Indium Microrod Tags for ElectrochemicalDetection of DNA Hybridization

Joseph Wang,* Guodong Liu, and Qiyu Zhu

Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico, 88003

The preparation and advantages of indium microrodtracers for solid-state electrochemical detection of DNAhybridization are described. The cylindrical metal par-ticles were prepared by a template-directed electrochemi-cal synthetic route involving plating of indium into thepores of a host membrane. The linear relationship be-tween the charge passed during the preparation and theresulting particle size allows tailoring of the sensitivity ofthe electrical DNA assay. The resulting micrometer-longrods thus offer a greatly lower detection limit (250 zmol),as compared to common bioassays’ spherical nanoparticletags. Indium offers a very attractive electrochemicalstripping behavior and is not normally present in biologi-cal samples or reagents. Solid-state derivative-chronopo-tentiometric measurements of the indium tracer havebeen realized through a “magnetic” collection of the DNA-linked particle assembly onto a thick-film electrode trans-ducer. Factors affecting the performance, including thepreparation of the microrods and pretreatment of thetransducer surface, were evaluated and optimized. Theresulting protocol offers great promise for other affinitybioassays, as well as for electrical coding and identifica-tion (through the plating of different metal markers andof multimetal redox-encoded tags).

The development of electrochemical DNA biosensors has beenthe subject of intense activity.1-3 Such detection of DNA hybridiza-tion has greatly benefited from the recent use of nanoparticletracers.4-9 Nanometer-sized metallic (Au, Ag) and semiconductor(CdS, PbS) spherical particles have, thus, been widely used forelectrochemical detection of DNA hybridization. Such nanopar-ticle-based electrical nucleic acid assays have commonly reliedon stripping-voltammetric detection of the dissolved tags4-6 orsolid-state chronopotentiometric measurements of a magneticallycollected DNA-linked particle assembly.7,8 The coupling of theamplification feature of nanoparticle/polynucleotide assemblieswith highly sensitive electrical transduction has facilitated assays

of DNA targets down to the picomolar level. Further sensitivityenhancement has been obtained by catalytic enlargement of themetal tracer in connection with nanoparticle-promoted precipita-tion of gold4 or silver.10 Yet, such enlargement is limited to thepreparation of 30-nm particles and requires an additional step.Since the sensitivity of such electrical bioassays depends on thesize of particle tag,5 a dramatic amplification of the hybridizationsignals is expected in connection with micrometer-size tracers(instead of nanometric ones).

We report here on the electrical detection of DNA hybridizationin connection with indium microrod tracers. These cylindricalmetal particles were prepared by the template-directed electro-chemical synthesis of Martin.11 Such a preparation route, involvingthe electrodeposition of metals into the pores of an aluminamembrane, permits a convenient and reproducible preparation ofcylindrical metal particles of a variety of sizes or compositions.The advantages of the template-directed electrochemical syntheticroute were documented for optical identification studies,12,13 butnot in connection with electrical DNA detection. As will beillustrated below, electrochemical bioassays greatly benefit fromthe ability to prepare a wide range of different metal tags with amore favorable electrochemical behavior (compared to commongold or silver spherical tracers). In particular, indium was selectedas the metal marker because it offers a very attractive electro-chemical stripping detection and is not normally present inbiological samples or as an impurity in related bioreagents.14 Theuse of micrometer-long wire tracers results in a greatly enhancedsensitivity (compared to commonly used spherical nanoparticletags) and with femtomolar detection limits. The template-directedpreparation offers great promise for multitarget electrical detection(through the plating of different metal tracers) and for improvedelectrochemical identification (in connection with “bar-coded”multimetal cylindrical particles with distinct voltammetric signa-tures). Detailed optimization and characterization of the microrod-based electrical DNA sandwich-hybridization assay are reportedin the following sections.(1) Palecek, E.; Fojta, M. Anal. Chem. 2001, 73, 75A.

(2) Mikkelsen, S. R. Electroanalysis 1996, 8, 15.(3) Wang, J. Anal. Chim. Acta 2002, 469, 63.(4) Wang, J.; Xu, D.; Kawde, A.; Polsky, R. Anal. Chem. 2001, 73, 5576.(5) Authier, L.; Grossiord, C.; Brossier, P.; Limoges, B. Anal. Chem. 2001, 73,

4450.(6) Cai, H.; Xu, Y.; Zhu, N.; He, P.; Fang, Y. Analyst 2002, 127, 803.(7) Wang, J.; Xu, D.; Polsky, R. J. Am. Chem. Soc. 2002, 124, 4208.(8) Wang, J.; Liu, G.; Polsky, R.; Merkoci, A. Electrochem. Commun. 2002, 4,

722.(9) Wang, J.; Liu, G.; Merkoci, A. J. Am. Chem. Soc. 2003, 125, 3214.

(10) Wang, J.; Polsky, R.; Xu, D. Langmuir 2001, 17, 5739.(11) Martin, C. R. Acc. Chem. Res. 1995, 28, 61.(12) Walton, I. D.; Norton, S. M.; Balasingham, He, L.; Oviso, D. F.; Gupta, J.

D.; Raju, P. A.; Natan, M. J.; Freeman, R. G. Anal. Chem. 2002, 74, 2240.(13) Nicewarner-Pena, S. R.; Freeman, R. G.; Reiss, B. D.; He, L.; Pena, D. J.;

Walton, I. D.; Cromer, R.; Keating, C. D.; Natan, M. J. Science 2001, 294,137.

(14) Doyle, M. J.; Halsall, H. B.; Heineman, W. R. Anal. Chem. 1982, 54, 2318.

Anal. Chem. 2003, 75, 6218-6222

6218 Analytical Chemistry, Vol. 75, No. 22, November 15, 2003 10.1021/ac034730b CCC: $25.00 © 2003 American Chemical SocietyPublished on Web 09/30/2003

EXPERIMENTAL SECTIONApparatus. Electroplating was accomplished using a CHI 440

Analyzer that was controlled by CHI 2.06 software (CH Instru-ments, Austin, TX). Derivative chronopotentiometric measure-ments were performed with a potentiometric stripping TraceLabPSU20 system (Radiometer), controlled by a PC using the TAP2software (Radiometer). In accordance with the TraceLab protocol,the derivative signal (dt/dE) was plotted against the potential, andthe peak area (following baseline correction and fitting) servedas the analytical signal (see Supporting Information).15,16 Thedetection was carried out with a mercury-coated screen-printedcarbon working electrode (Gwent ink, 2 × 4 mm) in the case ofsolid-state measurements or a 6-mm-long mercury-coated carbonfiber cylinder microelectrode (8 µm diameter, from Alfa Aesar,Ward Hill, MA) in measurements of the dissolved indium tag. AAg/AgCl reference electrode (CH Instruments) and a platinumwire counter electrode were dipped into a 100-µL droplet of acetatebuffer (0.2 M, pH 5.2) placed on the surface of the screen-printedworking electrode. The setup for the magnetic “collection”experiments was described elsewhere.7 Magnetic-bead DNAassays were performed on an MCB 1200 Biomagnetic ProcessingPlatform (Dexter, CA).17 All centrifugation steps were performedusing a Micromax centrifuge (Thermo IEC, MA). The microrodswere characterized using a Hitachi S-3200 scanning electronmicroscope. The silver film (on the alumina membrane) wasprepared by laser ablation of a solid silver target in connectionwith a YAG Laser (Quanta-Ray DCR-02A, Mountain View, CA).

Reagents. All stock solutions were prepared using deionizedand autoclaved water. The sodium acetate buffer (3 M, pH 5.2),nitric acid, sodium hydroxide, Tris-HCl buffer, lithium chloride,sodium phosphate, and sodium chloride were purchased fromSigma. The silver, gold, and indium standard atomic absorption(AA) solutions (1000 ppm) were obtained from Sigma as a platingsolution. Carbon ink was obtained from Gwent Electronic Materi-als Ltd (Rontypool, U.K.). Tween 20 was purchased from Aldrich.Alumina membranes (25-mm diameter, 10-µm thickness) werepurchased from Whatman (Clifton, NJ). Proactive streptavidin-coated microspheres (0.8-µm diameter, CMO1N) were purchasedfrom Bangs Laboratories.

The DNA oligonucleotides (target and probe 1) and thethiolated DNA (probe 2) oligonucleotide were obtained from LifeTechnologies (Grand Island, NY) and Alfa DNA (Montreal,Canada), respectively; these had the following sequences:

Target: 5′-AAA GTG TTT TTC ATA AAC CCA TTA TCC AGGACT GTT TAT AGC TGC TGT TGG AAG GAC TAG GTC-3′

Probe 1: 5′-GGG TTT ATG AAA AAC ACT TT-3′ biotinProbe 2: 5′-SH-(CH2)6-GAC CTA GTC CTT CCA ACA

GC-3′Stock solutions of the target oligouncleotide (1000 mg L-1)

were prepared in autoclaved water and diluted as required (inautoclaved water) during a given assay.

Preparation of Indium/Gold Rods. Indium/gold microrodswere prepared on the basis of a modified literature protocol.12,13

Alumina membranes with 200-nm pores and annular support ringswere used in these experiments. Before starting the electroplating,

a 0.5-1.0-µm silver layer was prepared by laser ablation of a solidsilver target under argon gas pressure of 0.3 mTorr (0.04 Pa) toprovide electrical contact for further electrodeposition. Themembrane was placed on a glass slide with the silver side up.Electrical contact to the silver layer was made using an aluminumfoil. Subsequently, silver was deposited to further seal themembrane using a constant current of -5 mA for 20 min in thepresence of a 100 mg L-1 silver (diluted AA) solution. This wasdone to prevent leakage of plating solution through the membrane.The membrane was placed on the aluminum foil, folding a glassslide, so that the silver film on the membrane contacted the foil.Additional silver was then plated into the membrane at -0.5 mAfor 20 min to fill in the branched sections of the membrane. Asilver AA solution was then replaced by a gold AA solution (100mg L-1). The galvanostatic gold deposition proceeded for 20 minat -5 mA. The membrane was then rinsed with distilled waterand was covered with 1 mL of the diluted indium AA solution(100 mg L-1). The indium deposition proceeded for 60 min usinga constant current of -5 mA (with the indium solution beingreplaced every 20 min).

Upon completing the indium plating, the membrane was rinsedwith distilled water, and the silver film backing was first dissolvedwith 30% HNO3 for 10 min. (Higher acid concentrations and longerperiods may result in partial dissolution of the indium.) Thealumina membrane was then rinsed with distilled water and placedin 3 M NaOH for 30 min to dissolve the alumina. The resultingsuspension was centrifuged at 3000 rpm to precipitate the particles.This process was repeated three times to remove the residualsalt.

Preparation of Indium/Gold-DNA Conjugate. About 0.1mg indium/gold microrods was dispersed in 2 mL of autoclavedwater. The thiolated oligonucleotide (2 OD in final OD value) wasadded to the above solution, and the mixture was stirred for 16 hat room temperature. Appropritate amounts of 0.5 M phosphatebuffer and 0.5 M sodium chloride solutions were added to theresulting mixture to generate a 0.1 M phosphate buffer solution(PBS). This was allowed to stand for 40 h, followed by a 20-mincentrifugation at 14 000 rpm to remove the unbound oligonucle-otides. The indium/gold-DNA precipitate was washed andseparated by centrifugation three times (4 min each) with a 0.1M phosphate buffer solution. Finally, the rod-DNA conjugate wasresuspended in a fresh 0.3 M phosphate buffer solution.

Preparation of Oligonucleotide-Coated Magnetic Beadsand Hybridization Protocol. DNA bioassays (and related wash-ing steps) were carried out on an MCB 1200 Biomagneticprocessing platform using a modified procedure recommendedby Bangs Laboratories.18 Briefly, 2 µL streptavidin-coated magneticbeads were transferred into a 1.5-mL centrifuge tube. The beadswere then washed with 95 µL of TTL buffer (100 mM Tris-HCl,pH 8.0, 0.1% Tween 20, and 1 M LiCl; one min magnetic stirringcoupled to removal of the supernatant) and were suspended in21 µL of TTL buffer. Subsequently, 4 µL of the biotinylated DNAprobe 1 (1000 mg L-1) was added, and the mixture was incubatedfor 20 min with gentle mixing. The probe-coated magnetic beads,were washed twice with 95 µL of TT buffer (250 mM Tris-HCl,0.1% Tween 20) and suspended in 45 µL of hybridization buffer(15) Jagner, D. Trends Anal. Chem. 1983, 2, 53.

(16) Wang, J.; Cai, X.; Wang, J.; Jonsson, C.; Palecek, E. Anal. Chem. 1995, 67,4065.

(17) Wang, J.; Xu, D.; Polsky, R.; Arzum, E. Talanta 2002, 56, 931. (18) Technote 101. Bangs Laboratories Inc.: Fishers, IN, August 29, 1999.

Analytical Chemistry, Vol. 75, No. 22, November 15, 2003 6219

(750 mM NaCl, 150 mM sodium citrate). The correspondingamount of the target was added and mixed for 20 min. Theresulting hybrid-conjugated microspheres were then washed twicewith 95 µL of TT and suspended again in 45 µL of hybridizationbuffer. Subsequently, 10 µL of the indium/gold rod-modified probe2 (containing 10 mg L-1 of DNA) was added and incubated for 20min. The magnetic beads bound to the sandwich-conjugate DNAwere then washed with 95 µL of TT buffer and resuspended in100 µL of acetate buffer (0.2 M, pH 5.2).

Magnetic “Collection” and Chronopotentiometric Detec-tion Protocols. “Magnetic” collection experiments were con-ducted using a mercury-coated screen-printed carbon electrode.The electrode was first preconditioned for 2 min at +1.8 V;the mercury deposition proceeded for 10 min at -1.10 V using a0.1 M HCl solution containing 100 mg L-1 of mercury (II) ion.The DNA-nanoparticle assembly was “collected” by placing anexternal magnet under the working electrode before placingthe 100 µL of the particle/DNA assembly for measurementsin a manner analogous to that described elsewhere (Figure 1Band C).7

Solid-state derivative chronopotentiometric measurements ofthe indium-rod tag were performed at the surface of the coatedscreen-printed electrode using 1-min preconditioning at -0.10 V,followed by 1 s at -1.1 V in a 100-µL acetate buffer solution (0.2M, pH 5.2) containing the DNA-linked particle assembly. Subse-quent measurement was carried out (after a 10-s rest period) usingan anodic current of +1.0 µA. The raw chronopotentiometricpotential-time curve was converted into a peak-shaped dt/dE vsE signal, and the data were filtered (by an 8-point “movingaverage” filter) and baseline-corrected using the TAP2 software.

Solution-phase measurements were conducted by dissolvingthe DNA-linked particle conjugate in 10 µL of 6 M nitric acid (for45 min) and transferring this solution to a 100-µL acetate buffer(0.2 M, pH 5.9) solution. Derivative chronopotentiometric mea-surements of the dissolved indium tracer were performed at amercury-coated carbon-fiber electrode prepared by preplatingmercury for 10 min at -1.1 V from a quiescent 0.1 M HCl solutioncontaining 100 mg L-1 mercury (II) ions. The indium deposition

proceeded for 2 min at -0.9 V; subsequent stripping was carriedout (after a 5-s rest period) using an anodic current of +1.0 µA.

RESULTS AND DISCUSSIONThe new protocol combines the amplification features of

particle/polynucleotide assemblies with a sensitive solid-statederivative chronopotentiometric detection of micrometer-longindium rods (Figure 1). It consists of the sandwich hybridization(A), followed by the magnetic collection (B) and a solid-statederivative chronopotentiometric detection (C). The latter combinesthe simplicity of solid-state DNA detection with the sophisticatedbaseline correction and noise filtration of computerized derivativechronopotentiometric detection (see Supporting Information).4,7

The use of long tracers leads to a dramatic sensitivity enhance-ment, as compared to common spherical nanoparticle tags. The“magnetic collection” of the DNA-linked particle assembly providesdirect electrical contact of the indium tracer, hence, obviating theneed for dissolving it. The success of the new electrical DNAassays requires optimal preparation of the rod tracers and of theelectrochemical transducer.

Figure 2 displays SEM images of the indium/gold microrods(A) and of the hybridization-induced DNA-linked particle assembly(B). The rods appear somewhat conical (rather than perfectcylinders), with a length of 3-5 µm. Their diameter varies from300 to 700 nm, reflecting variations in the pore size of themembrane template19 and slight dissolution of indium (on the gold-free edge). Such imperfect cylindrical shape does not affect thereproducibility and quality of the electrochemical data. Althoughthe gold and indium segments are indistinguishable (using thismagnification), XPS analysis confirmed the 1:6 composition ratioexpected from the different deposition times (not shown). TheSEM image of Figure 2B indicates that the hybridization eventresults in cross-linking of the indium tracer and the magneticbeads (B). A degree of cross-linking is low, with few indiummicrorods linked to the ∼1-µm magnetic beads. This is in contrastto the well-defined networks common to DNA-linked sphericalnanoparticles.4 Magnetic “collection” of the hybridization-inducedaggregates leads to direct contact of the indium rods with thethick-film transducer, hence, facilitating a solid-state electricaldetection. The degree of such contact depends on the length ofthe rods and surface porosity.

(19) Reiss, B. D.; Freeman, R. G.; Walton, I. D.; Norton, S. M.; Smith, P. C.;Stonas, W. G.; Keating, C. D.; Natan, M. J. J. Electroanal. Chem. 2002,522, 95.

Figure 1. Schematic representation of the analytical protocol: (A)sandwich hybridization linking the magnetic beads and microrodsthrough the DNA hybrid, (B) magnetic collection of the DNA-linkedparticle assembly onto the thick-film electrode transducer, and (C)solid-state derivative chronopotentiometric measurements of thecaptured indium rods. P1, DNA probe 1; T, DNA target; P2, DNA probe2; MR, microrods; MB, magnetic beads; and M, external magnet.

Figure 2. SEM image of indium/gold rods (A) and of the DNA-linkedparticle assembly (after sandwich hybridization assay) (B). Preparation(deposition) times, 60 min (indium) and 20 min (gold).

6220 Analytical Chemistry, Vol. 75, No. 22, November 15, 2003

The template-directed electrochemical synthesis avenue pro-vides precise control of the metal marker. Indium tracers can bereadily prepared in a variety of sizes. The length of the resultingrods and, hence, the sensitivity of the resulting bioassay, arestrongly dependent on the amount of the charge passed duringtheir preparation. Figure 3A shows solid-state derivative chrono-potentiograms for indium microrods prepared by plating the metalfor 20 (a), 40 (b), and 60 (c) min. Well-defined and sharp indiumpeaks are observed in connection with these preparation times(Ep ) 0.59 V; b1/2 ) 29 mV). The peak height is proportional tothe plating time. Such knowledge of the relationship between theplating time and the particle size allows controlled growth ofmicrorod tags to the desired length. The ability to tailor thesensitivity, through control of the preparation time, representsan attractive advantage of the redox microrod tracers (versuscommonly used spherical nanoparticles). In particular, such aroute offers a dramatic amplification of the hybridization signalsin view of the micrometer length of the rod markers. Assumingthat the sensitivity is proportional to the number of indium atomsin the microrod, a typical 1-µm-long/200-nm-diameter rod contains∼1.3 × 109 indium atoms vs 2.3 × 105 gold atoms in a typical20-nm spherical gold nanoparticle.5 This translates to a 5625amplification of the response, that is, lowering of the detectionlimit by 3-4 orders of magnitude. Longer rod tracers can beprepared in connection with thicker host membranes (althoughthe wires tend to bend or break above 10 µm).19 The implicationsof this signal amplification upon the sensitivity of the correspond-ing DNA bioassays are discussed below.

The reproducibility of the template-based preparation of metaltracers is indicated from Figure 3B. This figure shows derivativechronopotentiometric signals for six different aliquots of theindium microrods (a-f). Highly reproducible peaks are observedwith a mean peak area of 1102 ms. The resulting relative standarddeviation (8.7%) reflects the reproducibility of the plating process.

Conditions of the thick-film carbon electrode transducer havea profound effect upon the solid-state electrical indium response.It is known that the morphology, roughness, and composition ofscreen-printed transducers are strongly affected by the preparationand pretreatment of these electrodes.20 Such surface conditionsmay influence the efficiency of the magnetic collection of the DNA-

linked aggregate as well as the degree of contact of the indiumrods with the surface (essential for the solid-state detection), andhence, the overall analytical performance. Figure 4 examines theeffect of the electrode preparation/pretreatment upon the deriva-tive chronopotentiometric indium signal. A well-defined peak isobserved in connection with a 1-h curing (of the ink) at 100 °C(A). The indium response increases greatly (>3-fold) following a2-min anodic pretreatment at +1.8 V (B). Such preanodization isknown to increase the electrochemical activity of the printedsurface.20 A broader and distorted peak is observed in connectionwith a higher (250 °C) curing temperature that leads to graphite-rich porous composite strips (C). Apparently, an increased porositydoes not promote the intimate contact desired for the solid-statedetection (because some rods do not penetrate into the voids).As expected, no response is observed for gold rods containingno indium (D). Such a profile reflects also the absence of indiumimpurities in common supporting electrolytes. An initial goldplating was required for anchoring the indium wire to the thiolatedDNA probe.

Such control of the preparation of the microrod tracers and ofthe electrochemical transducer leads to highly sensitive DNAhybridization assays. Such bioassays were carried out in connec-tion with “magnetic collection” of the DNA-linked particle as-sembly and solid-state chronopotentiometry of the anchoredindium tracer. Figure 5A displays typical derivative chronopoten-tiograms for solutions containing increasing target concentrationsin 100 ng L-1 (ppt) steps (a-f). Well-defined indium peaks areobserved (Ep ) -0.59V) in connection with these extremely lowtarget concentrations. The indium signals are proportional to thetarget concentration; the resulting calibration plot (shown in theinset) is highly linear, with a sensitivity of 0.776 s V-1 L ng-1

(correction coefficient, 0.995). The response for a 50 ng L-1 targetsolution (shown in the second inset) indicates a detection limitof ∼4 ng L-1 (210 fM), that is, 200 fg (10 amol) in the 50-µLsolution (based on S/N ) 3). Notice also the small resolvablepeak at -0.47 V, which is attributed to a trace of lead in the blanksolution. Although this peak has no effect on the target indiumsignal, it indicates the problem of using common metals (e.g.,Pb) as tags. Further lowering of the detection limit down to 0.1ng L-1 (250 zmol) DNA target was achieved in connection withsolution-phase measurements of the dissolved indium tag and amicroelectrode detector (based on the response for 1.0 ng L-1

target shown in Figure 5C,a). This value corresponds to 5.4 fMand is considerably lower than the (2 nM, 5 pM, and 0.5 pM)

(20) Wang, J.; Pedrero, M.; Sakslund, H. Analyst 1996, 121, 345.

Figure 3. (A) Solid-state derivative chronopotentiograms for mi-crorods prepared with different electrodeposition times: 20 (a), 40(b), and 60 (c) min. (B) Reproducibility of the microrod preparation;derivative chronopotentiograms for six different aliquots of the indiumrods (a-f) prepared with 60-min electrodeposition. Measurements arebased on adding 20 µL of the 0.1 mg mL-1 indium/gold rod dispersedsolution into 100 µL of acetate buffer (0.2 M, pH 5.2). The indium/gold rods were collected by 30 min precipitation on the surface of amercury-coated screen-printed electrode. Electrode preconditioning,1 min at -0.1 V, followed by 1 s at -1.1 V; constant current, +1 µA.

Figure 4. Derivative chronopotentiograms for indium/gold microrodsat screen-printed electrodes, prepared and pretreated under differentconditions: (A) 1 h curing at 100 °C; (B) same as A with 2-minpretreatment at +1.8 V (before plating the mercury film); (C) 1 h curingat 250 °C; and (D) same as B with gold microrods (containing noindium) prepared with 20 min of plating; other conditions same as(B). Other conditions as in Figure 3.

Analytical Chemistry, Vol. 75, No. 22, November 15, 2003 6221

detection limits reported for analogous electrical protocols basedon dissolved CdS and colloidal gold and silver nanoparticle tags,respectively.8,4,6 Such substantial improvement reflects the dra-matically larger size of the indium tracer, along with its favorable(reversible) electrochemical behavior.14 The lower detection limitof the solution-phase detection protocol (vs the solid-state one)indicates that the indium rods are not fully accessible to thesurface during the solid-state detection The microelectrodedetector used in such solution-phase measurements allows en-hanced mass transport (by nonlinear diffusion) during the elec-trodeposition of indium from the 110-µL droplet.

In practice, the detectability of DNA hybridization methodswould be limited by nonspecific binding events. The coupling ofmicrorod tags with the magnetic separation and detection resultsin minimal nonspecific adsorption effects. Figure 5B examinesthese effects by comparing the response for 200 ng L-1 of theDNA target (a) with those of 50- (b) and 500-fold (c) excess of anoncomplementary oligonucleotide. These high levels of thenoncomplementary DNA yielded substantially smaller signalscompared to that of the 200 ng L-1 target. The minimization ofnonspecific binding is attributed to the efficient magnetic separa-tion, that is, removal of nonhybridized DNA. Such small contribu-tions facilitate ultratrace (ng L-1) measurements of the targetDNA. For example, a detection limit of ∼30 ng L-1 can beestimated by comparing the response of the complementary andnoncomplementary oligonucleotides (Figure 5B, a vs b). Theprecision was estimated from a series of six successive measure-ments of 250 ng L-1 DNA target that yielded reproducible indiumpeaks with a relative standard deviation of 9.8% (not shown).

CONCLUSIONSWe have demonstrated for the first time the use of metallic

wire tracers for electrochemical monitoring of DNA hybridization.The indium microrod tagging system has been shown to be

extremely attractive for electrochemical detection. The templatepreparation route allows control of the sensitivity, as desired forlowering the detection limits of particle-based electrical DNAassays. The high sensitivity and selectivity make this protocol apowerful addition to the armory of nanoparticle-based electro-chemical genetic testing schemes. Practical clinical applicationswould require the incorporation of relevant sample pretreatmentsteps. The attainment of such sensitivity and selectivity requiresproper attention to variables of the rod preparation and transducerpreparation/activation. Replacement of the mercury-film electrodewith an environmentally friendly bismuth-coated one21 wouldfurther enhance the power of the new protocol. Although solution-phase measurements of dissolved microrods offer slightly lowerdetection limits, the solid-phase detection leads to a greatlysimplified operation (obviating the need for acid dissolution) andis more suitable for DNA arrays. The template-directed preparationoffers great promise for multitarget electrical detection (throughthe plating of different metal tracers) and for improved electro-chemical identification (in connection with “bar-coded” multimetalcylindrical particles with distinct voltammetric signatures). Themetal rod tags should also be useful for other electrical affinitybioassays, for example, immunological ones.

ACKNOWLEDGMENTFinancial support from the National Science Foundation

(Grants nos. CHE-0209707 and OCE-332918) and NATO (Sciencefor Peace program) is gratefully acknowledged.

SUPPORTING INFORMATION AVAILABLERaw and treated derivative chronopotentiometric response of

the indium microrods. This material is available free of chargevia the Internet at http://pubs.acs.org.

Received for review July 2, 2003. Accepted August 26,2003.

AC034730B(21) Wang, J.; Lu, J.; Hocaver, S.; Farias, P.; Ogorevc, B. Anal. Chem. 2000, 72,

3218.

Figure 5. (A) Derivative chronopotentiograms for increasing concentration of the DNA target in 100 ng L-1 steps (a-f). Also shown (insets),the resulting calibration plot and the response for a 50 ng L-1 target solution. Amount of magnetic beads, 20 µg; hybridization time, 20 min. (B)Derivative chronopotentiograms for 200 ng L-1 target (a), 10 µg L-1 noncomplementary DNA (NC, b) and 100 µg L-1 noncomplementary DNA(c). (C) Derivative chronopotentiograms for 1 ng L-1 target (a) and 100 ng L-1 noncomplementary DNA (NC, b) at a mercury-film fiber electrode,following dissolution of the DNA-linked particle assembly in 10 µL of 6 M nitric acid and addition of 100 µL of acetate buffer. Details of themagnetic collection are described in the Experimental Section. Other conditions as in Figure 3.

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