DNA surface hybridization regimesPing Gong* and Rastislav Levicky†‡

*Department of Chemical Engineering, Columbia University, New York, NY 10027; and †Department of Chemical and Biological Engineering, PolytechnicUniversity, Brooklyn, NY 11201

Edited by Jacqueline K. Barton, California Institute of Technology, Pasadena, CA, and approved January 30, 2008 (received for review October 3, 2007)

Surface hybridization reactions, in which sequence-specific recog-nition occurs between immobilized and solution nucleic acids, areroutinely carried out to quantify and interpret genomic informa-tion. Although hybridization is fairly well understood in bulksolution, the greater complexity of an interfacial environmentpresents new challenges to a fundamental understanding, andhence application, of these assays. At a surface, molecular inter-actions are amplified by the two-dimensional nature of the immo-bilized layer, which focuses the nucleic acid charge and concen-tration to levels not encountered in solution, and which impactsthe hybridization behavior in unique ways. This study finds that, atlow ionic strengths, an electrostatic balance between the concen-tration of immobilized oligonucleotide charge and solution ionicstrength governs the onset of hybridization. As ionic strengthincreases, the importance of electrostatics diminishes and thehybridization behavior becomes more complex. Suppression ofhybridization affinity constants relative to solution values, andtheir weakened dependence on the concentration of DNA coun-terions, indicate that the immobilized strands form complexes thatcompete with hybridization to analyte strands. Moreover, anunusual regime is observed in which the surface coverage ofimmobilized oligonucleotides does not significantly influence thehybridization behavior, despite physical closeness and hence com-pulsory interactions between sites. These results are interpretedand summarized in a diagram of hybridization regimes that mapsspecific behaviors to experimental ranges of ionic strength andprobe coverage.

biosensor � ferrocene � ionic strength � microarray � probe coverage

Solid-phase or ‘‘surface’’ hybridization is the foundation ofmodern microarray and biosensing technologies widely used

in applied genomics for genotyping, drug discovery, gene ex-pression profiling, and related applications based on measure-ment of genomic information (1, 2). These tools functionthrough detection of interactions between nucleic acids immo-bilized on a solid support, or ‘‘probes,’’ with analyte ‘‘target’’nucleic acids present in solution. Binding, or hybridization,between probes and targets to form an immobilized duplex takesplace based on the degree of complementarity between theprobe and target base sequences. The tremendous growth inapplications of surface hybridization has been mirrorred byincreased emphasis on experimental and fundamental aspects ofthe assays that directly impact measurement and interpretationof data (3, 4).

As summarized in several recent reviews (5–8), physicalstudies under simplified experimental conditions have begun tounravel the rich phenomenology that occurs in diagnostic assays.Applications typically operate away from equilibrium and arefaced with a highly diverse pool of target sequences competingfor the probe sites. A variety of aids to enhance hybridizationperformance is used, including surfactants and blocking agentsto control nonspecific adsorption, washing procedures to de-velop contrast between fully and partially complementary se-quences, and variation of assay parameters such as ionic strengthor temperature. Physical studies (9–27) aim to distill down thiscomplexity into scenarios where specific features of surfacehybridization are brought out; for example, the impact of probesurface coverage on target hybridization (17, 20, 22, 23, 28).

A key ingredient in translating fundamental insight to appli-cations lies in understanding how experimental conditions en-gender specific hybridization behaviors. One relationship ofinterest is the cooperative influence of ionic strength (CB) andprobe coverage (SP). These parameters are linked through theirelectrostatic nature: ionic strength governs the magnitude ofelectrostatic interactions, whereas probe coverage determinesthe surface density of immobilized charge, from the probephosphate groups, that a hybridizing target must overcome. Inthis study, these two parameters are found to dominate hybrid-ization at low conversions, near the hybridization onset. Underconditions more typical of applications, when conversion ishigher, additional interactions become significant. Here, sup-pressed affinity for targets and a lowered sensitivity of probe-target hybridization to salt conditions indicate presence ofprobe-probe associations. Moreover, a curious regime is encoun-tered in which probes are physically close enough to interact yet,contrary to intuition, changes in their separation exert only weakeffects on target hybridization. These regimes define a map ofhybridization behaviors as a function of experimental conditions,and are discussed in the context of molecular level mechanisms.

ResultsGeneral Features of Hybridization in CB–SP Space. Experiments werecarried out using monolayers of 20mer single-stranded oligode-oxyribonucleotide probes immobilized to gold supports. A‘‘brush’’ type geometry, whereby the probes are tethered at oneend and dangle into solution at the other, was created by usingmercaptopropanol (MCP) as a surface passivant to decreasenonspecific interactions between the DNA and the surface (21,28). The probe sequence used was the retinoblastoma RB1marker 5� TTT TAA ATT CTG CAA GTG AT-(CH2)3-S-S-(CH2)3-OH 3�, with the 3� disulfide providing the surfaceimmobilization.

Hybridization experiments were carried out across a grid ofexperimental conditions that spanned probe coverages between2 � 1012 and 1.6 � 1013 strands per cm2, and sodium phosphatebuffer concentrations between 0.012 and 1 M in phosphategroups, at pH 7.0. This parameter space was selected to coverconditions typically encountered in applications (5, 7). Eachprobe layer of a given coverage SP (probes per area) underwenta series of hybridizations at different buffer strengths CB (molphosphate per liter) to 100 nM complementary target sequence5� ATC ACT TGC AGA ATT TAA-F2 3�, where F2 signifies anelectroactive N-(2-ferrocene-ethyl) maleimide moiety (29). Thetarget was complementary to the first 18 bases at the 3� end ofthe probe. Target coverage ST was determined from the totalcharge Q needed to switch the F2 tags of bound targets betweenferrocene and ferricinium states, F2 � e�7 F2�. Hybridization

Author contributions: P.G. and R.L. designed research; P.G. performed research; P.G. andR.L. analyzed data; and P.G. and R.L. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

‡To whom correspondence should be addressed. E-mail: rlevicky@poly.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0709416105/DC1.

© 2008 by The National Academy of Sciences of the USA

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was performed while stirring until ST reached a stationary value.Fig. 1 shows representative cyclic voltammetry (CV) data as afunction of time for SP � 5.7 � 1012 cm�2 and CB � 1 M. Aftersubtraction of the background at t � 0, the peak areas forcathodic and anodic scans were integrated to give Q and theaverage was converted to ST using ST � Q/(eA), where 1e isthe charge per tag (target) and A is the electrode area. Thesymmetric shape of the peaks, near equivalence of F2 oxidationand reduction potentials, and linear scaling of peak current withscan rate (not shown) confirmed that the signals originated fromhybridized targets, not diffusing species in solution.

Fig. 2 presents line and surface plots of the final coverage ofhybridized target molecules, ST, as a function of ionic strength CB

and probe coverage SP. For the lowest ionic strength, CB � 0.012M, hybridization was undetectable over the entire investigatedrange of 2 � 1012 � SP � 1.6 � 1013 strands per cm2. Regardlessof probe coverage, ST increased monotonically with CB, a trendthat results from screening of electrostatic penalties to hybrid-ization at higher ionic strengths. If SP was increased while CB wasfixed, a maximum in ST was observed at an intermediate probedensity SP

ST, consistent with prior measurements at high ionic

strengths (17, 20, 22, 23). The initial rise in target coverage withSP reflects an increasing capacity of the surface to bind target,whereas the subsequent decrease is attributed to overwhelmingsteric and electrostatic repulsions presented to targets bycrowded, dense probe films. SP

ST is seen to shift to highercoverages as CB increases. This dependence reflects compen-sating effects between probe coverage and ionic strengthwhereby stronger electrostatic screening (higher CB) is effectivein reducing electrostatic hybridization penalties to higher probecoverages.

An alternative way to visualize these data is as the fraction ofprobes that hybridized, represented by the hybridization con-version x � ST/SP. When ionic strength was high, 0.33 M and 1M data series, Fig. 3 shows that x was approximately independentof probe coverage at low values of SP. At higher probe coverages,x decreased. Implications of this behavior will be consideredbelow. At lower ionic strengths, CB � 0.11 M and 0.037 M, xexhibited a monotonic decrease as SP increased within theaccessible experimental window; whether a plateau existed atvery low probe coverages could not be ascertained due todifficulties with quantification of SP below �1 � 1012 cm�2. Asa practical note, the strongest hybridization signal was realizedat SP

ST when ST was at a maximum but where x was already indecline.

Hybridization at Low Conversion: Electrostatics and the Onset ofHybridization. What are the requirements for surface hybridiza-tion to proceed to some small value of x? From a purelyelectrostatic perspective, hybridization should cease at low ionicstrengths when solution ions do not provide sufficient chargescreening between the layer of immobilized probes and targetsin solution. Because the charge density of the probe layer isconcentrated if SP increases, high probe coverages are expectedto exacerbate the electrostatic barrier to hybridization. Can acriterion be formulated that integrates these expectations into atestable condition for onset of surface hybridization?

The immobilized negative DNA charge must retain an ele-vated countercharge CC,S of cations at the surface, CC,S � CC,B,where CC,B is the buffer concentration of cations. When a targetbinds, to maintain electroneutrality of the near surface regionadditional cations must transfer from solution to compensate thenewly added target charge. Because CC,S � CC,B this transfer willoccur uphill, against the cation concentration gradient. In thelimit of a very dilute buffer this gradient is large, because thenCC,B �� CP � CC,S, where CP is the concentration of immobilizedprobe charge. In this dilute limit the resultant osmotic penalty tohybridization will therefore be large and probe-target bindingwill be suppressed. In the other extreme, when CC,B �� CP, the

Fig. 2. Target coverage ST (targets per cm2) as a function of probe coverage SP (probes per cm2) and ionic strength CB (moles of phosphate per liter). (Left) Scatterplot of measured data. Lines are guides for the eye. (Right) An interpolated surface plot representation.

Fig. 1. CV time series showing progress of hybridization to complementarytarget at 5 min intervals from 0 to 40 min. (Right Inset) Target coverage as afunction of time. (Left Inset) Initial (t � 0) and final (t � 40 min) CV traces fora noncomplementary 5�-TTT TTT TCC TTC CTT TTT-F2–3� ferrocene-labeledtarget. Probe coverage, 5.7 � 1012 cm�2; buffer strength, 1 M; target concen-tration, 100 nM.

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cation concentration in the probe layer CC,S will be comparableto that in solution, CC,S � CC,B, and the penalty will be small.Onset of hybridization is expected between these two extremes,when CC,B exceeds CP. This stipulation is conveniently expressedby defining ' CP/CC,B, with the crossover between hybridizingand nonhybridizing conditions expected around � 1. For pH7 sodium phosphate buffer, CC,B equals CNa�,B � 1.61CB, whereCB is the buffer phosphate concentration. Thus, onset of hy-bridization is expected for

� � CP�1.61CB � 1 [1]

The arguments leading to expression 1 are similar to thoseinvoked in theoretical analyses of polyelectrolyte brushes (30,31). A quantity related to also arises in the hybridizationtheory of Halperin, Buhot and Zhulina (HBZ) (32), where itdelineates between different predictions for hybridization iso-therms. The HBZ parameter is HBZ � rD

2/H where rD is theDebye screening length in solution, H is the thickness of theprobe layer, and �1/� is the Gouy–Chapman length which actsas a metric of the immobilized areal density of charge �. HBZis readily shown to be proportional to by recognizing that rD

2

�1/CB and that 1/H � CP.The hypothesis that the onset transition should occur around

� 1 is tested in Fig. 4, where x data are plotted against calculated as described in the figure legend. The most strikingfeature is the changeover from hybridizing to nonhybridizingconditions at � 2.2, consistent with the above estimate of �1.§ Substitution of Na� by K� counterions produced a trendsimilar to that in Fig. 4 [supporting information (SI)]. Althoughthese results confirm that hybridization onset was governed by acooperative interplay of probe coverage and ionic strength, asreflected in the ratio CP/CB, the lack of collapse to a single curvedeeper in the hybridizing regime indicates that, at higher con-versions, additional influences are affecting x. This more com-plex behavior is considered below.

Hybridization at High Conversion. Assay applications tend to op-erate deeply in the hybridizing regime of Fig. 4, under conditionsof high ionic strength and well away from the onset of hybrid-ization. High ionic strength favors large values of x (i.e., x3 1),thus increasing the diagnostic signal. The equilibrium behaviorof x can be represented by an isotherm, a function that relates theextent of hybridization to target concentration and, in principle,to other parameters defining the equilibrium state including

probe coverage and ionic strength. The most common modelthat has been applied to surface hybridization is the Langmuirisotherm (18, 26, 27, 33, 34)

x1 � x

� CTK [2]

CT is the concentration of targets in solution. The equilibriumconstant K is given by exp(��GS

o/kT) where �GSo, the standard

free energy of surface hybridization, is defined for a reaction inwhich a target T binds to an immobilized probe P to yieldthe immobilized duplex D: Tsolution � Psurface7 Dsurface. T is theabsolute temperature, and k is the Boltzmann constant. In theLangmuir model, probe sites are assumed to be noninteractingand equivalent in their binding affinity toward target species.This scenario, in which probes do not interact, can only be strictlyvalid if probes are spaced far apart so that physical contact isimpossible and noncontact electrostatic interactions are atten-uated to insignificant levels, i.e., below the thermal energy kT.For such a ‘‘Langmuir scenario’’ K is independent of the probecoverage SP and, from Eq. 2, so is x.

From the experimental dependence of x on SP (Fig. 3) tworegimes can be discerned: (1) a ‘‘suppressed hybridization’’ (SH)regime where x decreases with SP, and a ‘‘pseudo-Langmuir’’

§The precise threshold value of is expected to vary somewhat with base sequence,temperature, and other parameters that influence hybridization equilibrium.

Fig. 3. Hybridization conversion x � ST/SP as a function of probe coverage SP (probes per cm2) and ionic strength CB (moles of phosphate per liter). (Left) Scatterplot. (Right) An interpolated surface plot representation.

Fig. 4. Correlation of the extent of hybridization x with the dimensionlessgroup . Onset of hybridization was observed at � 2.2. was calculatedusing Eq. 1 and CP � SPNP/HNA, where SP is the experimentally determinedprobe coverage, NP � 20 is the number of phosphate charges per probe, NA isAvogadro’s number, and H is thickness of the probe layer. H was approximatedby the probe contour length, H � 10 nm, based on polyelectrolyte brushtheory, which predicts the probes to be highly stretched at the hybridizationcross-over due to the still significant osmotic pressure of small ions (31).

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(PL) regime that occurs at lower probe coverages and withinwhich x achieves an approximately constant, plateau value. ThePL regime is only resolved for the 1 M and 0.33 M traces. Thereason for the ‘‘pseudo’’ nomenclature will become evidentbelow. The SH regime clearly violates the Langmuir model,because the required constancy of x with SP does not hold. ThePL regime, on the other hand, is consistent with Langmuirbehavior in that x does not vary, at least not strongly, with probecoverage. These trends were reproduced in a second set ofexperiments using potassium instead of sodium as the counte-rion (SI). Attempts to fit theoretical hybridization isothermsincorporating repulsive site interactions (7, 32, 35, 36) to the datawere able to approximately account for the SH regime but notfor the ionic strength-dependent PL plateau.

The average distance between probe sites in the experimentswas at most 7.5 nm (SP � 1.7 � 1012 cm�2), and was thus alwayscomfortably spanned by 20mer probes with a contour length of10 nm. At these coverages, it appears impossible for the probesto not interact. The suppression of hybridization as SP increasesin the SH regime is consistent with presence of site-site inter-actions, but the invariance of x with probe coverage in the PLregime is not. Although the plateau for the 1 M series for whichx is close to unity could simply reflect saturation of the surfacecapacity to bind target, such an explanation is untenable for the0.33 M series for which the data are clearly below saturation, andyet a plateau was observed.

Valuable insight into the PL regime can be obtained byconsidering the salt dependence of the x plateau. In solution, theequilibrium constant K depends on salt concentration (37)because hybridization involves a change in the number ofcounterions that are strongly associated (‘‘condensed,’’ refs. 38and 39) with the polyelectrolyte DNA backbone. Formally,counterion condensation can be included in the hybridizationreaction as T � P � J C 7 D, where a number J of cations C,initially free in solution, associate to the duplex D to screen itsincreased linear charge density. The role of the cations manifeststhrough dependence of K on their concentration CC,B as K � K1MCC,B

J, where K1M is the value of K when CC,B � 1 M. Treating theK for surface hybridization similarly rearranges Eq. 2 to

x1 � x

� CTK1MCNa�,BJ [3]

The measured plateau values of x for CB � 1 M (x � 0.88) and0.33 M (x � 0.68) are fit by Eq. 3 when K1M � 4.2 � 107 and J �1.1. The K1M value of 4.2 � 107 falls between those reportedpreviously for surface hybridization of 18mer targets (of differ-ent sequences) under 1 M Na� strength; e.g., K1M � 3 � 107

reported by Georgiadis and coworkers (18) and K1M � 6.8 � 107

reported by the Corn group (26), after correcting for differencesin ionic strength using J � 1.1 from above. Interestingly, irre-spective of sequence the surface K1M values are similar in thesestudies and orders of magnitude smaller than those for solutionhybridization under same conditions (5). For the particularsequence of this study, the surface K1M of 4.2 � 107 is nine ordersof magnitude below the solution value of 8 � 1016 at 25°C (37).A second significant result is that J � 1.1 is much smaller thanJ � 3.1 estimated from known salt dependence of solutionhybridization for 18mer oligonucleotides (37).

The PL regime can thus be summarized by three primaryobservations. First, x is approximately independent of probecoverage, consistent with Langmuir behavior. Second, the K1Mvalue is greatly lowered relative to solution, indicating decreasedaffinity of target hybridization. Third, the apparent number ofcounterions condensed when target hybridizes is reduced. Whatmolecular level mechanisms can account for these observations?The first observation, constancy of x, indicates that duplexformation at a probe site is not affected by the spacing between

sites. Given that sites are in sufficient proximity to interact, thisis possible only if a mechanism exists that regulates interactionsof a hybridized (duplex) site with its surroundings so as tomaintain them approximately independent of site-site spacing.Such a mechanism presumably involves structural relaxation. Forexample, interactions between duplexes could be moderated byadoption of a more perpendicular helix orientation to the surfaceas duplex coverage increases. Transition to the SH regime wouldthen signal exhaustion of the available relaxation mode; forexample, once the helices have realized a maximal degree ofalignment. Thereafter, at yet higher probe coverages, the frac-tion x of probes undergoing hybridization would thus be increas-ingly suppressed.

Significantly, the proximity of surface sites also enables un-hybridized probes to interact and, if preferred, to exist in anassociated state formed through attractive base stacking andhydrogen bonding interactions (40). Probe associations would bemost favored at high ionic strengths when electrostatic repul-sions between probes are weak. Existence of probe complexeshas been postulated earlier by Forman et al. (24) as a possiblecause of suppressed hybridization on commercial DNA chipsunder high (1 M) salt conditions. The lowering of K1M and J,relative to solution hybridization, is consistent with this inter-pretation. Probe association would decrease K1M because pre-existing probe complexes would have to be disrupted duringhybridization of target, thus making hybridization less favorable.Moreover, the backbone charge density of a probe complexshould be higher than that of unassociated probes, with morecounterions condensed before target hybridization. Then theadditional condensation from subsequent target binding wouldbe lowered, reducing J as indeed observed experimentally.Interestingly, insertion of a 1.1 nm poly(ethylene glycol) (PEG)linker between the solid support and the probe, keeping otherconditions fixed, raised K1M eightfold to 3.5 � 108 and J to,approximately, 2.7 (SI). These changes suggest that probe asso-ciations can be destabilized by PEG spacers.

While evidence for probe association came from analyzing thePL regime, these interactions are expected whenever conditionsare favorable, including in the SH regime. Finally, it is worthnoting that probe association can also explain suppression ofsurface K values relative to those in solution found in otherphysical studies of solid phase hybridization (5, 18, 26, 27).

A Map of Hybridization Regimes. The experimental results can besummarized in the form of a hybridization map, illustrated in Fig.5. A non-hybridizing (NH) regime was observed at high probe

Fig. 5. A map of surface hybridization regimes in CB–SP space. L, Langmuirregime; PL, pseudo-Langmuir regime; SH, suppressed hybridization regime;NH, non-hybridizing regime. Points represent conditions sampled in thisstudy. Dotted curves separate distinct regimes, with solid portions taken fromexperimental data.

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coverages and low ionic strengths. In the NH regime, the targetcoverage ST was below the detection limit of 2 � 1011 cm�2. Allpoints in 0.012 M buffer, and some points measured under 0.037M and 0.11 M conditions, belonged to the NH regime. The NHto SH transition, corresponding to the onset of hybridization, wasdefined by the condition that concentration of solution salt, CB,be comparable to the concentration of immobilized probecharge, CP. This condition is expected to apply as long aselectrostatics dominate the NH to SH transition. If, however,probe coverage approaches the sterically-limited maximum ofabout 5 � 1013 cm�2, hybridization will become impossible. Thisphysical constraint is indicated as an upturn in the NH to SHtransition. In the SH regime x depended on the coverage ofprobe sites, a clear signature of non-Langmuirian behaviorindicating that steric and/or electrostatic interactions betweensites were influencing hybridization. From the SH regime, adecrease in coverage at higher ionic strengths led to the PLregime. The defining characteristic of the PL regime was nearindependence of x on the coverage of probe sites, despitecloseness of the sites and thus compulsory presence of site-siteinteractions. It was argued that this independence requires thatthe pliable nature of the DNA layer moderate site-site interac-tions through a structural reorganization (e.g., through a reori-entation of helices). With further decrease in probe coverageeventually a true ‘‘Langmuir’’ (L) regime must exist whereprobes are so far apart that they do not interact. Although thisputative regime was not accessible to the experiments it isincluded in Fig. 5 as an expected limiting condition. The grayarea between the L and PL regimes may consist of additionalregimes, depending on which interactions between sites domi-nate. These interactions can comprise probe association as wellas interactions involving both hybridized and unhybridized sites.This region also was not studied due to inability to quantify probecoverage at these low values.

Fig. 5 suggests directions for future inquiry. Most immediatelyit would be intriguing to extend measurements to lower probecoverages, down to the L regime, to characterize the onset ofsite-site interactions and to fill in the largely unexplored area tothe left of the SH and PL regimes. As probe and target lengthsvary in applications, experimental studies over ranges in strandlengths and polydispersities are also urgent and would providevaluable test of theoretical predictions (41).

Materials and MethodsMaterials. All DNA oligonucleotides were purchased from MWG Biotech andwere HPLC purified. Synthesis and characterization of N-(2-ferrocene-ethyl) ma-leimide and its conjugation to target oligonucleotides are described in the SI.

Electrode Preparation. The studies used a 1.6 mm diameter polycrystalline goldworking electrode (WE), a platinum wire counter electrode, and a Ag/AgCl/3MNaCl reference electrode. The working electrode was mechanically and elec-trochemically polished as previously described (42, 43), and its roughnessfactor r (r � true area/geometric area; r � 1) was measured from the doublelayer capacitance (42, 43). Values of r ranged from 1.39 to 1.69, with anaverage of 1.56 0.09, and were used to correct probe and target coverages.Probe deposition on the WE was for 30 min from concentrations ranging from

0.05 to 1 �M probe in 1 M MgCl2, followed by 90 min immersion in 1 mMsolution of MCP in deionized water. During all transfer steps the electroderemained covered by a droplet of solution to prevent ambient contact.

Determination of Probe Coverage. Probe coverage was determined using anexisting protocol (23, 43, 44). After a background cyclic voltammetry (CV)measurement at 0.08 V/s in deoxygenated 10 mM Tris base, pH 7.4, the probelayer was immersed in 1 �M hexaamine ruthenium(III) (RuHex3�) in the sameelectrolyte and a CV scan was repeated after 3 min of equilibration. Thebackground was subtracted and the total charge due to reduction of RuHex3�

(RuHex3� � e-3 RuHex2�) associated with the probe layer was determined byintegration. The total charge was converted to probe coverage using a pre-viously derived calibration against coverages measured with x-ray photoelec-tron spectroscopy for the same probe sequence. Processing of the XPS dataused photoionization cross-sections from Wagner (45) and effective attenu-ation lengths calculated from the NIST SRD-82 v.1.0 database (46). The analysiswas as previously described (43, 47).

Hybridization and Regeneration of Probe Films. Consecutive hybridizations tocomplementary target were performed for five ionic strengths of pH 7.0sodium phosphate or, alternately, of pH 7.4 potassium phosphate buffer: 1 M,0.33 M, 0.11 M, 0.037 M and 0.012 M, expressed in molarity of buffer phos-phate. Control hybridizations to noncomplementary target were carried outat each probe coverage at 1 M and 0.012 M ionic strength. After 5 min ofpreconditioning in buffer a background CV was taken. Next, ferrocene-labeled target was added to a 100 nM final concentration. CV measurementswere collected 5 min apart at 0.08 V/s scan rate between 0 and 0.35 V until thelast several traces overlapped. During hybridizations in sodium phosphate thepotential was continuously held at 0 V except during CV scans, and the targetsolutions were stirred at all times. For the potassium phosphate series, fol-lowing each CV scan the WE potential was initialized at 0 V, and then theelectrochemical cell was shut off until the next CV scan. A double liquidjunction setup was used for the sodium phosphate, but not for the potassiumphosphate, measurements. Between hybridizations at different ionicstrengths the WE was rinsed with deionized water for 15 s to remove boundtarget, followed by preconditioning in fresh background electrolyte beforethe next hybridization.

Experimental Controls. Control experiments were carried out to test for non-specific adsorption of solution DNA to probe films, for stability of the probelayer over time, for effectiveness of probe layer regeneration between hy-bridizations at different values of CB, and for effect of the direction in whichCB was varied (1 M to 0.012 M, or 0.012 M to 1 M). Exposure of probe films tosolutions of noncomplementary target sequences 5�-TTT TTT TCC TTC CTT-TTT-F2-3�, under otherwise identical conditions, yielded signals below detec-tion limit (Fig. 1, left inset). Measurement of probe coverage before and afteran ionic strength series confirmed that probe coverage stayed within 12%.Effectiveness of the regeneration procedure was verified by absence of targetsignal after a deionized water rinse (e.g., the background trace in Fig. 1followed regeneration after a hybridization at CB � 0.33 M). Varying CB from1 M to 0.012 M, or from 0.012 M to 1 M, produced points that fell on the sameoverall trends. Data from both types of CB series were combined and presentedtogether.

ACKNOWLEDGMENTS. We thank Drs. Xiang Wang and Matt Tremblay fortheir advice with synthesis of N-ferrocene-ethyl maleimide. Dr. Jingyue Ju ismuch appreciated for providing access to MALDI-TOF MS characterization andHPLC purification facilities, and we thank Drs. Zengmin Li and Xiaoxu Li fortheir expert technical assistance with these techniques. The project was sup-ported by Grant Number HG003089 from the National Human Genome Re-search Institute.

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