Electrochemical Detection of DNA Hybridization via Bis-Intercalation of

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Anal. Chem. 2007, 79, 6922-6926

Electrochemical Detection of DNA Hybridization via Bis-Intercalation of a Naphthylimide-Functionalized Viologen Dimer Eli G. Hvastkovs† and Daniel A. Buttry*

Department of Chemistry (3838), University of Wyoming, 1000 East University Avenue, Laramie, Wyoming 82070

The synthesis and DNA binding properties of a bisnaphthyl imide tetracationic diviologen compound NI(CH2)3V2+(CH2)6V2+(CH2)3NI (where V2+ ) 4,4′-bipyridinium and NI ) naphthyl imide, NIV) are described. Binding to thiolated ssDNA and dsDNA immobilized at Au electrodes was characterized using the electrochemical response for reduction of the V2+ state to the V+ (viologen radical cation) state. Isotherms and binding constants for this molecule to both forms of immobilized DNA were generated in this fashion. The character of the binding isotherm for dsDNA suggests bis-intercalation. Under high saline conditions, the diviologen molecule dissociated 160 times slower from dsDNA compared to ssDNA. Slow dissociation kinetics from dsDNA (kd )7.0 × 10-5 s-1) allow this molecule to be used as an effective DNA hybridization indicator. Selective and sensitive detection of DNA hybridization has important applications in the areas of gene identification, molecular diagnostics, and drug development.1,2 Electrochemical transduction of DNA detection offers speed, miniaturization, and cost benefits over optical means.2,3 Several types of electroactive molecules have been used to detect DNA hybridization including minor groove binders,4 intercalators,5-8 and covalent modifiers.9-11 Intercalators represent the most selective molecules for doublestranded (dsDNA) hybridization indication.7 These are molecules that insert a planar moiety between base pairs of DNA. Many are cationic to allow for electrostatic attraction to DNA. Enhanced * To whom correspondence should be addressed. E-mail: [email protected]. † Current address: Department of Chemistry, U-3060, 55 North Eagleville Rd., University of Connecticut, Storrs, CT 06269-3060. (1) Ramsey, G. Nat. Biotechnol. 1998, 16, 40-45. (2) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 21, 11921199. (3) Palecek, E.; Jelen, F. Crit. Rev. Anal. Chem. 2002, 32, 261-270. (4) Hvastkovs, E. G.; Buttry, D. A. Langmuir 2006, 22, 10821-10829. (5) Erkkila, K. E.; Odom, T. D.; Barton, J. K. Chem. Rev. 1999, 99, 27772795. (6) Wong, E. L. S.; Gooding, J. J. Anal. Chem. 2006, 78, 2138-2144. (7) Takenaka, S.; Yamashita, K.; Takagi, M.; Uto, Y.; Kondo, H. Anal. Chem. 2000, 72, 1334-1341. (8) Tansil, N. C.; Xie, H.; Xie, F.; Gao, Z. Anal. Chem. 2005, 77, 126-134. (9) Baruah, H.; Day, C. S.; Wright, M. W.; Bierbach, U. J. Am. Chem. Soc. 2004, 126, 4492-4493. (10) Keppler, B. K. Metal Complexes in Cancer Chemotherapy; Verlag Chemie: Weinham, 1993. (11) Jacquet, L.; Davies, R. J. H.; Mesmaeker, A. K.-D.; Kelly, J. M. J. Am. Chem. Soc. 1997, 119, 11763-11768.

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dsDNA binding selectivity arises from hydrophobic interactions with properly spaced duplex nucleobases,7,8 allowing the possibility of electrochemical detection of DNA hybridization in situ.12 Although this represents the optimal case, often intercalators show significant affinity for single-stranded DNA (ssDNA) based on similar interactions.13-15 Synthetic modifications to basic DNA intercalators can endow them with greater specificity for dsDNA versus ssDNA. For instance, bis-intercalation, employing two intercalation groups tethered by a linker,16-18 and threading intercalation, using a larger intercalator linking external redox species,7,8,19,20 have been shown to be very effective in detecting hybridization. We describe in this paper the synthesis and electrochemical behavior of an electroactive bis-intercalator, NI(CH2)3V2+(CH2)6V2+(CH2)3NI (where V2+ ) 4,4′-bipyridinium and NI ) naphthyl imide, NIV) (Chart 1) that effectively discriminates between dsDNA and ssDNA. The electroactive tether consists of two viologen subunits. Naphthyl imide functionalities serve as intercalation moieties. Viologen groups have several characteristics that make them useful in this application, including reversible electrochemistry,4 an ability to bind in the minor groove of dsDNA,4,21-23 and facile strategies for synthetic modifications. Naphthyl imide groups are well-established DNA intercalators24-28 (12) Wong, E. L. S.; Gooding, J. J. Aust. J. Chem. 2005, 58, 280-287. (13) Kapuscinski, J.; Darzynkiewicz, Z. Nucleic Acids Res. 1983, 11, 7555-7568. (14) Kapuscinski, J.; Darzynkiewicz, Z.; Melamed, M. R. Biochem. Pharm. 1983, 32, 3679-3694. (15) Rye, H. S.; Glazer, A. N. Nucleic Acids Res. 1995, 23, 1215-1222. (16) Takenaka, S.; Ihara, T.; Takagi, M. Chem. Commun. 1990, 14851487. (17) Spielmann, H. P.; Wemmer, D. E.; Jacobsen, J. P. Biochemistry 1995, 34, 8542-8553. (18) Petersen, M.; Jacobsen, J. P. Bioconjugate Chem. 1998, 9, 331-340. (19) Takenaka, S. Bull. Chem. Soc. Jpn. 2001, 74, 217-224. (20) Guelev, V.; Sorey, S.; Hoffman, D. W.; Iverson, B. L. J. Am. Chem. Soc. 2002, 124, 2864-2865. (21) Bunkenborg, J.; Stidsen, M. M.; Jacobsen, J. P. Bioconjugate Chem. 1999, 10, 824-831. (22) Bunkenborg, J.; Gadjev, N. I.; Deligeorgiev, T.; Jacobsen, J. P. Bioconjugate Chem. 2000, 11, 861-867. (23) Takenaka, S.; Sato, H.; Ihara, T.; Takagi, M. J. Heterocycl. Chem. 1997, 34, 123-127. (24) Bailly, C.; Brana, M. F.; Waring, M. J. Eur. J. Biochem. 1996, 240, 195208. (25) Bousquet, P. F.; Brana, M. F.; Conlon, D.; Fitzgerald, K. M.; Perron, D.; Cocchairo, C.; Miller, R.; Moran, M.; George, J. Cancer Res. 1995, 55, 11761180. (26) Carrasco, C.; Joubert, A.; Tardy, C.; Maestre, N.; Cacho, M.; Brana, M. F.; Bailly, C. Biochemistry 2003, 42, 11751-11761. (27) Stevenson, K. A.; Yen, S.-F.; Yang, N.-C.; Boykin, D. W.; Wilson, W. D. J. Med. Chem. 1984, 27, 1677-1682. 10.1021/ac070358e CCC: $37.00

© 2007 American Chemical Society Published on Web 08/15/2007

Chart 1

and exhibit characteristics similar to the threading intercalators that were shown to be extremely effective in dsDNA detection by Takenaka et al. 7,19 and Tansil et al.8 Due to its ability to intercalate and the tetracationic nature of the viologen linker, this molecule binds extremely tightly to dsDNA, offering definitive electrochemical DNA hybridization detection. EXPERIMENTAL Synthesis of NIV. The synthesis and characterization of NIV followed Scheme S1 as described in the Supporting Information. 1H NMR and mass spectral (MALDI-TOF) analysis were used to confirm the presence and purity of all intermediates and the final product. Materials. All solutions were made with 18-MΩ deionized water from a Millipore MilliQ system. Potassium phosphate and TRIS buffer were purchased from Sigma. 6-Mercapto-1-hexanol (MCH) was purchased from Aldrich. All other chemicals were of reagent grade or better. DNA oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). A 25-mer with a 6-mercaptohexyl linker at the 5′-end was used as the immobilized ssDNA. Its sequence was 5′-CAC GAC GTT GTA AAA CGA CGG CCA G-3′. It is abbreviated HS-ssDNA. The complementary singlestrand target was a 25-mer with the sequence 5′-CTG GCC GTC GTT TTA CAA CGT CGT G-3′. Electrode Preparation. DNA-modified Au electrodes (with surface area of 8 × 10-2 cm2) were prepared following a protocol described previously.4 The amount of ssDNA probes and dsDNA hybrids immobilized at the surface was assayed using the method of Tarlov and co-workers.29 Briefly, chronocoulometry was performed in the presence of Ru(NH3)63+ to determine the amount of DNA phosphate groups present before and after hybridization. Ru(NH3)63+ binds in a 1:3 ratio with phosphate groups on the electrode surface. Based on this protocol, the number of ssDNA probes and dsDNA hybrids present on the Au electrode was determined to be 2.62 ((0.38) × 1012 strands cm-2 and 2.42 ((0.52) × 1012 duplexes cm-2, respectively. Probe density at this value has previously been shown to promote complete hybridization.30 Mercaptohexanol was used as a diluent thiol after ssDNA immobilization to passivate the remaining exposed electrode area, eliminating 85-90% of the charging current.29 Passivation toward nonspecific adsorption of DNA by MCH modification also was previously demonstrated.4,29 Electrochemical Assay. ssDNA- or dsDNA-modified electrodes were placed in 4 mL of 5 mM Tris + 10 mM NaCl buffer at pH 7.1 and exposed to increasing concentrations of NIV. (28) Yen, S.-F.; Gabbay, E. J.; Wilson, W. D. Biochemistry 1982, 21, 2070-2076. (29) Steel, A. B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70, 4670-4677. (30) Peterlinz, K. A.; Georgiadis, R. M.; Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 3401-3402.

Figure 1. CV of 2 µM NIV at a dsDNA-modified gold electrode, ν ) 0.1 V s-1, 5 mM Tris, and 10 mM NaCl, pH 7.1. Inset shows scan rate dependence on reduction wave peak current.

Chronocoulometry and cyclic voltammetry (CV) were performed using a CH Instruments model 900 potentiostat. Ag/AgCl reference and Pt wire counter electrodes were used. Solutions were purged with N2 for several minutes before measurements. For measurement of the rate of NIV desorption/loss from either ssDNA- or dsDNA-modified electrodes, the electrodes were placed in a solution of 10 µM NIV for 30 min to achieve equilibrium surface coverage and then transferred into the Tris buffer + 0.5 M NaCl solution containing no NIV. Chronocoulometry was used to monitor the surface coverage of the NIV as a function of time after transfer to the high-saline buffer solution. RESULTS AND DISCUSSION Figure 1 shows a CV for a dsDNA-modified electrode immersed in a 2 µM solution of NIV. At this concentration, the contribution of the dissolved NIV to the electrochemical response is negligible, as was demonstrated in a previous study of a different diviologen derivative.4 The CV shows that the NIV is strongly adsorbed at the dsDNA-modified surface. The symmetric wave shape is characteristic of a surface-bound species, and the peak current is proportional to the first power of scan rate, as expected for a surface-bound species (data shown in inset). Also of note is the single reduction or oxidation wave denoting that the hexyl spacer allows the viologen moieties within the NIV to be reduced or oxidized independently of each other. This behavior of viologens is well-known31 and has been previously demonstrated in the presence of DNA.4 The quantity of NIV bound to electrodes modified with MCH, ssDNA, and dsDNA was assayed using chronocoulometric intercept analysis. This method has been shown to be effective at quantifying electroactive molecules present at DNA-modified surfaces.29 Figure 2 shows the binding isotherms of NIV at the various surfaces. The data show very weak adsorption at MCHmodified electrodes, as expected. Binding at ssDNA is characterized by a single Langmuir isotherm (fit shown as solid line) with uptake occurring in the concentration range 0-4 µM and saturation near 1.5 × 10-11 mol cm-2. In contrast, NIV binds to dsDNA in two distinct steps. A first plateau is observed in the concentra(31) DeLong, H. C.; Buttry, D. A. Langmuir 1992, 8, 2491-2496.

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Figure 2. Binding isotherms for NIV on (a) ssDNA (circles) and MCH (squares) and (b) dsDNA (circles) modified gold electrodes. Solid lines are Langmuir fits. Conditions: 5 mM Tris, 10 mM NaCl, pH 7.1, N2 purge. 0 to -0.75 V, and 0.1-s pulse. Table 1. Data for NIV Binding to ssDNA- and dsDNA-Modified Gold Surfaces

surface

Γsata (×10-11 mol cm-2)

n (NIV/DNA)

Keq (×106 M-1)

kdes(s-1)

ssDNA dsDNA (1) dsDNA (2)

1.50((0.040) 0.40((0.045) 2.6((0.12)

3.4((0.094) 0.90((0.091) 6.1((0.27)

1.6((0.20) 14((0.42) 0.23((0.24)

0.011 7.0 × 10-5

a

Corrected for nonspecific adsorption of NIV to MCH.

tion range 0-250 nM with a saturation binding of 0.4 × 10-11 mol cm-2. Uptake toward a second saturation plateau occurs up to ∼6 µM with saturation binding of 2.6 × 10-11 mol cm-2. This twostep binding behavior is not seen on the ssDNA-modified electrode. The solid lines in Figure 2a and b represent Langmuir fits, with two separate binding events used to fit the data at the dsDNA surface. Table 1 gives the data derived from the Langmuir fits shown in Figure 2, including the saturation coverage for each plateau (Γsat), the number of NIV molecules bound per ssDNA strand or dsDNA duplex (n), and the binding constant for each saturation process, Keq (i.e., one binding constant for ssDNA and two for dsDNA). The binding data for NIV at ssDNA are similar to those recently reported for a structurally similar diviologen derivative bearing n-dodecyl chains in place of the naphthyl imide moieties.4 The only significant difference is that NIV binds with a slightly higher binding constant and a lower saturation coverage. The higher binding constant may be due to the somewhat lower solubility of NIV compared to the dodecyl derivative and its ability to interact with the nucleic acid bases via hydrophobic or π stacking interactions. The origin of the lower saturation coverage for the NIV compound is less clear, though there are several possibilities, including the larger steric bulk and the possibility of more favorable hydrophobic interactions between dodecyl chains than between naphthyl imide groups, thereby promoting better aggregation along the ssDNA chain for the dodecyl derivative. Given these small differences between NIV and the C12 derivative, we attribute the binding of NIV with ssDNA to a combination of electrostatic interactions with the phosphate groups on ssDNA, as well as hydrophobic or π stacking interac6924

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tions with the exposed bases. This is similar to the previous assessment of the interactions driving binding of the dodecyl derivative with ssDNA.4 The biphasic binding of NIV to dsDNA is unique to this viologen derivative. As shown in Table 1, the initial saturation event results in binding of a single NIV molecule to each dsDNA duplex, i.e., n ≈ 1. This binding process is attributed to bisintercalation of the NIV to the dsDNA duplex. The binding constant for this first step is roughly 1 order of magnitude higher than that seen for binding to ssDNA, consistent with the much stronger binding expected for a bis-intercalator. There are a few binding constants available in the literature to which the value for the bis-intercalated state in Table 1 can be compared. Qian et al. synthesized and characterized bis-isoquinolino[4,5-bc]acridines binding to calf thymus DNA.32 They reported binding constants in the range 3.6 × 104-2.4 × 105 M-1, depending on the length of the linker. McFayden and co-workers reported similar binding constants in the range 1.2 × 104-12 × 104 M-1 for a series of binuclear 4-aminoquinolines bridged via their 4-amino group.33 In contrast, Pavlov et al. reported binding constants for bis-intercalation of bis-naphthylimide spermine and spermidine derivatives.34 They gave values in the range 10.5 × 107-18 × 107 M-1 depending on the structure of the linker. Thus, the value given in Table 1 is within the range of previously reported values for structurally similar bis-intercalators, though the range is quite large due to the considerable structural differences between the various systems. The origin of saturation for the bis-intercalated state at a single NIV per dsDNA duplex is attributed to geometrical constraints as follows. Each NIV has an intra-naphthyl distance of 35 Å for the fully extended molecule, while each dsDNA hybrid has a total length of ∼80 Å.4 Neighbor exclusion suggests that each intercalation site prevents a second intercalation event any closer than two base pairs away.35 With a distance per base pair of 3.4 Å for (32) Yang, P.; Yang, Q.; Qian, X. Tetrahedron 2005, 61, 11895-11901. (33) McFadyen, W. D.; Sotirellis, N.; Denny, W. A.; Wakelin, L. P. G. Biochem. Biophys. Acta 1990, 1048, 50-58. (34) Pavlov, V.; Kong, Thoo, Lin, P.; Rodilla, V. Chem.-Biol. Int. 2001, 137, 1524. (35) Veal, J. M.; Li, Y.; Zimmerman, S. C.; Lamberson, C. R.; Cory, M.; Zon, G.; Wilson, W. D. Biochemistry 1990, 29, 10918-10927.

Figure 3. Desorption of NIV from ssDNA (blue) and dsDNA (red) modified electrodes vs time. Conditions: presaturation from 10 µM NIV; 5 mM Tris, 0.5 M NaCl, pH 7.1, N2 purge. 0 to -0.75 V, and pulse 0.1 s.

dsDNA, this suggests that the total distance along the duplex occupied by a bis-intercalated NIV is 49 Å, i.e., 35 Å plus two base pairs on each side. Thus, only a single bis-intercalated molecule can be accommodated per duplex. Mono-intercalation may be possible, but not with the higher binding constant characteristic of the bis-intercalated state. After the initial saturation binding at n ) 1, increased concentration of NIV leads to a second saturation plateau. Just as for NIV binding to ssDNA, this second binding process is attributed to a combination of electrostatic and hydrophobic/π stacking interactions. However, mono-intercalation is also possible. The binding constant for the second binding process is Keq ) 2.34 × 105 M-1 (Table 1), which compares favorably with the binding of similar viologen molecules to dsDNA.4,29,36 The desorption of NIV from ssDNA and dsDNA also was investigated, with the motivation that bis-intercalation should result in far slower desorption than would electrostatic and hydrophobic/π stacking interactions. Figure 3 shows the results of such experiments in which NIV was first equilibrated with either a ssDNA- or dsDNA-modified surface under saturating conditions, followed by transfer to a solution containing only supporting electrolyte. Chronocoulometry was used to monitor the NIV surface population, with short negative potential excursions being used to avoid spending large amounts of time at potentials where the insoluble radical cation state would be present, since this would perturb the desorption of the NIV. As can be seen, immediately after transfer to pure supporting electrolyte, NIV begins to desorb from ssDNA (blue points), reaching near-zero levels after 8-10 min. NIV desorption from dsDNA (red points) shows a significantly different behavior. An initial fraction is rapidly lost on a time scale very similar to that for loss from ssDNA. (36) Millan, K. M.; Mikkelsen, S. R. Anal. Chem. 1993, 65, 2317-2323.

However, in contrast to the behavior at ssDNA surfaces, the initial rapid desorption stops at a surface coverage of ∼0.45 × 10-11 mol cm-2, after which the desorption rate decreases dramatically. Thus, the desorption from dsDNA is biphasic. The data at both ssDNA and dsDNA surfaces were fit using a first-order desorption kinetics model to give first-order rate constants for the desorption process. The results of the desorption experiments are summarized in Table 1 as desorption rate constants, kdes (s-1). There are two salient features of the data in Figure 3. First, for NIV desorption from dsDNA, the surface coverage at which the rapid loss terminates is 0.42 × 10-11 mol cm-2. This is the same surface coverage at which the first, small plateau was observed in the adsorption isotherm in Figure 2b. Thus, we attribute the material remaining after the rapid desorption to NIV in this much more strongly bound state. Second, as can be seen by inspection in Figure 3, the first-order desorption rate constant for NIV from ssDNA is quite similar to that for the rapidly lost fraction of NIV from dsDNA. These data suggest that the initial rapidly desorbing fraction at dsDNA and the NIV that desorbs at the same rate from ssDNA are likely bound in similar environments. As discussed above, we attribute this binding to a combination of electrostatic and hydrophobic/π stacking interactions. Whatever the origin of the binding, it is clear that these two components behave substantially differently than the slowly desorbing component at dsDNA. The desorption rate constants given in Table 1 show that the strongly bound state of NIV at dsDNA desorbs 160 times more slowly from dsDNA than does NIV from ssDNA. This is consistent with a more tightly bound, bis-intercalated structure. Some literature comparisons are available for dissociation rate constants for structurally similar bis-intercalators. Bailly and co-workers reported a dissociation rate constant of 2.48 × 10-3 s-1 for a structurally similar dimeric pyrazinonaphthalimide bis-intercalating agent dissociating from [C‚G]4.26 Gallego used NMR measurements to obtain values in the range 1.1-5 s-1 for dissociation of the bis-intercalator elinafide, a dimeric naphthyl imide with a -(CH2)2NH(CH2)3NH(CH2)2- linker.37 Thus, the desorption rate constant in Table 1 is substantially lower than those reported for several structurally similar dimeric naphthyl imides, consistent with a quite stable bis-intercalated state in the present case. The considerably slower dissociation for NIV than these comparator compounds is attributed to its tetracationic charge. The data in Table 1 also can be used to calculate adsorption rate constants for NIV bound at ssDNA and NIV bound in the strongly adsorbed state at dsDNA. Equation 1 shows the relationship between these quantities:

Keq ) kads/kdes

(1)

Use of the values in Table 1 gives adsorption rate constants for NIV at ssDNA and dsDNA of 1.8 × 104 and 1.0 × 103 M-1 s-1, respectively. The slower adsorption rate constant for the bisintercalated state of NIV is consistent with the need for reorganization of the duplex in order to accommodate the bis-intercalated state. These values are considerably lower than those for several (37) Gallego, J. Nucliec Acids Res. 2004, 32, 3607-3614.

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Figure 4. CV comparison of NIV on ssDNA (blue) and dsDNA (red) after 480-s exposure to saline buffer solution. Conditions: 5 mM Tris, 0.5 M NaCl, pH 7.1, N2 purge, and 100 mV/s.

other bis-intercalators, which range from 105 to 107 M-1 s-1.26,38 These differences may be related to different structures, as well as to variations in the local environments for the dsDNA, i.e., in solution37,38 versus in polymeric films confined at an interface26 versus on a Au surface in the present case. The very low value of kdes for NIV from dsDNA allows for a kinetic differentiation between ssDNA and dsDNA. This could be used to detect a target DNA strand that is complementary to the thiolated DNA strand immobilized at the electrode surface. In such a scenario, the electrode is first exposed to a solution that may or may not contain the target. Then, the electrode is rinsed and exposed to the NIV compound for a sufficient time to achieve equilibrium binding on the bis-intercalator. Finally, the electrode (38) Markovits, J.; Garbay-Jaureguiberry, C.; Roques, B. P.; Le, Pecq, J. B. Eur. J. Biochem. 1989, 180, 359-366. (39) Heaton, R. J.; Peterson, A. W.; Georgiadis, R. M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 3701-3704. (40) Theil, A. J.; Frutos, A. G.; Jordan, C. E.; Corn, R. M.; Smith, L. M. Anal. Chem. 1997, 69, 4948-4956. (41) Glazer, A. N.; Peck, K.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 3851-3855.

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is transferred to pure supporting electrolyte solution and left there just long enough for complete desorption of NIV from the ssDNA. Under these conditions, any NIV remaining is bound to dsDNA, thereby allowing detection of the complementary target strand. Figure 4 shows the results of such an experiment. The blue and red curves provide a cyclic voltammetric comparison of the NIV response on ssDNA- and dsDNA-modified electrodes, respectively, after loading with NIV followed by an 8-min exposure to 0.5 M NaCl. The significant amount of NIV that remains bound to dsDNA versus ssDNA after this time can be easily seen. The signal from the NIV bound to dsDNA is stable toward repetitive cycling. In this case, the complementary target strand was provided in excess, driving the dsDNA coverage to saturation. Thus, integration of the reduction peak in the dsDNA CV gives Γ ) 0.42 × 10-11 mol cm-2, as shown in Figures 2 and 3. In addition, Figure 3 demonstrates that NIV remains bound to dsDNA under these conditions at least as long as 30 min. This would potentially allow for single-nucleotide polymorphism detection in conjunction with an application of a slight negative potential39 or buffer temperature increase,40 both of which have been employed to detect DNA hybridization/dehybridization within similar time limits in situ. Also, the ability of the NIV compound to discriminate ssDNA from dsDNA is analogous to the fluorescence-based discrimination provided by the ethidium homodimer.41 CONCLUSION We have described the synthesis and electrochemical behavior of an electroactive tetracationic bis-intercalating viologen derivative for detection of DNA hybridization. The NIV compound was shown to have a binding constant, association rate constant, and dissociation rate constant comparable to various other bisintercalators described in the literature. The large difference in dissociation rate between ssDNA and dsDNA was shown to provide the basis for detecting the hybridization of target strands with immobilized probe DNA strands. SUPPORTING INFORMATION AVAILABLE Experimental procedures, 1H NMR, and MALDI-TOF NIV characterization data. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review February 20, 2007. Accepted June 14, 2007. AC070358E