Use of Polymeric Indicator for Electrochemical DNA Sensors: Poly(4

A poly(4-vinylpyridine) (PVP) derivative bearing redox-active osmium complexes, PVP-[Os(5,6-dmphen)2Cl]2+ (5,6-dmphen = 5,6-dimethyl-1 ...
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Anal. Chem. 2004, 76, 2975-2980

Use of Polymeric Indicator for Electrochemical DNA Sensors: Poly(4-vinylpyridine) Derivative Bearing [Os(5,6-dimethyl-1,10-phenanthroline)2Cl]2+ Aihua Liu and Jun-ichi Anzai*

Graduate School of Pharmaceutical Sciences, Tohoku University, Aramaki, Aoba-ku, Sendai 980-8578, Japan

A poly(4-vinylpyridine) (PVP) derivative bearing redoxactive osmium complexes, PVP-[Os(5,6-dmphen)2Cl]2+ (5,6-dmphen ) 5,6-dimethyl-1,10-phenanthroline), was employed as a hybridization indicator for electrochemical DNA sensors. PVP-[Os(5,6-dmphen)2Cl]2+ exhibited ∼1000 times higher sensitivity than the corresponding monomeric analogue, [Os(5,6-dmphen)3]2+, in DNA determination due to polymeric effects. The detection limit of the present sensor was ∼0.5 amol. Another merit of the polymeric indicator is that the redox potential was found to be +360 mV (vs Ag/AgCl), which is significantly lower than that reported for the monomeric analogue (+672 mV). The polymeric indicator was applicable to the discrimination of single- and double-base-mismatched DNAs from fully matched target DNA. The polymeric indicator can be removed from the electrode surface by rinsing the electrode in a high-temperature buffer for 6 min, and thus, the polymeric indicator-based DNA sensor can be used repeatedly. Electrochemical DNA sensors have received much attention due to the high sensitivity, rapid response, easy handling, compatibility with miniaturization technology, and low cost.1-5 Electrochemical DNA sensors can be classified into two categories depending on the signal transduction mode on the electrode. The direct signal transduction sensors rely on the electrooxidation of guanine or adenine residues in the DNA chains hybridized on the electrode surface.6 In some cases, to amplify the signal of guanine or adenine oxidation, Ru(bpy)32+ ion has been employed as a redox mediator for the electrocatalytic oxidation.7-9 Indirect detection of hybridization, on the other hand, uses enzyme * To whom correspondence should be addressed. E-mail: junanzai@ mail.pharm.tohoku.ac.jp. (1) Thorp, H. H. Trends Biotechnol. 1998, 16, 117-121. (2) Palecek, E.; Fojta, M. Anal. Chem. 2001, 73, 75A-83A. (3) Wang, J. Anal. Chim. Acta 2002, 469, 63-71. (4) Palecek, E. Talanta 2002, 56, 809-819. (5) Lucarelli, F.; Marrazza, G.; Turner, A. P. F.; Mascini, M. Biosens. Bioelectron. 2004, 19, 515-530. (6) Wang, J.; Rivas, G.; Fernandes, J. R.; Paz, J. L. L.; Jiang, M.; Waymire, R. Anal. Chim. Acta 1998, 375, 197-203. (7) Johnston, D. H.; Glasgow, K.; Thorp, H. H. J. Am. Chem. Soc. 1995, 117, 8933-8938. (8) Palecek, E.; Billova, S.; Havram, L.; Kizek, R.; Miaulkova, A.; Jelen, F. Talanta 2002, 56, 919-930. 10.1021/ac0303970 CCC: $27.50 Published on Web 04/17/2004

© 2004 American Chemical Society

labels10-14 or such redox indicators as metal complexes,15-20 daunomycin,21 and methylene blue22,23 that selectively bind to double-stranded DNA (ds-DNA) chains. Metal complexes such as [Co(bpy)3]Cl2 (bpy ) 2,2′-bipyridine),15 [Co(phen)3]Cl2 (phen ) 1,10-phenanthroline),16 [Os(bpy)3]Cl2,17 and [Os(5,6-dmphen)3]Cl2 (5,6-dmphen ) 5,6-dimethyl-1,10-phenanthroline)18 have often been used for this purpose because of their high stability and reversibility in the redox reactions. In practice, DNA hybridization is detected by measuring the redox current for redox indicators adsorbed on the ds-DNA chains on the electrode. In this protocol, it is a prerequisite for the redox indicators to be adsorbed more efficiently on ds-DNA chains than on single-stranded DNA (ssDNA) for effecting sensitive determination of DNA hybridization. From this point of view, the binding efficiency of the metal complexes above should be improved because their binding affinity is still inadequate. This originates from the fact that the metal complexes bind to ds-DNA chains through the electrostatic force of attraction between the positive charges in the metal complexes and negative charges in the DNA backbone or through hydrophobic interactions. Recently, metal complexes have been (9) Gore, M. R.; Szalai, V. A.; Ropp, P. A.; Yang, I. V.; Silverman, J. S.; Thorp, H. H. Anal. Chem. 2003, 75, 6586-6592. (10) de Lumly, T.; Campbell, C.; Heller, A. J. Am. Chem. Soc. 1996, 118, 55045505. (11) Patolsky, F.; Katz, E.; Bardea, A.; Willner, I. Langmuir 1999, 15, 37033706. (12) Alfonta, L.; Singh, A. K.; Willner, I. Anal. Chem. 2001, 73, 91-102. (13) Zhang, Y.; Kim, H.-H.; Heller, A. Anal. Chem. 2003, 75, 3267-3269. (14) Kim, E.; Kim, K.; Yang, H.; Kim, Y. T.; Kwak, J. Anal. Chem. 2003, 75, 5665-5672. (15) Millan, K. M.; Saraullo, A.; Mikkelesen, S. R. Anal. Chem. 1994, 66, 29432948. (16) Wang, J.; Rivas, G.; Cai, X.; Chicharro, M.; Parrado, C.; Dontha, N.; Begleiter, A.; Mowat, M.; Palecek, E.; Nielsen, P. E. Anal. Chim. Acta 1997, 344, 111-118. (17) Mishima, Y.; Motonaka, J.; Maruyama, K.; Minagawa, K.; Ikeda, S. Sens. Actuators, B 2000, 65, 340-342. (18) Maruyama, K.; Motonaka, J.; Mishima, Y.; Matsuzaki, Y.; Nakabayashi, I.; Nakabayashi, I. Sens. Actuators, B 2001, 76, 215-219. (19) Takenaka, S.; Yamashita, K.; Takagi, M.; Uto, Y.; Kondo, H. Anal. Chem. 2000, 72, 1334-1341. (20) Nakayama, M.; Ihara, T.; Nakano, K.; Maeda, M. Talanta 2002, 56, 857866. (21) Marrazza, G.; Chiti, G.; Mascini, M.; Anichini, M. Clin. Chem. 2000, 46, 31-37. (22) Erdem, A.; Kerman, K.; Meric, B.; Akarce, U. S.; Ozsoz, M. Anal. Chim. Acta 2000, 422, 139-149. (23) Ozkan, D.; Kara, P.; Kerman, K.; Meric B.; Erdem, A.; Jelen, F.; Nielsen, P. E.; Ozsoz, M. Bioelectrochemistry 2002, 58, 119-126.

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modified with intercalative ligands to improve the binding affinity to ds-DNA.24,25 In this context, Maruyama and co-workers have studied the electrochemical properties of various kinds of Os complexes as redox indicators for electrochemical DNA sensors and found that dipyridophenazine (DPPZ) complexes of Os showed a high affinity for ds-DNA chains through intercalation.26 It was also reported that the redox potential of the Os complexes can be tuned by changing the type of substituent of the ligands.26 Thus, they developed a [Os(DA-bpy)2DPPZ]2+ complex (DA-bpy ) 4,4′-diamino-2,2′-bipyridine) as an excellent redox indicator that shows high affinity for ds-DNA chains and low redox potential. These findings suggest that molecular design of the ligand plays a key role in developing a high-performance redox indicator for electrochemical DNA sensors. We report here an alternative way to develop a highperformance redox indicator for electrochemical DNA sensors based on a polymeric complex of Os. We used poly(4-vinylpyridine) (PVP) derivatives bearing [Os(5,6-dmphen)2Cl]2+ or [Os(bpy)2Cl]2+ in the side chains. The PVP-[Os(5,6-dmphen)2Cl]2+ exhibited ∼1000 times higher sensitivity than the monomeric analogue, [Os(5,6-dmphen)3]2+, in DNA determinations due to polymeric effects. The redox potential of this indicator was found to be +360 mV (vs Ag/AgCl), which is significantly lower than the value reported for the monomeric analogue (+672 mV). The polymeric indicator induced no problem in repeated use of the DNA sensor; the polymeric indicator was easily removed from the surface of the DNA sensor by rinsing the sensor surface in a high-temperature buffer (pH 8.0), and the sensor can thus be used repeatedly. EXPERIMENTAL SECTION Reagents. Potassium hexachloroosmate(IV) (K2OsCl6) and 6-mercapto-1-hexanol were purchased from Aldrich Chemical Co. (Milwaukee, WI). 5,6-Dimethyl-1,10-phenanthroline (5,6-dmphen) and 2,2′-bipyridine (bpy) were obtained from Tokyo Kasei Kogyo Co. (Tokyo, Japan). PVP (average molecular weight, 60 000, Aldrich) was purified by reprecipitation from methanol solution into ether twice. A 21-mer deoxyoligonucleotide tagged with a mercaptohexyl group at the 5′-phosphate end [HS(CH2)6-5′-d(TAA GGG AAT GGT TAG GAA GGC)-3′], fully matched oligonucleotide [5′-d(GCC TTC CTA ACC ATT CCC TTA)-3′], single-basemismatched oligonucleotide [5′-d(GCC TTC CTA ACT ATT CCC TTA)-3′], double-base-mismatched oligonucleotide [5′-d(GCC TTC CTA ACT CTT CCC TTA)-3′], and fully mismatched oligonucleotide [5′-d(CTT GAT ACG GAT CAG AGT CAT)-3′] were purchased from Nihon Gene Research Laboratory (Sendai, Japan). HS(CH2)6-poly(dT25), poly(dA25), and poly(dT25) were also from Nihon Gene Research Laboratory. All other reagents were of the highest grade available and used as received. All solutions were prepared in Milli-Q water. Preparation of Polymeric Indicators. Os(5,6-dmphen)2Cl2 and Os(bpy)2Cl2 were prepared from K2OsCl6 and 5,6-dmphen or bpy, respectively, according to the reported procedure.27 Two (24) Wilhelmsson, M. L.; Westerlund, F.; Lincoln, P.; Norde´n, B. J. Am. Chem. Soc. 2002, 124, 12092-12093. (25) Metcalfe, C.; Adams, H.; Haq, I.; Thomas, J. A. Chem. Commun. 2003, 1152-1153. (26) Maruyama, K.; Mishima, Y.; Minagawa, K.; Motonaka, J. Anal. Chem. 2002, 74, 3698-3703.

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Figure 1. Chemical structures of indicators 1 and 2.

kinds of polymeric indicators were prepared by modifying PVP with Os(5,6-dmphen)2Cl2 and Os(bpy)2Cl2. PVP (0.5-mmol monomer unit) and Os(5,6-dmphen)2Cl2 (0.1 mmol) or Os(bpy)2Cl2 (0.11 mmol) were refluxed in ethylene glycol (18 mL) for 2 h under N2. After cooling, 6 mL of ethanol and 4 mL of water were added to the reaction mixture, which was then dialyzed against an ethanol/water mixture (6:4 by volume) to remove ethylene glycol. The polymeric indicators were purified using a size exclusion gel chromatography on Sephadex G-25. The contents of Os(5,6dmphen)2Cl2 and Os(bpy)2Cl2 residues in the polymer chains were determined to be 18 and 21%, respectively, by UV-visible absorption spectra. The chemical structures of the polymeric indicators 1 and 2 are illustrated in Figure 1. Preparation of the DNA Biosensor. The DNA biosensor was prepared according to the reported procedure with minor modification.26 The surface of an Au disk electrode (1.6-mm diameter) was polished with alumina slurry and rinsed in a mixed acid (concetrated HNO3:concentrated HCl:water ) 1:3:5 by volume) for 2 min. The Au electrode was further treated electrochemically in a 0.5 M H2SO4 solution by scanning the potential from -0.2 to 1.7 V at a scan rate of 10 V s-1 for 15 min or until an ideal voltammogram for a clean Au was observed. The surface of the electrode was first modified with a 5′-mercaptohexyl-tagged oligonucleotide (ss-DNA) by exposing the surface to 5′-mercaptohexyl-tagged oligonucleotide solution for 3 h at 25 °C. After rinsing, the ss-DNA-modified electrode was treated with 1.0 mM 6-mercapto-1-hexanol solution for 2 h to mask the unmodified Au sites. The thiol-tagged probe DNA and 6-mercapto-1-hexanol were thus immobilized on the surface of the Au electrode through thiol-Au binding. Hybridization. A 2-µL aliquot of target DNA solution (in 10 mM Tris-HCl buffer containing 1 mM EDTA and 1.0 M NaCl, pH 8.0) was pipetted onto the surface of the ss-DNA-modified electrode as uniformly as possible and incubated at 25 °C for the (27) Lay, P. A.; Sargeson, A. M.; Taube, H.; Chou, M. H.; Creutz, C. Inorg. Synth. 1986, 24, 293-295.

Figure 2. Cyclic voltammograms of Fe(CN)63- ion (1 mM) on ssDNA-modified (a) and a bare Au electrode (b) in 0.1 M KCl. Scan rate, 50 mV s-1. The Au electrode was modified with 0.1 µM HS(CH2)6-5′-d(TAA GGG AAT GGT TAG GAA GGC)-3′.

desired time for hybridization. During incubation, the electrode was covered with a rubber cap to avoid evaporation. The ds-DNAmodified electrode thus prepared was subjected to measurement of the electrochemical response. Measurements of Electrochemical Response.The ss-DNAor ds-DNA-modified electrode was immersed in a polymeric indicator solution (0.1 mM in a water/ethanol mixture in 9:1 by volume) for 10 min to deposit the indicator on the electrode and rinsed to remove any nonspecifically adsorbed indicator. Electrochemical measurements were carried out using a CS2000 electrochemical system (Cypress Systems, Inc., Lawrence, KS) in a conventional three-electrode cell consisting of the ssDNA- or ds-DNA-modified electrode as the working electrode, a platinum wire as the counter electrode, and a Ag/AgCl electrode (3.3 M KCl) as a reference electrode. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurements were performed at room temperature in 10 mM Tris-HCl buffer containing 0.1 M NaClO4 (pH 7.4). RESULTS AND DISCUSSION Prior to hybridization of the target DNA on the surface of the probe DNA-modified electrodes, we examined the surface coverage of probe DNA on the electrode by CV in the presence of Fe(CN)63- marker ion in solution, according to the reported procedure.18,20,26,28 The CVs of Fe(CN)63- ion on the Au electrodes before and after being modified with probe DNA [HS(CH2)6-d(TAA GGG AAT GGT TAG GAA GGC)-3′] are shown in Figure 2. A symmetric, reversible voltammogram was obtained for a bare Au electrode, whereas the DNA-modified electrode showed an asymmetric, irreversible wave. Both the anodic and cathodic currents are severely reduced for the DNA-modified electrode, due to the electrostatic repulsion between Fe(CN)63- anion and negatively charged DNA chains on the electrode surface. These results suggest that the electrode surface is well covered with the probe DNA under the modification conditions. Electrochemical Response of the Indicator-Adsorbed Electrodes before and after Hybridization. The electrochemical response of the present DNA sensor relies on the redox reaction (28) Aoki, H.; B×c4hlmann, P.; Umezawa, Y. Electroanalysis 2000, 12, 12721276.

Figure 3. Schematic illustration of surface modification of an Au electrode and determination of DNA hybridization.

of the indicator adsorbed on the electrode surface after hybridization, as schematically illustrated in Figure 3. In this protocol, the magnitude of the redox current would depend on the amount of Os complex adsorbed on the electrode. Therefore, to realize sensitive detection of DNA, greater amounts of indicator molecules have to be adsorbed on the electrode after hybridization than before hybridization. To evaluate this, we carried out DPV on the indicator-adsorbed electrodes before and after hybridization. Figure 4 illustrates the response of the electrodes using polymeric indicators 1 (Figures 4A) and 2 (Figure 4B) before Analytical Chemistry, Vol. 76, No. 10, May 15, 2004

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Table 1. Effects of the Concentration of Probe DNA in the Solution Used for Surface Modification on the Response of the Sensors before and after Hybridizationa

concn of probe DNA for surface modification/µM before hybridizationb after hybridizationc

response current in DPV/ µA 0.01 0.1

1.0

0.19 0.95

0.30 1.10

0.19 1.00

a The probe and target DNAs used are HS(CH ) -5′-d(TAA GGG 2 6 AAT GGT TAG GAA GGC)-3′ and 5′-d(GCC TTC CTA ACC ATT CCC TTA)-3′, respectively. The concentration of the target DNA in sample is 1.0 nM. b These values contain (5% error. c These values contain (10% error.

Figure 4. Differential pulse voltammograms of 1 (A) and 2 (B) adsorbed on the sensor before (a) and after hybridization (b). The Au electrode was modified with 0.1 µM HS(CH2)6-5′-d(TAA GGG AAT GGT TAG GAA GGC)-3′ and hybridized with 1.0 nM target DNA. Differential pulse voltammetry conditions: scan rate, 50 mV s-1; pulse height, 50 mV; pulse width, 100 ms; pulse period, 200 ms. The voltammograms were obtained in 10 mM Tris-HCl buffer containing 0.1 M NaClO4 (pH 7.4).

and after hybridization with the target DNA. Figure 4A shows that a highly enhanced response was observed after hybridization, while the response was rather low before hybridization. This result suggests a much higher affinity of 1 for the ds-DNA chain than for the ss-DNA on the electrode surface. Thus, the indicator 1 seems promising for electrochemical detection of hybridization on the electrode. It should be noted here that indicator 1 shows a peak potential (Ep) at +360 mV, which is significantly lower than the reported value for the monomeric analogue, [Os(5,6dmphen)3]2+, (+672 mV) used as an electrochemical indicator for a DNA biosensor under similar experimental conditions.18 This probably results from replacement of one of the 5,6-dmphen ligands of the monomeric analogue with a Cl atom for 1. It has been reported that ligand modifications alter the electron density at the metal center of the Os complex, resulting in shifts in the redox potential.29 The operating potenatial of electrochemical DNA sensors should be as low as possible to circumvent possible interference arising from partial oxidation of adenine and guanine bases (The redox potential: guanine, ∼+1.05 V; adenine, ∼+1.19 V vs Ag/AgCl).30 In contrast to 1, indicator 2 was not adsorbed selectively on the ds-DNA chains but adsorbed on the electrode weakly both before and after hybridization, implying that 2 is less effective for detecting hybridization. It is likely that this difference comes from possible stacking and intercalating interactions for 1, while 2 binds to the ss-DNA and ds-DNA simply by an electrostatic force (29) Kober, E. K.; Caspar, J. V.; Sullivan, B. P.; Meyer, T. J. Inorg. Chem. 1988, 27, 4587-4598. (30) Holmberg, R. C.; Thorp, H. H. Anal. Chem. 2003, 75, 1851-1860.

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due to the lack of a planar aromatic ligand. It has been reported that tris-chelated metal complexes with phenanthroline ligands bind to DNA by intercalation31,32 in contrast to the electrostatic binding of metal complexes with bipyridine ligands.33 The different binding mode of indicators 1 and 2 may be responsible for the positive shift in the peak potential of 1 in the voltammogram after hybridization (Figure 4A), which is not the case for indicator 2 (Figure 4B). It is likely that the response characteristics of DNA sensors depend on the density of the probe DNA on the electrode surface, because the surface coverage of the probe DNA may alter the efficiency of DNA binding on the surface. For this reason, we evaluated the effects of the density of the probe DNA on the electrochemical response by modifying the electrode in 0.01, 0.1, and 1.0:M probe DNA solutions. Table 1 lists the response current of the DNA sensors based on indicator 1 before and after hybridization. For all cases tested, the response current was highly enhanced after hybridization. The response current was rather small before hybridization even when the electrode was modified in the highest concentration solution of probe DNA (1.0 µM). These results also suggest the selective binding of indicator 1 to the ds-DNA on the electrode surface. From these results, the Au electrode hereafter was modified with 0.1 µM probe DNA solution. Calibration Graphs of the DNA Sensors. We used two kinds of probe DNAs, HS(CH2)6-poly(dT25) and HS(CH2)6-5′-d(TAA GGG AAT GGT TAG GAA GGC)-3′, to modify the Au electrode for preparing DNA sensors. The sensors were incubated in the target or a related DNA sample solution for 25 min to complete hybridization on the electrode surface, and the DPV response was measured after being treated with indicator 1 (0.1 mM).34 The response of the HS(CH2)6-poly(dT25)-modified sensor to poly(dT25) and poly(dA25) is depicted in Figure 5. The sensor exhibited amperometric response to complementary poly(dA25) while no response was observed for poly(dT25), suggesting that indicator 1 was adsorbed selectively on the hybridized ds-DNA chains on (31) Barton, J. K. Comments Inorg. Chem. 1985, 3, 321-348. (32) Carter, M. T.; Rodriguez, M.; Bard, A. J. J. Am. Chem. Soc. 1989, 111, 8901-8911. (33) Kumar, C. V.; Barton, J. K.; Turro, N. J. J. Am. Chem. Soc. 1985, 107, 5518-5523. (34) We carried out optimizatiion of the experimental variables in hybridization and in the adsorption of indicator 1. The response current of the sensor increased linearly with increasing hybridization time up to 21 min and thereafter reached a constant value. We used a 0.1 mM solution of 1 due to the limited solubility of 1.

Figure 5. Typical calibration graphs of the sensor modified with HS(CH2)6-poly(dT25) to poly(dA25) (b) and poly(dT25) (O). The differential pulse voltammetry was carried out under the same conditions as in Figure 4. Reproducibility of the data was ∼(10%.

Figure 6. Typical calibration graphs of the sensor modified with HS(CH2)6-5′-d(TAA GGG AAT GGT TAG GAA GGC)-3′ to fully matched (b), one-base-mismatched (O), two-base-mismatched (9), and fully mismatched DNA (0). The differential pulse voltammetry was carried out under the same conditions as in Figure 4. Reproducibility of the data was ∼(10%.

the electrode surface. The calibration graph is linear over the poly(dA25) concentration of 0.1 pM-1.0 nM (0.8 pg mL-1-8 ng mL-1), which corresponds to 0.5 amol-5 fmol of DNA because the volume of the sample solutions used for hybridization is 2 µL. It is important to discriminate single-base- or double-basemismatched DNAs from fully matched DNA in diagnostic testing. For this reason, we evaluated the response characteristics of the sensor modified with the HS(CH2)6-5′-d(TAA GGG AAT GGT TAG GAA GGC)-3′ probe to its single-base-mismatched, double-basemismatched, and full-base-mismatched DNAs together with its complementary DNA. Figure 6 depicts the typical DPV response of the sensor to the samples, showing that the sensor discriminates explicitly the double-base mismatch in the DNA chains. The sensor also discriminated single-base-mismatched DNA from fully matched DNA to some extent. It should be noted here that the lower detection limit of the sensor was highly improved by using polymeric indicator 1; the calibration graph for the fully matched DNA is linear down to 0.1 pM (0.6 pg mL-1 or 0.5 amol). This detection limit is ∼1000 times lower than the reported value for a similar DNA sensor based on

the monomeric analogue [Os(5,6-dmphen)3]2+ (the lower detection limit of the [Os(5,6-dmphen)3]2+-based sensor was reported to be 690 pg mL-1 for 20-mer DNA).18 Thus, polymeric indicator 1 proved to be superior to the monomeric analogue for highly sensitive determination of DNA. This is probably due to polymeric effects; the loading of the Os complex on the electrode surface should be higher for 1 than for the monomeric analogue because all the Os complexes in 1 are connected to the polymer chain. In other words, the adsorption of the first Os complex in 1 to the ds-DNA chain would facilitate the adsorption of adjacent complexes in the same polymer chain through entropic effects, which cannot be expected for monomeric indicators. The average polymerization degree of indicator 1 is ∼650 and 18% of the monomer units are substituted with the Os complex. Therefore, ∼120 residues of Os complex are connected to a single polymer chain. This is the reason indicator 1 exhibited higher sensitivity than the monomeric analogue. Recent papers report DNA sensors capable of picomolar or femtomolar DNA determination based on enzyme-linked amperometry,13,35 a high-affinity redox indicator,19 nanoparticle labeling,36 molecular imaging with a CCD camera,37 nanoparticle-coupled quartz-crystal microbalance,38 dry-reagent strip sensor using nanoparticles,39 etc. However, these sytems often require multistep treatments and need costly equipment. Therefore, the electrochemical DNA sensor based on indicator 1 is still useful for the sensitive determination of DNA hybridization. Reusability. It has been reported that electrochemical DNA sensors can be used repeatedly for DNA determination after rinsing the electrode surface in a high-temperature buffer to remove the target DNA and redox indicators. Thus, the original ss-DNA-modified surface can be restored for its repeated use. It is interesting to study whether the polymeric indicator 1 can be removed from the electrode surface by the same treatment as for monomeric indicators. In fact, the electrode surface loaded with ds-DNA and indicator 1 was immersed in high-temperature TrisHCl buffer containing 1 mM EDTA (∼100 °C, pH 8.0) for 6 min, and after cooling, the electrode was used again for detecting target DNA. The response current of the sensor modified with HS(CH2)65′-d(TAA GGG AAT GGT TAG GAA GGC)-3′ hybridized with 1 nM fully matched DNA was measured 10 times repeatedly, after regeneration of ss-DNA. The response currents were 1.1 ( 0.1 µA, and virtually no deterioration of the response was observed. These results clearly show that indicator 1 and target DNA can be removed from the electrode surface by this treatment and that the probe DNA-modified surface is regenerable repeatedly. CONCLUSIONS A PVP derivative-bearing [Os(5,6-dmphen)2Cl]2+ complex has proved to be a useful redox indicator for electrochemical DNA sensors. The polymeric indicator exhibited ∼1000 times higher sensitivity than the corresponding monomeric analogue in the (35) Patolsky, F.; Lichtenstein, A.; Willner, I. Nat. Biotechnol. 2001, 19, 253257. (36) Cao, Y. C.; Jin, R.; Mirkin, C. A. Science 2002, 297, 1536-1540. (37) Anazawa, T.; Matsunaga, H.; Yeung, E. S. Anal. Chem. 2002, 74, 50335038. (38) Tao, L.; Tang, J.; Jiang, L. Biochem. Biophys. Res. Commun. 2004, 313, 3-7. (39) Glynou, K.; Ioannou, P. C.; Christopoulos, T. K.; Syriopoulou, V. Anal. Chem. 2003, 75, 4155-4160.

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determination of a 21-mer oligonucleotide due to polymeric effects. The detection limit of the present sensor is 0.1 pM or 0.5 amol. Another merit of the polymeric indicator is that the redox potential (+340 mV) is significantly lower than that reported for the monomeric analogue (+672 mV). The polymeric indicator can be removed from the surface of the sensor by the same treatment as for the monomeric indicators, enabling the repeated use of the

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sensor. Thus, the present work has demonstrated a novel strategy based on polymers for developing useful indicators for electrochemical DNA sensors. Received for review December 1, 2003. Accepted March 11, 2004. AC0303970