Electrochemical Quantitation of DNA Immobilized on Gold - Analytical

Oct 8, 1998 - We have developed an electrochemical method to quantify the surface density of DNA immobilized on gold. The surface density of DNA, more...
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Anal. Chem. 1998, 70, 4670-4677

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Electrochemical Quantitation of DNA Immobilized on Gold Adam B. Steel,*,† Tonya M. Herne, and Michael J. Tarlov*

Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-0001

We have developed an electrochemical method to quantify the surface density of DNA immobilized on gold. The surface density of DNA, more specifically the number of nucleotide phosphate residues, is calculated from the amount of cationic redox marker measured at the electrode surface. DNA was immobilized on gold by forming mixed monolayers of thiol-derivitized, single-stranded oligonucleotide and 6-mercapto-1-hexanol. The saturated amount of charge-compensating redox marker in the DNA monolayer, determined using chronocoulometry, is directly proportional to the number of phosphate residues and thereby the surface density of DNA. This method permits quantitative determination of both single- and double-stranded DNA at electrodes. Surface densities of single-stranded DNA were precisely varied in the range of (1-10) × 1012 molecules/cm2, as determined by the electrochemical method, using mixed monolayers. We measured the hybridization efficiency of immobilized single-stranded DNA to complementary strands as a function of the immobilized DNA surface density and found that it exhibits a maximum with increasing surface density. The development of DNA chips, miniaturized arrays of immobilized single-stranded DNA, is motivated by their potential for applications in disease diagnosis and genome sequencing.1 In an array-based sensor, DNA probes of varying sequence are immobilized on a surface where the location of each probe is known. DNA diagnostics are performed by exposing the array to a solution containing single-stranded DNA of unknown sequence, the target, that can be labeled with a fluorescent or radioactive marker. The sequence of the target is determined by imaging the array and correlating the position of “hot spots” † Current address: Gene Logic Genomics Co., Gaithersburg, MD 20878. (1) (a) O’Donnell, M. J.; Tang, K.; Koster, H.; Smith, C. L.; Cantor, C. R. Anal. Chem. 1997, 69, 2438-2443. (b) Jacobs, J. W.; Fodor, S. P. A. Trends Biotechnol. 1994, 12, 19-26. (c) Mirzabekov, A. D. Trends Biotechnol. 1994, 12, 27-32. (d) Southern, E. M.; Case-Green, S. C.; Elder, J. K.; Johnson, M.; Mir, K. U.; Wang, L.; Williams, J. C. Nucleic Acids Res. 1994, 22, 1368-1373. (e) Williams, J. C.; Case-Green, S. C.; Mir, K. U.; Southern, E. M. Nucleic Acids Res. 1994, 22, 1365-1367. (f) Pease, A. C.; Solas, D.; Sullivan, E. J.; Cronin, M. T.; Holmes, C. P.; Fodor, S. P. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5022-5026.

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with a probe sequence. DNA chips must offer lower cost and greater material efficiencies to gain acceptance over traditional DNA diagnostic methods. Further reduction in the cost of performing DNA diagnostics can be realized by implementing less expensive detection methods. In this paper we report a novel electrochemical method to quantify the amount of DNA immobilized on the surface. We have quantified the surface density of DNA by taking advantage of the electrostatic attraction of specific redox cations with the nucleotide phosphate backbone. The interactions of redox-active complexes and DNA have been studied widely due to their importance as strand scission mediators, chemotherapeutic agents, and structural probes.2 There are two types of interactions for small redox molecules with DNA: electrostatic attraction and intercalation. The mode of interaction for a given redox molecule is typically determined by its molecular structure and is often ionic strength dependent.3 In the method described here, a DNA-modified electrode is placed in a low ionic strength electrolyte containing a cationic redox marker. Redox cations exchange for native counterions associated with the nucleotide phosphate residues of the probe. The amount of redox marker “electrostatically trapped” at the DNA-modified electrode is then determined using chronocoulometry. At saturation coverage of the redox marker, the surface density of the probe is calculated assuming complete charge compensation of the DNA phosphate residues by redox cations. A strong appeal of this approach is that this measurement, unlike measurements using other noncovalent labels,4 is insensitive to both the base composition and the chain order (single-stranded vs duplex). The measured redox marker is directly proportional to the number of phosphate groups present at the surface. We have used this method to quantify (2) (a) Carter, M. T.; Rodriguez, M.; Bard, A. J. J. Am. Chem. Soc. 1989, 111, 8901-8911. (b) Arkin, M. R.; Stemp, E. D. A.; Turro, C.; Turro, N. J.; Barton, J. K. J. Am. Chem. Soc. 1996, 118, 2267-2274. (c) Holmlin, R. E.; Stemp, E. D. A.; Barton, J. K. J. Am. Chem. Soc. 1996, 118, 5236-5244. (d) Ho, P. S.; Frederick, C. A.; Saal, D.; Wang, A. H.-J.; Rich, A. J. Biomol. Struct. Dynam. 1987, 4, 521-534. (e) Murphy, C. J.; Arkin, M. R.; Ghatlia, N. D.; Bossmann, S.; Turro, N. J.; Barton, J. K. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 53155319. (3) Hard, T.; Norden, B. Biopolymers 1986, 25, 1209-1228. (4) (a) Chen, A. Y.; Yu, C.; Gatto, B.; Liu, L. F. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 8131-8135. (b) Meyer-Almes, F. J.; Porschke, D. Biochemistry 1993, 32, 4246-4253. (c) Piehler, J.; Brecht, A.; Gauglitz, G.; Zerlin, M.; Maul, C.; Thiericke, R.; Grabley, S. Anal. Biochem. 1997, 249, 94-102. 10.1021/ac980037q CCC: $15.00

© 1998 American Chemical Society Published on Web 10/08/1998

both the surface density of probe as a function of the deposition procedure and the hybridization of target to those surfaces. In addition, electrochemical capacitance and cyclic voltammetry were used to examine the microscopic organization of the DNAcontaining monolayers. MATERIALS AND METHODS Materials. All reagents were obtained from Aldrich Chemical, unless otherwise noted. All solutions were made with deionized water (18 MΩ‚cm resistivity) from a Millipore MilliQ system. DNA oligonucleotides were purchased from Research Genetics (Huntsville, AL). Two different target sequences containing a 6-mercaptohexyl linker were utilized: a mixed-base sequence and a homooligomer. Both probes contained 25 bases. The mixed-base sequence was 5′-CAC GAC GTT GTA AAA CGA CGG CCA G-3′ (P1), and the homooligomer was all T’s (P2). The complementary single-stranded targets were a 25mer with the sequence 5′-CTG GCC GTC GTT TTA CAA CGT CGT G-3′ (T1) for P1 and the 20mer homooligomer of all A’s (T2) for P2. The noncomplementary controls contained the same sequence as the probes but lacked the 6-mercaptohexyl linker and are designated NT1 for P1 and NT2 for P2. In addition, the noncomplementary target NT2 was just 20 bases in length. 6-Mercapto-1-hexanol (MCH) and 2-mercapto-1-ethanol (MCE) were purified using flash chromatography (silica, chloroform) to remove traces of the corresponding dithiols. Hexaammineruthenium(III) chloride (99%) (Strem Chemical, Newburyport, MA) and potassium ferricyanide (Aldrich, 99+%) were used as received. Tris(2,2′-bipyridyl)cobalt(III) perchlorate was prepared using the procedure of Dollimore and Gillard and recrystallized twice from water prior to use.5 Four buffers were employed for specific tasks in this investigation. DNA deposition was performed from 1.0 M potassium phosphate buffer, 0.5 M KH2PO4, and 0.5 M K2HPO4, pH 7 (DBFR). Electrodes were rinsed in 10 mM NaCl with 5 mM Tris buffer, pH 7.4 (R-BFR). Hybridization reactions were performed in 1.0 M NaCl with 10 mM Tris buffer, pH 7.4, and 1 mM EDTA (H-BFR). Electrochemical characterization was performed in 10 mM Tris buffer, pH 7.4 (E-BFR). Electrode Preparation. Gold electrodes were prepared by rf sputter deposition of ∼200 Å of a chromium adhesion layer followed by ∼2000 Å of gold (each 99.99% purity) onto glass microscope slides through a special mask. Electrodes were cleaned by exposure to warm piranha solution (70% concentrated sulfuric acid, 30% peroxide solution (30%); WARNING: piranha solution reacts violently with organic solvents) for 15 min and 5% aqueous hydrofluoric acid for 30 s. The electrodes were rinsed thoroughly with deionized water after each exposure and placed in monolayer deposition solutions while still wet. DNA-modified surfaces were prepared using our previously described procedure.6 Briefly, mixed monolayer surfaces containing thiolated target DNA and MCH were prepared by immersing the clean gold substrate in a 1.0 µM solution of probe oligonucleotide7 in D-BFR for a specific time period, rinsing with R-BFR for 5 s, immersing in a 1.0 mM MCH solution in deionized water for (5) Dollimore, L. S.; Gillard, R. D. J. Chem. Soc., Dalton Trans. 1973, 933. (6) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916-8920. (7) Solution concentrations of DNA were determined from the optical density at 260 nm. Reichmann, M. E.; Rice, S. A.; Thomas, C. A.; Doty, P. J. Am. Chem. Soc. 1954, 76, 3047-3053.

1 h, and finally rinsing with R-BFR for 5 s and drying under a stream of nitrogen. Hybridization was performed at 35 °C for 60 min in H-BFR. The concentration of complementary target and noncomplementary target, for nonspecific adsorption controls, was 1.0 µM. Background blanks were placed in H-BFR containing no DNA under conditions identical to those of the specific hybridization experiments. Upon removal from the hybridization reaction solution, the electrodes were rinsed with R-BFR for 5 s and dried under a stream of nitrogen prior to characterization. Instrumentation. Cyclic voltammetry (CV) and chronocoulometry (CC) were performed with a CH Instruments (Cordova, TN) model 660 electrochemical analyzer.8 The following parameters were employed: CV, sweep rate ) 100 mV/s; CC, pulse period ) 500 ms, pulse width ) 500 mV. Ultraviolet-visible spectra were obtained on a Perkin-Elmer Lambda Bio 20 spectrophotometer. Procedures. All electrochemical characterizations were performed in a single-compartment cell with a 10-mL volume. The working electrodes were prepared as described above. A saturated calomel electrode (SCE) and platinum wire gauze served as the reference and counter electrodes, respectively. Supporting electrolyte, E-BFR, was deoxygenated via purging with nitrogen gas for 10 min prior to measurements, and the cell was blanketed with nitrogen for the duration of the experiments. All experiments were carried out at laboratory ambient temperature (21-24 °C). The electrode capacitances were measured using cyclic voltammetry and chronocoulometry. In the former method, the capacitances were calculated from cyclic voltammograms performed from +100 to -100 mV at 500 mV/s in E-BFR. In the latter method, the capacitances were calculated for a 500-mV potential step experiment (+100 to -400 mV) in E-BFR. The capacitance values determined by each method were within 5% of each other for a single electrode. Quantitation of DNA. The probe DNA coverage at the electrode surface was calculated from the number of cationic redox molecules electrostatically associated with the anionic DNA backbone. Cations provide charge compensation for the anionic phosphate groups in DNA. In solution these cations are labile and readily exchange with other cations in solution.9 The association constant between cations and DNA phosphate increases with the cation charge.10 When an electrode modified with DNA is placed in a low ionic strength electrolyte containing a multivalent redox cation, the redox cation exchanges with the native charge compensation cation (potassium ion in our preparation) and becomes electrostatically trapped at that interface. The amount of cationic redox marker can then be measured using chronocoulometry, a current integration technique, under equilibrium conditions.11 An attractive aspect of chronocoulometry is that the doublelayer charge and the charge due to reaction of species adsorbed on the electrode surface can be differentiated from the charge (8) Certain commercial products and instruments are identified to adequately specify the experimental procedure. In no case does such identification imply endorsement by the authors or NIST. (9) Rose, D. M.; Bleam, M. L.; Record, M. T. J.; Bryant, R. G. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 6289-6292. (10) Taquikhan, M. M.; Martell, A. E. J. Am. Chem. Soc. 1967, 89, 5585-5590. (11) Bard, A. J.; Faulkner, L. R. Electrochemical Methods; John Wiley & Sons: New York, 1980; pp 199-206.

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due to reaction of redox molecules that diffuse to the electrode surface. Consequently, measurements of surface-confined redox species can be made in the presence of solution redox marker so that the system is observed under equilibrium conditions. The integrated current, or charge Q, as a function of time t in a chronocoulometric experiment is given by the integrated Cottrell expression,

Q)

2nFAD01/2C0* π1/2

t1/2 + Qdl + nFAΓ0

(1)

where n is the number of electrons per molecule for reduction, F the Faraday constant (C/equiv), A the electrode area (cm2), Do the diffusion coefficient (cm2/s), Co* the bulk concentration (mol/ cm2), Qdl the capacitive charge (C), and nFAΓo the charge from the reduction of Γo (mol/cm2) of adsorbed redox marker. The term Γo designates the surface excess and represents the amount of redox marker confined near the electrode surface. The chronocoulometric intercept at t ) 0 is then the sum of the doublelayer charging and the surface excess terms. The surface excess is determined from the difference in chronocoulometric intercepts for the identical potential step experiment in the presence and absence of redox marker. Meaningful conversion of the measured surface excess to DNA surface density is reasonable only under the condition of saturation. Solution redox marker concentrations that provide a saturation condition, i.e., complete charge compensation of DNA by redox marker, were determined from adsorption isotherms. The saturated surface excess of redox marker is converted to DNA probe surface density with the relationship,

ΓDNA ) Γ0(z/m)(NA)

(2)

where ΓDNA is the probe surface density in molecules/cm2, m is the number of bases in the probe DNA, z is the charge of the redox molecule, and NA is Avogadro’s number. A similar strategy was used for monitoring hybridization. A batch of electrodes was processed simultaneously to ensure uniformity within a sample set. In each batch, all electrodes were exposed to probe deposition solution for the same period; a subset was then exposed to target DNA and the remainder exposed to blank hybridization solutions. Probe surface densities were determined from electrodes that were placed in blank hybridization solutions (i.e., no DNA) during the sample preparation sequence. Hybridized target surface densities were determined from the increase in saturated surface excess of redox marker over the probe-only saturated surface excess determination. To ensure that the measured surface excess of redox marker is the saturated value, a second determination of surface-confined redox marker was made after the addition of more redox species to the E-BFR. In a typical experiment, the double-layer charge term was determined first in E-BFR. The redox marker was then introduced to the E-BFR solution at a concentration that provides saturation of the probe DNA layer with redox cation and stirred. The electrode was allowed to equilibrate with the redox-containing solution for 30 s prior to performing the potential step experiment. 4672 Analytical Chemistry, Vol. 70, No. 22, November 15, 1998

The surface excess of redox marker was calculated as the difference in the chronocoulometric intercepts in the absence and presence of redox marker. RESULTS AND DISCUSSION Electrochemical measurements have long been used to characterize modified electrodes, whether the modification is a polymer coating12 or a self-assembled monolayer.13 Electrodes modified with DNA have been electrochemically characterized as well.14 Electrochemical measurements are useful in assessing the microscopic integrity and average behavior of such assemblies. The mixed DNA/MCH system here comprises both polymer (DNA) and self-assembled monolayer (MCH) modifications, so the electrochemical behavior will reflect both. Electrochemical capacitance measurements provide a probe of the average structure of the DNA/MCH assembly on the gold surface. The voltammetric response for solution redox species is influenced by the presence of defects15 (e.g., pinholes or grain boundaries) and by the polyanionic nature of the surface-confined probe DNA.16 Monolayer Capacitance. For an electrochemical doublelayer, the capacitance increases with decreasing separation between the electrode surface and the plane of closest approach for ionic charge and also increases with increasing dielectric constant for the material in the intervening medium.17 Miller et al. have shown that the behavior of hydroxythiol monolayers on gold is well described by an ideal parallel plate capacitor.18 The capacitance data for electrodes prepared from each of the probes, with and without MCH treatment, along with pure MCH samples are given in Table 1. In the absence of MCH treatment, the DNAmodified electrode capacitance is similar to that of a bare gold electrode. Comparison of the mixed DNA/MCH capacitances to pure MCH monolayer values shows that the mixed systems have solvent and ionic permeabilities that are quite similar to those of a pure MCH monolayer. The small increase in the mixed system capacitances compared to the pure MCH values indicates a slightly enhanced ionic permeability in the mixed system; however, a detailed interpretation of film structure on the basis of the capacitance data for mixed monolayers is unwarranted. The capacitance data for DNA samples with and without treatment with MCH support the contention that the MCH treatment largely (12) (a) Oyama, N.; Anson, F. C. Anal. Chem. 1980, 52, 1192-1198. (b) Peerce, P. J.; Bard, A. J. J. Electroanal. Chem. 1980, 112, 97-115. (c) Schneider, J. R.; Murray, R. W. Anal. Chem. 1982, 54, 1508-1515. (d) Guadalupe, A. R.; Abruna, H. D. Anal. Chem. 1985, 57, 142-149. (13) (a) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559-3568. (b) Song, S.; Clark, R. A.; Bowden, E. F.; Tarlov, M. J. J. Phys. Chem. 1993, 97, 6564-6572. (c) Sinniah, K.; Cheng, J.; Terrattaz, S.; Reutt-Robey, J. E.; Miller, C. J. J. Phys. Chem. 1995, 99, 1450014505. (14) (a) Millan, K. M.; Mikkelsen, S. R. Anal. Chem. 1993, 65, 2317-2323. (b) Hashimoto, K.; Ito, K.; Ishimori, Y. Anal. Chem. 1994, 66, 3830-3833. (c) Wang, J.; Cai, X.; Rivas, G.; Shiraishi, H.; Faria, P. A. M.; Donta, N. Anal. Chem. 1996, 68, 2629-2634. (d) Mucic, R. C.; Herrlein, M. K.; Mirkin, C. A.; Letsinger, R. L. Chem. Commun. 1996, 555-557. (e) Zhao, Y.; Pang, D.; Wang, Z.; Cheng, J.; Qi, Y. J. Electroanal. Chem. 1997, 431, 203-209. (f) Kelley, S. O.; Barton, J. K.; Jackson, N. M.; Hill, M. G. Bioconjugate Chem. 1997, 8, 31-37. (15) (a) Finklea, H. O.; Snider, D. A.; Fedyk, J. Langmuir 1990, 6, 371. (b) Becka, A. M.; Miller, C. J. J. Phys. Chem. 1992, 96, 2657-2668. (16) Oyama, N.; Shimomura, T.; Shigehara, K.; Anson, F. C. J. Electroanal. Chem. 1980, 112, 271-280. (17) Bard, A. J.; Faulkner, L. R. Electrochemical Methods; John Wiley & Sons: New York, 1980; pp 500-515. (18) Miller, C. J.; Cuendet, P.; Gratzel, M. J. Phys. Chem. 1991, 95, 877-886.

Table 1. Capacitance of DNA-Modified Electrodes monolayer

capacitance (µF/cm2)

na

bare Au MCH P1 P2 P1/MCH P2/MCH

43 ( 15 3.9 ( 0.1 25 ( 8 33 ( 7 4.8 ( 0.2 4.9 ( 0.2

6 10 4 4 8 8

a The number of independent measurements on different monolayer preparations.

Figure 1. Schematic of a mixed probe-DNA/MCH monolayer in solution containing single-stranded target DNA.

lifts the DNA from the surface to a conformation where each probe is immobilized solely through the thiolate,6 as depicted in Figure 1. Voltammetry at DNA-Modified Electrodes. The voltammetry for a variety of redox molecules at our DNA-modified electrodes provides additional qualitative information about the system’s organization. The voltammetric reversibility of highly charged redox ions is markedly influenced by the attractive/ repulsive interactions with the polyanionic layer that the ions must penetrate to reach the electrode surface.12,16 The voltammetry for two redox markers of opposite charge, ferricyanide(3-/4-) and ruthenium hexaammine(3+/2+), at MCE, MCH, P1/MCH, and P2/MCH electrodes is given in Figures 2 and 3, respectively. The MCE electrode is used in place of a bare gold electrode because it prohibits adsorption of redox marker at the bare metal surface while still providing reversible (diffusion-limited) voltammograms for these fast redox markers.19 The voltammetry for the redox markers at DNA/MCH electrodes is clearly affected by electrostatic interactions with the polyanionic overlayer. The reduction of ferricyanide is less reversible at both DNA/MCH surfaces than at the MCH surface, as evidenced by the increased peak splitting, ∆Ep ) 400 vs 200 mV. The increase in irreversibility for the redox couple is due to repulsive electrostatic interactions impeding the ability of anions to reach the electrode surface. Such behavior has been described previously by Oyama and Anson for the oxidation of Fe2+ at a polyvinyl pyridine (a polycation)-coated electrode.12a Conversely, the reversibility of the reduction of ruthenium hexaammine (RuHex) is enhanced at DNA/MCH electrodes relative to that at an MCH electrode. In fact, the reduction proceeds at the diffusion(19) Terrattaz, S.; Cheng, J.; Miller, C. J.; Guiles, R. D. J. Am. Chem. Soc. 1996, 118, 7857-7858.

Figure 2. Voltammetric behavior of ferricyanide(3-/4-) at DNAmodified electrodes. The data correspond to electrode modifications of MCE (solid line), MCH (dash line), P1/MCH (dot line), and P2/ MCH (dash-dot line). The concentration of ferricyanide was 2.5 mM in E-BFR. The electrode area was 0.13 cm2, and the sweep rate was 100 mV/s.

Figure 3. Voltammetric behavior of ruthenium hexaammine(3+/ 2+) at DNA-modified electrodes. The data correspond to electrode modifications of MCE (solid line), MCH (dash line), P1/MCH (dot line), and P2/MCH (dash-dot line). The concentration of ruthenium hexaammine was 1.8 mM in E-BFR. The electrode area was 0.13 cm2, and the sweep rate was 100 mV/s.

limited rate, suggesting that the RuHex is able to approach the electrode surface more closely in the mixed DNA/MCH film than in the pure MCH monolayer. A surface wave for RuHex is Analytical Chemistry, Vol. 70, No. 22, November 15, 1998

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observed as a postwave shoulder when the voltammetry is measured in solutions with lower redox molecule concentrations, e.g., 50 µM. While the reduction of RuHex is enhanced, the return oxidation is still diminished, as evidenced by the broader diffusive wave shape and lower peak current. The voltammetry for the oxidation of a neutral probe, ferrocenedimethanol, is nearly identical for DNA/MCH- and MCE-modified electrodes. Our data suggest a monolayer architecture that is made up of two layers: a densely packed underlayer formed by MCH and a much more disperse layer of oligonucleotide extending out into solution. Measuring Surface Density of Probe DNA. The electrostatic trapping of cationic redox molecules in polyanionic media has been used as a means to quantify the number density of anionic sites at electrode surfaces.12,16 More recently, the association of cationic redox molecules with DNA has been applied as a hybridization indicator, but no absolute determination of probe density was reported.14a,c We have outlined our approach to develop a quantitative, electrochemical method to determine probe densities above. The validity of the approach hinges upon the following three assumptions: (1) the redox marker associates with DNA strictly through electrostatic interactions; (2) the amount of electrostatically trapped redox marker can be determined accurately, that is, all redox molecules are electrochemically accessible; and (3) charge compensation for the DNA phosphate functionalities is provided solely by the redox marker. As the fulfillment of these three assumptions forms the basis of our quantitative results, we will present evidence that each assumption is satisfied in the following paragraphs. There can be a remarkable variety of association modes between a redox molecule and DNA.2 The variability is manifested in binding site size differences (number of bases per redox molecule) and the specificity of the interaction (either selectivity for certain base pairings or for single vs duplex DNA). The interaction between small molecules and DNA generally demonstrates ionic-strength-dependent binding with interplay between electrostatic and intercalation (or hydrophobic) interactions. The degree to which a particular mode of binding predominates is dictated by the structural, geometric, and charge properties of the binding molecule.2,20 Carter et al. report that 2,2′-bipyridine metal complexes, M(bpy)33+/2+, bind via electrostatic interactions, whereas the analogous 1,10-phenanthroline complexes bind via intercalative interactions.2 In the case of electrostatic binding, they report a binding site size of 3 base pairs per triply charged cationic redox molecule in 50 mM sodium chloride, whereas the binding site size of intercalators is seen to vary with redox molecule charge and chemical composition. Crystal structures of ruthenium(III) hexaammine with DNA show that this metal complex associates through electrostatic interactions.21 To avoid complications due to intercalation of redox marker into the hydrophobic region of DNA, we have selected redox markers that interact solely through electrostatic interactions. We have employed both cobalt(III) trisbipyridyl (CoBPY) and ruthenium(III) hexaammine (RuHex) and observe that both interact with surface-immobilized DNA in a similar manner, as discussed below. (20) Palanti, S.; Marrazza, G.; Mascini, M. Anal. Lett. 1996, 29, 2309-2331. (21) Ho, P. S.; Frederick, C. A.; Saal, D.; Wang, A. H.-J.; Rich, A. J. Biomol. Struct. Dynam. 1987, 4, 521-534.

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Figure 4. Chronocoulometric response curves for MCH (squares) and P1/MCH (circles) modified electrodes in the absence (open) and presence (closed) of 50 µM RuHex. The lines represent the fit to the data used to determine the intercept at t ) 0. The solid lines are the fits for the MCH curves, and the dashed lines are the fits for the P1/ MCH curves.

The determination of adsorbed redox species is governed by eq 1. For the equation to hold, the initial potential in the step experiment must be at a potential where insignificant electrolysis occurs and the final potential at a value where the electrolysis occurs at the diffusion-limited rate. The appropriate initial and final potentials were determined for each redox marker on the basis of cyclic voltammetry data at DNA-modified electrodes. Critical to the success of this method is the ability to determine accurately the double-layer charge, Qdl. The capacitance of bare gold can range from 20 to 50 µF/cm2, and this variability can severely limit the accuracy of DNA determinations. As can be seen from the data in Table 1, an advantage of using the MCH diluent is that it dramatically lowers the electrode capacitance to a reproducible value. Typical chronocoulometric responses for RuHex at a MCH and a P1/MCH electrode are given in Figure 4. The data for the MCH electrode shows that there is negligible nonspecific adsorption of RuHex at the electrode surface in the absence of DNA because the intercepts are nearly identical in the absence and presence of redox marker. The surface excess of RuHex at a DNA-modified electrode is determined from the difference in intercepts for the response in the absence and presence of redox marker. From the reproducibility of the double-layer charge determination, we can estimate a detection limit of 1 × 1011 molecules/cm2 for this system. Accurate determination of the surface excess requires that all of the surface-confined redox species are electroactive on the time scale of the experiment. While this has been shown not to be true for thick polymer films,12c Guadalupe and Abruna have shown it to be valid for thin, monolayer regime films.12d They based this

conclusion on the observation that the amount of charge consumed in the voltammetric experiment is independent of sweep rate, and the voltammetric and chronocoulometric charge determinations are equivalent. We observe similar behavior: at low solution redox marker concentrations, integrated currents are independent of sweep rate, and the integrated voltammetric current and the chronocoulometric intercept are equivalent. Hence, we are confident that chronocoulometry is providing an accurate measure of the surface excess of redox marker at a DNAmodified electrode due to the reproducibility of double-layer charge determinations and the agreement with voltammetry data. The condition of saturation was determined by measuring the adsorption isotherms for each of the redox markers at DNAmodified electrodes. Here, saturation is taken to mean that charge compensation of the nucleotide phosphates is provided solely by the redox marker; that is, one 3+ redox marker is electrostatically trapped for every three nucleotide phosphate groups at the electrode surface. The binding site size, s, of CoBPY with doublestranded DNA was given by Carter et al. as 3, or 1 redox marker per 6 phosphates.2a Welch and Thorp report that the binding constant of CoBPY increases with decreasing ionic strength of the electrolyte,22 as would be expected for an electrostatic interaction. We observe that the binding site size of electrostatically bound redox markers decreases with decreasing ionic strength in DNA titration experiments similar to those of the Bard and Thorp groups.23 The association constant, K, and the binding site size, s, were determined for the interaction of RuHex with calf thymus DNA. In E-BFR containing 10 mM NaCl, the data give K ) 4.7 × 105 M-1 and s ) 2.5 base pairs, while in E-BFR with no added salt the association constant increases to K ) 1.2 × 106 M-1 and the binding site size decreases to s ) 1.9 base pairs. Thus, under the conditions used herein, the saturation assumption is very nearly satisfied. The binding site size determined in the absence of excess NaCl with calf thymus DNA approaches the binding site size assumed in our calculation of DNA surface density. Note that, in these binding experiments, the sodium ion concentration is equal to the nucleotide phosphate concentration, ∼1 mM, so there is appreciable competition between RuHex and sodium ions, resulting in a binding site size that is somewhat larger than the theoretical value of 1.5. In the DNA monolayer experiments, RuHex is the only cation present in appreciable concentration, so we would expect that complete saturation is achieved. The surface excess adsorption isotherms for both CoBPY and RuHex at a P1/MCH electrode in E-BFR are given in Figure 5. Adsorption isotherms have been measured as a function of ionic strength on DNA-modified electrodes, and we find, not suprisingly, that increasing the salt concentration changes the shape of the isotherm. With increasing potassium chloride concentration in the buffer, higher solution redox marker concentrations are required to saturate the DNA layer. We note that the surface excess of both redox markers on a pure MCH surface is negligible (22) (a) Welch, T. W.; Corbett, A. H.; Thorp, H. H. J. Phys. Chem. 1995, 99, 11757-11763. (b) Welch, T. W.; Thorp, H. H. J. Phys. Chem. 1996, 100, 13829-13836. (23) Titrations were performed by addition of calf thymus DNA (Sigma) to a 50 µM RuHex solution. The bound fraction of RuHex was determined using normal pulse voltammetry. Association constants and binding site sizes were determined using a nonlinear curve fit of the noncooperative, discrete site binding model of Carter and Bard to the data, ref 2a.

Figure 5. Binding isotherms for cobalt trisbipyridyl(3+) (solid circles) and ruthenium hexaammine(3+) (open circles) on identically prepared P1/MCH electrodes. The inset is a fit of the binding data to the Langmuir adsorption isotherm. The data have been plotted in a linearized version of the Langmuir isotherm expression.

over the entire concentration range. The data have been fit to a Langmuir adsorption isotherm expression with favorable results (see inset in Figure 5). The saturated coverage values of both redox markers are nearly identical, indicating a similar interaction mode with DNA, as was assumed above. The isotherms do show that the association constant, K, for each of the redox markers is different. The K for CoBPY, 3 × 105 M-1, is nearly an order of magnitude smaller than that for RuHex, 2 × 106 M-1. We note that the K determined at the monolayer-modified electrode is in good agreement with the K determined for DNA in solution and would be anticipated given the distance in closest approach of the two redox species (CoBPY is larger, hence a worse ion-pairer than RuHex). The fact that redox markers of the same charge (3+) yet different association constants give the same limiting surface excess values is additional evidence that charge compensation is provided solely by the redox marker. Based on the foregoing discussion, we are confident that the experimental conditions fulfill the assumptions necessary to validate the use of eqs 1 and 2 to quantify the amount of DNA immobilized on a electrode surface as a result of the procedure detailed herein. Determination of Probe Surface Density. The surface densities of P1 and P2 as a function of immersion time in DNA deposition solution are given in Figure 6. The shapes of the isotherms are in good agreement with the qualitative isotherm determined by XPS previously in our group.6 The electrochemically determined probe surface densities are also in good agreement with surface densities calculated from surface plasmon Analytical Chemistry, Vol. 70, No. 22, November 15, 1998

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Figure 6. Probe surface density as a function of the exposure to probe solution in the deposition process. The data for P1 are given by the solid circles and P2 by the open circles. At least three determinations were made for each exposure time.

resonance data for this same system.24 The saturated coverage of the homooligomeric P2 is significantly larger than that for P1. The difference in probe surface density at long exposure times for these two DNA oligomers implies that there is a difference in the affinity for gold between the individual bases. We are currently investigating this phenomenon further using a larger probe DNA set. We note that, at higher oligonucleotide surface densities, it may not be possible to realize complete saturation of redox cations in the DNA layer due to steric limitations. Hybridization Monitoring as a Function of Probe Surface Density. The hybridization behavior of surface-immobilized DNA was determined as a function of probe surface density for both P1 and P2 probes. Relatively long hybridization times, 60 min, and high salt, 1.0 M NaCl, were employed to maximize duplex yield.25 Hybridization experiments with noncomplementary targets, NT1 and NT2, showed no increase in measured redox marker, indicating that nonspecific adsorption is negligible on these DNA/MCH surfaces, confirming earlier radiolabeling experiments.6 In the electrochemical determination of hybridized DNA, an additional potential step experiment was performed at higher redox marker concentration to ensure that the measured surface excess value was the saturated value. The resultant chronocoulometric response curves exhibited an increased slope but the same intercept, indicating that saturation had been achieved. The target surface densities acquired by hybridization measured as a function of probe surface density are given in Figure (24) Peterlinz, K. A.; Georgiadis, R. M.; Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 3401-3402. (25) Tsuruoka, M.; Yano, K.; Ikebukuro, K.; Nakayama, H.; Masuda, Y.; Karube, I. J. Biotechnol. 1996, 48, 201-208.

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Figure 7. Hybridized target surface density as a function of the probe surface density. The amount of target incorporated on the surface by hybridization is given for T1/P1 by the closed circles and T2/P2 by the open circles. The error bars represent the standard deviation on at least four determinations per probe coverage. The dotted line represents the theoretical maximum target surface density for complete hybridization of the probes.

7. The dashed line in the figure represents the theoretical maximum surface density of target attainable by hybridization, or the case where all probes on the surface are hybridized. The data for both P1 and P2 follow the 100% hybridization efficiency line for coverage values less than roughly 4 × 1012 molecules/ cm2 for both P1 and P2. This surface density of probe corresponds to an area per probe molecule of 2500 Å2/molecule, which is roughly the footprint area (width × length) of a 25mer duplex.26 It should be noted that similar hybridized target surface densities were observed in surface plasmon resonance studies.24 The hybridization efficiencies for the two probe-target pairs differ considerably. Target surface densities for the mixed-base pair (P1/T1) fall off sharply for probe surface densities in excess of 4 × 1012 molecules/cm2. In the case of the homooligomers (P2/T2), the target surface density is observed to plateau in the range of probe surface densities studied. We believe that this may be due to the difference in binding specificity between the two pairs. Target coverage values are limited in the P1/T1 case, where the restrictions on duplex formation are very rigid. For the P2/T2 pair, where the target is shorter and there are no restrictions as to the alignment of bases because both probe and target are homooligomers, larger target coverage values are possible. (26) Using 34 Å/10 bases as the length and 20 Å as the diameter of doublestranded DNA, we calculate a footprint of 1700 Å/molecule for a 25mer duplex DNA lying flat on the surface, compared to a footprint of 314 Å/molecule for DNA oriented normal to the surface.

CONCLUSION We have developed an electrochemical method to quantitatively determine DNA, either single-stranded or duplex, on a gold electrode based upon the electrostatic attraction between a cationic redox marker and the anionic DNA phosphate backbone. From the plot of target uptake as a function of probe surface density, it is apparent that hybridization is quite efficient up to a threshold probe surface density value. We believe that there is a dependence of this critical value on geometric factors of the probe and the probe/target duplex. It may be possible to infer orientation information about surface-immobilized DNA in solution from the threshold probe surface density; however, the current data set is too limited to warrant any conclusions at this time. Our group is undertaking a directed effort to elucidate the structure of these DNA monolayer assemblies in solution using surface plasmon

spectroscopy and neutron reflectivity to better understand the factors that will provide optimal chips for DNA diagnostics. ACKNOWLEDGMENT The authors gratefully acknowledge numerous discussions with Dr. Cary Miller and the use of the sputtering chamber in his laboratory for electrode preparation. A.B.S. acknowledges an NRC postdoctoral fellowship at NIST. We would also like to thank Dr. Stanley Abramowitz of the Advanced Technology Program at NIST for his support.

Received for review January 12, 1998. Accepted August 26, 1998. AC980037Q

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