisting of a 7-base-pair (bp) stem and 25-nt loop,with the MB situated at the center of the loop. The signalingcurrent arises from electron transfer between the MB redox tagand the gold electrode surface (Figure 1), and the magnitude ofthe faradaic current depends on the configuration of the probe.

(31) Sando, S.; Sasaki, T.; Kanatani, K.; Aoyama, Y. J. Am. Chem. Soc. 2003,125, 15720–15721.(32) Sando, S.; Narita, A.; Sasaki, T.; Aoyama, Y. Org. Biomol. Chem. 2005, 3,

1002–1007.(33) Fan, C. H.; Plaxco, K.W.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2003,

100, 9134–9137.(34) Lubin, A. A.; Lai, R. Y.; Baker, B. R.; Heeger, A. J.; Plaxco, K. W. Anal.

Chem. 2006, 78, 5671–5677.(35) Ricci, F.; Lai, R. Y.; Plaxco, K. W. Chem. Commun. 2007, 3768–3770.(36) Xiao, Y.; Qu, X. G.; Plaxco, K. W.; Heeger, A. J. J. Am. Chem. Soc. 2007,

129, 11896–11897.(37) Gooding, J. J. Electroanaylsis 2002, 14, 1149–1156.(38) Wang, J. Anal. Chim. Acta 2002, 469, 63–71. (39) Xiao, Y.; Lai, R. Y.; Plaxco, K. W. Nat. Protoc. 2007, 2, 2875–2880.

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In the absence of the target, the rigid stem-loop probe configura-tion fixes the MB tag away from the electrode, generating arelatively small faradaic current (Figure 1, step A), presumablyarising from limited, long-range electron transfer fromMB to theelectrode40 or from short-range electron transfer from the probesthat are not in the stem-loop conformation. Upon addition oftarget DNA (29-nt) and lambda exonuclease (Figure 1, step B),the target DNA hybridizes with the signaling probe to form a 29-bpDNAduplex at the 50 end and the 30 end retains a 23-nt single-stranded structure. The lambda exonuclease in turn preferentiallybinds to the duplex region and selectively hydrolyzes the 50

phosphate-modified probe strand in the 50-to-30 direction41-43

(Figure 1, step C), with digestion terminating after the duplex isfully consumed. The probe is thereby transformed into a flexible23-nt single-stranded fragment with MB affixed to its distal end(Figure 1, step D), allowing MB to transfer electrons efficientlyand generate an increased faradaic current.33,35 Digestion releasesthe intact target DNA, which is now able to hybridize with a new,undigested ExTCT probe and catalyze a new cycle of probetransformation (Figure 1, steps E-G). In this way, a single DNAtarget is able to trigger the permanent transformation of multiplesignaling probes from the rigid stem-loop conformation to theflexible linear structure and thereby generate an amplified fara-daic current (Figure 1, step H).

Upon challenging the ExTCT sensor with a 200 nM perfectlymatched target (2) and 50 units of exonuclease, we obtained a170% signal gain (Figure 2A, with target). This signal gainpersisted even after washing the sensor with 6.0 M guanidinehydrochloride (Figure 2A, post washing), confirming that the

electrochemical signal is indeed the result of a permanent changein the structure of the signaling probe (1). Furthermore, when thetransformed sensor was rechallenged with 200 nM target DNA(2), it showed a negligible change in electrochemical signal,indicating that all immobilized hairpin probes became digestedand the target DNA can no longer hybridize to the digestedprobes (data not shown). The ExTCT sensor is also sequence-specific; when challenged with 200 nM noncognate 29-nt target(3), the resulting signal was 26% of the signal obtained with theperfectly matched target (2) (Figure 2B). When we furtherinvestigated the specificity of our sensor withmismatched targets,we found that theExTCT sensorwas limited to the discriminationof five or more mismatches (data not shown) as a result of thethermodynamic characteristic that is inherent in the stem-loopstructure, consistent with previous work on the hybridization-based electrochemical stem-loop DNA sensor.33,34

To explore the limits of the signal-amplificationmechanism,weperformed three control experiments (Figure 2C). First, weincubated the sensor in the reaction buffer alone and observed∼10% of the faradaic current obtained from the positive controlreaction with the matched target DNA (2) and exonuclease. Thisbackground signal could not be regeneratedwith 6.0Mguanidinehydrochloride washing (data not shown), suggesting that it mayresult fromthe nonspecific adsorptionof bovine serumalbumin inthe reaction buffer onto the sensor surface. Second, we challengedthe sensorwith 200 nM target DNA in the absence of exonucleaseand observed a signal equivalent to∼29%of the positive control,which presumably originates from direct hybridization betweenthe signaling probe and target.We estimate that the open hairpinsconstituted approximately 0.35 pmol/cm2 because the surfacecoverage of the total immobilized probes was ∼1.2 pmol/cm2.Finally, when we challenged the sensor with exonuclease withoutthe target, the observed signal was ∼26% of the positive control(Figure 2C). The signal change observed in the third control

Figure 1. Overview of the ExTCT sensor scheme. The ExTCT sensor consists of surface-bound, stem-loop DNA probes modified with a50 terminal phosphate, a 30 terminal thiol group, and an internal MB tag. In the absence of the target, the relatively rigid stem-loop probeproduces a small faradaic current (stepA). In the presence of targetDNA, a target-probe complex is formed that consists of a single-strandedregion and a double-stranded duplex region (step B). Lambda exonuclease subsequently binds to the duplex and selectively hydrolyzes thephosphate-modified probe in the 50-to-30 direction (stepC), effectively digesting the probe. Because the nuclease preferentially digests double-strandedDNA, the enzyme activity is halted as it reaches the single-stranded region.Digestion releases the intact targetDNA, leaving behinda permanently “transformed” MB-labeled single-stranded probe (step D). The released target DNA can then hybridize and initiate thedigestion of another probe (steps E-G). As such, a single target DNA can trigger the transformation of multiple DNA probes, resulting insignal amplification (step H).

(40) Kelly, S. O.; Barton, J. K. Bioconjugate Chem. 1997, 8, 31–37.(41) Sriprakash, K. S.; Lundh, N.; Mooonhuh, M.; Radding, C. M. J. Biol.

Chem. 1975, 250, 5438–5445.(42) Little, J. W. Gene Amplif. Anal. 1981, 2, 135–145.(43) Mitsis, P. G.; Kwagh, J. G. Nucleic Acids Res. 1999, 27, 3057–3063.

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experiment suggests that undesired digestion of the nativeExTCTprobe occurs under the experimental conditions, presumably dueto imperfect selectivity of lambda exonuclease. This parasiticphenomenon was also confirmed by gel electrophoresis experi-ments (Supporting Information Figure S1).Effect of Probe Density on ExTCT Sensor Performance.

The surface density of the ExTCT probes is an importantparameter contributing to signal gain, and it can be controlledby varying the probe concentration during sensor fabrication.39,44

To optimize the signal gain, we characterized the performanceof sensors fabricated with various concentrations of signalingprobe (1) under standard reaction conditions with 200 nMperfectly matched target DNA (2). The observed probe densityincreased monotonically with increasing probe concentration,

and an optimal signal gain was obtained from sensors fabricatedwith a 1 μMsignaling probe, which translates to a packing densityof ∼1.2 pmol/cm2 (Supporting Information, Figure S2). Abovethis concentration, no further increases in signal gain wereobserved, presumably because the probe is saturated on thesurface.Efficiency of Target Recycling and Signal Amplification.

Transformation of the surface-boundExTCTprobe in the presenceof target DNA and exonuclease is relatively rapid, as illustrated bytime-course experiments (Figure 3). Under standard reaction con-ditions with 200 nM target DNA (2), we observed signal gainsaturation at ∼160% within ∼60 min. However, in the absence ofexonuclease or target DNA, the sensor achieved saturation after10min at amuch lower signal gain (Figure 3A). The observed delayin signal saturation in the presence of both the target and theexonuclease suggests that multiple cycles of target hybridization,exonuclease digestion, and probe transformation are indeed takingplace during the reaction. Because direct DNA hybridizationreaches saturation within ∼10 min, we hypothesize that theexonuclease reaction is the rate-limiting step. Furthermore, differ-ences in the reaction rates during the initial stage of the reactions arealso evident. For example, during the first 10 min, the exonuclease-amplified enzymatic reaction yielded an ∼9% signal gain perminute. However, smaller rates of signal gain were observed withreactions containing target only (5% increase per minute) orexonuclease only (4% per minute) (Figure 3A). After the first 10minutes of digestion, the velocity of exonuclease/target-catalyzedprobe transformation began to decelerate, presumably because theconcentration of the undigested, surface-bound probe had de-creased. In Figure 3B, we collected time courses of the ExTCTsensor at different target concentrations (5, 20, 100, and 200 nM).We observe that the enzymatic reaction slowed down with decreas-ing target concentration. The reaction time required to reach 50%of the saturated signal increased from 8.20 to 8.76 and 12.3 minwhen the target concentration decreased from 100 to 20 and 5 nM,respectively (Figure 3B). Because the DNA hybridization step isrelatively rapid,45 we estimated the rate constants of the enzymaticreaction by fitting the time course data to the Michaelis/Mentenequation. We note that the background signal caused by nonspe-cific cleavage was subtracted from the total signal for this calcula-tion (Figure 3B).Assumingadiffusion layer thickness of 5μm,46 the

Figure 2. Selective response of the ExTCT sensor to its targetDNA. (A) AC voltammograms reveal a 170% signal gain infaradaic current after enzymatic amplification. This increasedcurrent persists after washing the sensor in 6 M guanidine hydro-chloride (GuHCl) solution, verifying that the probe’s structuraltransformation is permanent. (B) When the ExTCT sensor ischallenged with a noncognate target and exonuclease, we observeonly 26% of the signal obtained with the perfectly matched target.(C) Compared to faradaic currents obtained with a perfectlymatched target and exonuclease, negative-control reactions withreactionbufferonly, lambdaexonuclease only, or targetDNAonlyyield significantly smaller signals (10, 29%, and 26%of the positivecontrol, respectively).

Figure 3. Time-course experiments reveal the kinetic response ofthe ExTCT sensor to the perfectly matched target and 50 units ofexonuclease. (A) Sensors challenged with only target DNA(200 nM) or exonuclease (50 units) reached saturation in approxi-mately 10 min. In contrast, delayed signal saturation (∼60 min) isobservedwhenboth target (200 nM) and exonuclease (50 units) arepresent, presumably because multiple probes are being trans-formed by a single DNA target. (B) Time courses of the ExTCTsensor at different target concentrations (5, 20, 100, and 200 nM).

(44) Ricci, F.; Lai, R. Y.; Heeger, A. J.; Plaxco, K. W.; Sumner, J. J. Langmuir2007, 6827–6834.

(45) Gao, Y.; Wolf, L. K.; Georgiadis, R. M.Nucleic Acids Res. 2006, 34, 3370–3377.

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calculated rate constants of lambda exonuclease for our ExTCTsensor were as follows: the surface Michaelis-Menten constant(Km)=9.13 ( 2.7 nM, the apparent turnover number of targetDNA on the surface (kcat)=0.030 s-1, and the efficiency (kcat/Km)of exonuclease = 3.29 � 106 M-1 s-1. The apparent turnovernumber of target DNA on the surface is∼100-fold lower than thatobserved in the solution reaction of the lambda exonuclease,43

suggesting that the efficiency of the enzyme is significantly ham-pered by the steric and electrostatic hindrance as well as by theconformational restriction of the surface-tethered strands.

The ExTCT sensor responds sensitively and reproducibly to itstarget, as shown in the dose-response curve (Figure 4A). With-out any background subtraction, the limit of detection (LOD) ofthe ExTCT sensor (∼2 nM) marks a 5-fold improvement over thehybridization-based sensor without the amplification mechanism(∼10 nM). In an effort to characterize the amplification effect ofthe ExTCT sensor quantitatively, we define the amplificationfactor as the ratio of the net signal gain obtainedwith the enzyme/target-catalyzed reaction to that obtained with target only aftersubtraction of the background current. Thus, a larger amplifica-tion factor is indicative of greater amplification and sensitivity.We observed that amplification factors of 16 and 8 were obtainedat 10 and 20 nM target concentrations, respectively (Figure 4B).Of note, although the amplification factor could not be calculatedbelow a 10 nM target concentration, it is evident that an evengreater signal amplification factor could be attained. The ampli-fication factor is limited to ∼4 at high target concentrations(above 20 nM) presumably because of the enzyme-limited natureof the reaction.

Conclusions

Wehave demonstrated a single step, single-reagent, isothermal,label-free DNA sensor architecture that utilizes a target recyclingmechanism to achieve amplified electrochemical signals. In thisassay, a single target DNA interacts with multiple probes totransform their structure permanently, yielding a large increase insignal amplitude. Interestingly, this mechanism operates moreefficiently at low target concentrations (<10 nM) where we

observe over 16-fold signal amplification. The sensitivity of thecurrent sensor cannot achieve sufficient sensitivity for the directdetection of genomicDNAderived by phenol/chloroform extrac-tion from physiological samples (typically ∼30 ng/μL).47 How-ever, we have identified a number of limiting factors that can beoptimized to improve the performance. For example, the sensi-tivity of our sensor is primarily limited by the stability of theemployed signaling probe, the exhaustion of available probes onthe surface, and the limited selectivity of the lambda exonuclease,which produces a background signal through the nonspecificdigestion of probes even at single-stranded segments.41-43 Withfurther optimization of the probe structure to incorporate differ-ent types of modified nucleotides;for example, peptide nucleicacids (PNA) or locked nucleic acids (LNA);and through the useof more selective exonucleases or the utilization of a solution-based, rather than a surface-based, reaction, we believe thatthe ExTCT architecture could offer an interesting alternativeapproach for convenient electrochemical DNA detection at thepoint of care.

Acknowledgment. This work was supported by the Office ofNaval Research, the National Institutes of Health, and theInstitute for Collaborative Biotechnologies through the U.S.Army Research Office. We acknowledge Dr. Xinhui Lou forinspiring conversations. We are especially grateful to Dr. RyanWhite,KareemAhmad,Dr.AndrewCsordas,KoryPlakos, ScottFerguson, Steven Buchsbaum, Jonathan Adams, and Seung SooOh for their assistance and support.

Supporting Information Available: Experimental details ofprobe density optimization and exonuclease and target-catalyzed transformation of the probe in solution. Experi-mental results including gel image of enzymatic digestionand probe transformation reaction products and optimiza-tion of probe packing density on the electrode. This materialis available free of charge via the Internet at http://pubs.acs.org.

Figure 4. Sensitive, reproducible responseof theExTCT sensor to its targetDNA. (A)TargetDNAconcentration calibration data (with andwithout exonuclease) show that the addition of lambda exonuclease greatly improves the limit of detection. The inset shows the calibrationcurve at low target concentrations. (B) The signal amplification factor, defined as the ratio of the net signal gain obtained with the enzyme/target-catalyzed reaction to that obtained with target only after subtraction of the background current, is∼16 at low target concentrations(<10 nM) but decreases to ∼4 at high target concentrations (>20 nM).

(47) Pilcher, K. E.; Pascale, G.; Fey, P.; Kowal, A. S.; Chisholm, R. L. Nat.Protoc. 2007, 2, 1329–1332.