DNA Sensor with a Dipyridophenazine Complex of Osmium(II) as an

DNA electrochemical sensors have been reported by many investigators.1-5 ... in targeted DNA by potentiometric stripping analysis (PSA)7 or conducting...
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Anal. Chem. 2002, 74, 3698-3703

DNA Sensor with a Dipyridophenazine Complex of Osmium(II) as an Electrochemical Probe Kenichi Maruyama,*,† Yuji Mishima,† Keiji Minagawa,† and Junko Motonaka†

Department of Chemical Science and Technology, Faculty of Engineering, The University of Tokushima, Minami-josanjima 2-1, Tokushima, 770-8506 Japan, and Faculty of Pharmaceutical Sciences, The University of Tokushima, Shomachi 1-78, Tokushima, 770-8505 Japan

Application of a dipyrido[3,2-a:2′,3′-c]phenazine (DPPZ)type metal complex as an DNA electrochemical probe was studied. The introduction of electron-donating groups (NH2) was effective for controlling the redox potential and binding affinities of the DPPZ-type osmium complex. The [Os(DA-bpy)2DPPZ]2+ complex (DA-bpy; 4,4′-diamino2,2′-bipyridine) had a lower half-wave potential (E1/2) of 147 mV (vs Ag|AgCl) and higher binding affinity with DNA (binding constant, K ) 3.1 × 107 M-1) than those of other complexes. With a single-stranded DNA immobilized gold electrode, the hybridization signal (∆I) of the [Os(DAbpy)2DPPZ]2+ complex was linear in the concentration range of 1.0 pg mL-1-0.12 µg mL-1 for the targeted DNA with a regression coefficient of 0.999. The detection limit was 0.1 pg mL1. The 400-bp yAL3 gene was also detected with good sensitivity and selectivity using the [Os(DAbpy)2DPPZ]2+ complex. DNA electrochemical sensors have been reported by many investigators.1-5 Electrochemical detections have been used to monitor DNA hybridization due to the high sensitivity, small dimensions, low cost, and compatibility with microfabrication technology.6 Most DNA electrochemical sensors can be classified as direct and the indirect types for monitoring DNA hybridization. With a typical direct-type sensor, Wang and co-workers reported direct monitoring of guanine oxidation in targeted DNA by potentiometric stripping analysis (PSA)7 or conducting polymer films.8 On the other hand, indirect-type monitoring of DNA hybridization uses electroactive molecules such as cationic metal com* Corresponding author. Current address: Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohokuku, Yokohama, Kanagawa 223-8522 Japan; (phone, fax) +81-45-566-1566; (e-mail) [email protected]. † Department of Chemical Science and Technology, Faculty of Engineering. ‡ Faculty of Pharmaceutical Sciences. (1) Hashimoto, K.; Ito, K.; Ishimori, Y. Anal. Chem. 1994, 66, 3830-3833. (2) Millan, K. M.; Mikkelsen, S. R. Anal. Chem. 1993, 65, 2317-2323. (3) Wang, J.; Rivas, G.; Cai, X.; Palecek, E.; Nielsen, P.; Shiraishi, H.; Dontha, N.; Luo, D.; Parrado, C.; Chicharro, M.; Farias, P.; Valera, F. S.; Grant, D. H.; Ozsoz, M.; Flair, M. N. Anal. Chim. Acta 1997, 347, 1-8. (4) Wang, J.; Cai, X.; Rivas, G.; Shiraishi, H.; Farias, P.; Dontha, N. Anal. Chem., 1996, 68, 2629-2634. (5) Kelly, S. O.; Boon, E. M.; Barton, J. K.; Jackson, N. M.; Hill, M. G.Nucleic Acids Res. 1999, 27, 4830-4837. (6) Wang, J.; Pumera, M.; Chatrathi, M. P.; Escarpa, A.; Korad, R.; Griebel, A.; Dorner, W.; Lowe, H. Electrophoresis 2002, 23, 596-601.

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plexes,3 intercalating organic compounds,2,9,10 and anticancer drugs.11 The electroactive molecules interact in different ways with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). The electrochemical response increases with the specific interaction of the electroactive molecules and the DNA hybrid. The use of transition metal complexes is a good method for electrochemical DNA probing because of advantages such as reversibility of the redox reaction, chemical stability, and simple functionalization. Some transition metal complexes as such ([Co(bpy)3]3+ (bpy ) 2,2′-bipyridine),12 [Co(phen)3]3+ (phen ) 1,10phenanthroline),13 [Os(bpy)3]3+,14 [Ru(bpy)3]3+,15 and [Os(5,6dmphen)3]2+ (5,6-dmphen ) 5,6-dimethyl-1,10-phenanthroline)16) have been applied to the electrochemical probe. However, these metal complexes have a low ability and sensitivity to recognize DNA hybridization because of the external binding mode of the DNA duplex. These complexes are bound by electrostatic interaction to the sugar-phosphate backbone of DNA or by hydrophobic interaction along the groove of DNA.17 Therefore, their binding modes result in relatively low binding affinities (e.g., binding constant, K ) 9.4 × 103 M-1 for [Co(bpy)3]2+/3+).18 The binding affinities of metal complexes with DNA are generally in the following order: intercalation (K > 106 M-1) > hydrophobic interaction (K >105 M-1) > electrostatic interaction (K > 103 M-1).18,19 Friedman et al.19 and Stoeffler et al.20 reported (7) Wang, J.; Rivas, G.; Fernandes, J. R.; Paz, J. L. L.; Jiang, M.; Waymire, R. Anal. Chim. Acta 1998, 375, 197-203. (8) Wang, J.; Jiang, M.; Fortes, A.; Mukherjee, B. Anal. Chim. Acta 1999, 402, 7-12. (9) Kelley, S. O.; Barton, J. K.; Jackson, N. M.; Hill, M. G. Bioconjugate Chem. 1997, 8, 31-37. (10) Boon, E. M.; Ceres, D. M.; Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2000, 18, 1096-1100. (11) Marrazza, G.; Chianella, I.; Mascini, M. Anal. Chim. Acta 1999, 387, 297307. (12) Steel, A. B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70, 4670-4677. (13) 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. (14) Mishima, Y.; Motonaka, J.; Ikeda, S. Anal. Chim. Acta, 1997, 345, 45-50. (15) Sistare, M. F.; Codden, S. J.; Heimlich, G.; Thorp, H. H. J. Am. Chem. Soc. 2000, 122, 4742-4749. (16) Maruyama, K.; Motonaka, J.; Mishima, Y.; Matsuzaki, Y.; Nakabayashi, I.; Nakabayashi, Y. Sens. Actuators, B 2001, 76, 215-219. (17) Dandliker, P. J.; Nunez, M. E.; Barton, J. K. Biochemistry 1998, 37, 64916502. (18) Carter, M. T.; Rodriguez, M.; Bard, A. J. J. Am. Chem. Soc. 1989, 111, 8901-8911. (19) Friedman, A. E.; Chambron, J. C.; Sauvage, J. P.; Turro, N. J.; Barton, J. K. J. Am. Chem. Soc. 1990, 112, 4960-4962. 10.1021/ac011148j CCC: $22.00

© 2002 American Chemical Society Published on Web 06/20/2002

Figure 1. Structural formula of [Os(DA-bpy)2DPPZ]2+.

the use of [Ru(bpy)2DPPZ]2+/3+ and [Os(bpy)2DPPZ]2+/3+ complexes as luminesence probes, where DPPZ ) dipyrido[3,2-a:2′,3′c]phenazine. These complexes show higher binding affinities (e.g., a binding constant of 4.0 × 106 M-1 for [Os(bpy)2DPPZ]2+/3+) than other metal complexes because the DPPZ ligand can intercalate between the base pairs of a DNA helix. These findings suggest that the metal complex as an electrochemical probe is desirable to intercalate between base pairs of DNA for precise detection of DNA hybridization. In particular, application of a DPPZ-type metal complex as an electrochemical probe should increase the detection limits in targeted DNA determinations because the DPPZ-type metal complex binds a larger amount per DNA sequence than an external binding-type metal complex (e.g., [Co(bpy)3]2+/3+).18,21 However, a DPPZ-type metal complex is difficult to use as an electrochemical probe because the high redox potential often causes a serious problem on the electrode surface; i.e., it destroys the complementary DNA immobilized on the electrode surface (adenine and guanine oxidation.).22,23 This problem can be resolved by the introduction of an electrondonating ligand (DA-bpy) for controlling the negative direction of the redox potential of the DPPZ-type osmium complex. In this work, we propose a novel DPPZ-type osmium complex (Figure 1) as an electrochemical probe for targeted DNA. This complex was characterized electrochemically and then applied for targeted DNA and yAL3 gene detection using a ssDNA modified gold electrode. EXPERIMENTAL SECTION Reagents. The following chemicals were used: calf thymus DNA (CT-DNA) was from Sigma Chemical Co. (St. Louis, MO); A 20-mer deoxyoligonucleotide probe, having a mercaptohexyl group at the 5′-phosphate end (HS-5′pdTGC AGT TCC CGG TGG CTG ATC-3′), targeted oligonucleotide (5′-GAT CAG CCA CCG CAA CTG CA-3′), 400-bp yAL3 gene, and mismatched oligonucleotide (mDNA) were from Nisshinbo Co. (Tokyo, Japan; (NH4)2OsCl6, bpy, and 4,4′-dimethyl-2,2′-bipyridine (DM-bpy) were from (20) Stoeffler, H. D.; Thornton, N. B.; Temkin, S. K.; Schanze, K. S. J. Am. Chem. Soc. 1995, 117, 7119-7128. (21) Welch, T. W.; Corbett, A. H.; Thorp, H. H. J. Phys. Chem. 1995, 99, 1175711763. (22) Pace, G. F.; Dryhurst, G. J. Electrochem. Soc. 1970, 117, 1259-1264. (23) Palecek, E. Bioelectrochem. Bioenerg. 1986, 15, 275-279.

Aldrich (St. Louis, MO). CT-DNA,24 4,4′-diamino-2,2′-bipyridine (DA-bpy),25 4,4′-dicarboxyl-2,2′-bipyridine (DC-bpy),26 and DPPZ27 were prepared and purified according to a reported procedure. DNA concentrations were determined spectrophotometrically in a UV-160A spectrophotometer (Shimadzu Co., Tokyo, Japan). All solutions were prepared using ultrapure water (18 Ω cm). Synthesis of Osmium Complexes. Osmium complexes and DPPZ were prepared according to published methods28,29 with some modification. Detailed procedures are given below for [Os(DA-bpy)2Cl2] and [Os(DA-bpy)2DPPZ]Cl2. (1) [Os(DA-bpy)2Cl2]. The (NH4)2OsCl6 salt (0.2 g, 0.46 mmol) was dissolved in 10 mL of ethylene glycol with 2 molar equiv of DA-bpy (0.17 g, 0.92 mmol). The solution was refluxed under argon for 45 min. After cooling to room temperature, 5 mL of aqueous sodium dithionite (0.87 g, 1.0 mol) was added to the reaction mixture to reduce Os(III) to Os(II). The precipitate formed was isolated by filtration, washed with water and a large amount of diethyl ether, and dried under vacuum for 1 day. (2) [Os(DA-bpy)2DPPZ]Cl2. The bis complex of [Os(DAbpy)2Cl2] (0.20 g, 0.33 mmol) was dissolved in 10 mL of ethylene glycol, together with 1.2 molar eqiv of DPPZ. The solution was refluxed at 180 °C under nitrogen for 4 h and then cooled to room temperature. The mixture was added to excess NH4PF6 to give a dark brown precipitate. The crude product was filtered off, washed with water and diethyl ether, dried, and then purified by column chromatography on silica gel with acetonitrile + toluene (1:1) as eluent. After evaporation, the precipitate was dissolved in acetonitrile + water (1:1, v/v) and changed to the dichloride salt by anion exchange chromatography on Sephadex QAE-25. After evaporation, the precipitate was dissolved in a small volume of anhydrous ethanol and filtered. The solvent was removed, and then the complex was recrystallized from ethanol + ether (yield, 20.3%). Anal. Calcd for C38H34N12OsCl2‚2H2O: C, 42.56; H, 4.08; N, 15.19. Found: C, 42.61;H, 4.10;N, 15.50. Apparatus. All electrochemical experiments were carried out using a BAS-CV50W electrochemical analyzer (Bioanalytical Systems) with a traditional three-electrode cell: a | Ag | AgCl | KCl (saturated) reference electrode (Bioanalytical Systems) and a platinum wire counter electrode. A plastic formed carbon (PFC) electrode (inner diameter 3.0 mm; outer diameter 6.0 mm) (Bioanalytical Systems) and a gold electrode (inner diameter 1.6 mm; outer diameter 6.0 mm) (Bioanalytical Systems) were used as working electrodes. Before use, the working electrode was polished mechanically with 0.05-µm alumina slurry to a mirror finish and then cleaned by sonication in water. The individual electrode surface conditions was confirmed with a cyclic voltammogram in 1.1 mmol dm-3 potassium hexacyanoferrate(III) solution with 0.1 mol dm-3 KCl. A thermostat (Coolnics CTE22W circulator, Yamato-Komatsu) was used for thermostatic control of sample solutions. Electrochemical titrations of osmium (24) Marmur, J. Mol. Biol. 1961, 3, 208-213. (25) Maerker, G.; Case, F. H. J. Am. Chem. Soc, 1958, 80, 2745-2748. (26) Anderson, S.; Constable, E. C.; Seddon, K. R.; Turp, J. E.; Baggott, J. E.; Pilling, M. J. J. Chem. Soc., Dalton Trans. 1985, 2247-2253. (27) Dickeson, J. E.; Summers, L. A. Aust. J. Chem. 1970, 23, 1023-1028. (28) Kober, E. M.; Caspar, J. V.; Sullivan, B. P.; Meyer, T. J. Inorg. Chem. 1988, 27, 4587. (29) Kuwana, T.; Bublitz, D. E.; Hoh, G. J. Am. Chem. Soc. 1960, 82, 58115816.

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Table 1. Electrochemical Properties of Osmium Complexesa osmium complex

E1/2/mV vs Ag|AgCl

∆E/ mV

106Dapp/ cm2 s-1

[Os(bpy)3]3+ [Os(bpy)2DPPZ]2+ [Os(DA-bpy)2DPPZ]2+ [Os(DM-bpy)2DPPZ]2+ [Os(DC-bpy)2DPPZ]2+

654 719 147 586 483

63.0 64.2 65.0 64.3 98.0

7.60 4.89 1.30 1.84 0.72

a Redox potentials (E 1/2/mV vs Ag|AgCl) were determined in TrisHCl buffer (pH 7.76) with 50 mmol dm-3 NaCl as the supporting electrolyte, Scan rate, 50 mV s-1. ∆E is the difference between Eox and Ered in Tris-HCl buffer (pH 7.76) with 0.1 mol dm-3 NaCl.

complexes with CT-DNA were performed as previously described.30 Preparation of the DNA Biosensor. DNA-immobilized electrodes were prepared according to published methods31 with some modifications. A polished gold electrode was reversibly cycled in 0.5 mol dm-3 H2SO4 solution from 0 to 1.7 V (E/mV vs Ag|AgCl) until the ideal redox wave of gold in H2SO4 was observed.32 The electrode was immersed in a solution of ssDNA with a mercaptohexyl group in 1.0 mol dm-3 phosphate buffer for 4 h at 25 °C and washed with purified water to remove adsorbed probes. Then the DNA-modified electrode (ssDNA electrode) was immersed in 1.0 mmol dm-3 6-mercapto-1-hexanol (MCH) solution for 1 h because of masking of the unmodified gold site. The electrode was washed with purified water and stored in Tris-HCl buffer containing 1 mmol dm-3 EDTA at 4 °C. Hybridization on the ssDNA Electrode and Detection of dsDNA. The ssDNA electrode was immersed in a hybridization buffer solution (Tris buffer (pH 8.5) containing 1.0 mol dm-3 sodium chloride and targeted oligonucleotide), and the solution was heated to 80 °C. Then it was cooled slowly to 37 °C with shaking for 1.5 h. Specific hybrids (dsDNA) were formed on the ssDNA electrode. The dsDNA electrode was immersed in 5.0 mmol dm-3 osmium complex solution in 10 mmol dm-3 Tris-HCl, 10 mmol dm-3 sodium chloride for 5 min and then washed with phosphate buffer to remove the nonspecifically adsorbed complexes. The anodic current, derived from the interaction between the osmium complex and the specific hybrids was determined by differential pulse voltammetry (DPV). RESULTS AND DISCUSSION Electrochemical and DNA-Binding Properties of Osmium Complexes in Solution. Table 1 shows electrochemical data on 1.0 mmol dm-3 osmium complexes in Tris-HCl buffer. The halfwave potential (E1/2) and ∆E values were determined by cyclic voltammetry (CV) at 50 mV s-1, and the apparent diffusion coefficient (Dapp) value was calculated by chronocoulometry (CC). The E1/2 and ∆E values of complexes evaluated were stable with pH variation of test solutions, except for those of the DC-bpytype complex. When the osmium complexes, are coordinated with bpy ligands with electron-donating groups (NH2, CH3) they have significantly lower E1/2 values than the [Os(bpy)2DPPZ]2+ com(30) Johnston, D. H.; Thorp, H. H. J. Phys. Chem. 1996, 100, 13837-13843. (31) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916-8920 (32) Sabatani, E.; Rubinstein, I. J. Phys. Chem. 1987, 91, 6663-6669

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Figure 2. Cyclic voltammograms of 0.25 mmol dm-3 [Os(DA-bpy)2DPPZ]2+ . Sample in 10 mL of Tris-HCl buffer (pH 7.76) containing 0.1 mol dm-3 NaCl at 25 °C. Scan rate, 50 mV s-1.

plex, which has a decreased back-donation effect in which the electron of the nonbonding d orbital for osmium metal is drawn to the empty p orbital. As a result, the osmium complex is usually stable in the Os(II) state because of the bias of the electron density. The introduction of an electron-donating group to bpy inhibits the above mechanism, and the corresponding osmium complex is relatively destabilized in the Os(II) state. The E1/2 of osmium complex-coordinated bpy ligands with electron-donating groups resulted in a shift to the negative direction. The [Os(DA-bpy)2DPPZ]2+ complex had one pair of welldefined redox peaks (∆E ) 65 mV, E1/2 ) 147 mV vs Ag|AgCl) in Tris-HCl buffer (Figure 2). The ∆E values of the [Os(DAbpy)2DPPZ]2+ complex increased slightly with an increase in the scan rate. The redox peak current was proportional to the square of the scan rate. These results suggest that mass transfer of the complex is under diffusion control and that the electrode reaction proceeds as a quasi-reversible one-electron reaction. Substitution of the DC-bpy ligand led to an increase in the ∆E value and a decrease in the Dapp value. The electrochemical properties (∆E ) 98 mV, Dapp ) 7.2 × 10-7 cm2 s-1) of the [Os(DCbpy)2DPPZ]2+ complex differed appreciably from those of the [Os(DA-bpy)2DPPZ]2+ complex despite the similar molecular structures of the two. The voltammetric response of [Os(DCbpy)2DPPZ]2+ as a function of pH was linear in the range of pH 1.5-9.0 with a slope of 25.7 mV/pH and a stable E1/2 value at pH >9.0. This behavior suggests that the complex has a comprehensive negative charge in pH 7.0 buffer. As a result, the electrostatic repulsion between the DC-bpy ligand and some functional groups (CO, COOH, OH, etc.) of the PFC electrode surface causes the high ∆E and low Dapp values. Evaluation of the binding constant (K) allows detailed determination of the binding mode of the osmium complexes. The normal pulse voltammetry technique (NPV) was applied for determination of the K values because the limiting current of NPV can precisely reflect the mass transfer between free metal complexes and metal complexes bound to CT-DNA.33 The limiting

Table 2. Binding Constants of Metal Complexesa metal complex

K/M-1

s

Kb/M-1

[Os(bpy)3]3+ [Os(bpy)2DPPZ]2+ [Os(DA-bpy)2DPPZ]2+ [Os(DM-bpy)2DPPZ]2+ [Os(DC-bpy)2DPPZ]2+

9.4 × 102 4.2 × 106 3.1 × 107 5.2 × 106 5.6 × 105

3.1 0.7 0.4 0.6 1.9

5.8 × 102 4.0 × 106 -

a Samples were in Tris-HCl buffer (pH 7.76) with 50 mmol dm-3 NaCl at 25 °C. a Reference 35.

current of NPV decreased significantly with addition of CT-DNA. In conditions of CT-DNA saturation, the limiting current decreased to 33.8% of that without CT-DNA. The theoretical equations have been reported by Thorp and co-workers24 to be as follows:

Xb ) {b - (b2 - 2K 2ct[DNA]/s)1/2}/2Kct

(1)

Xb )(I2 - Io2)/(Isat2 - Io2)

(2)

b ) 1 + Kct + K[DNA]/2s

(3)

where Xb is the molar binding fraction, I the observed current with addition of CT-DNA, Io the initial current observed before the addition of CT-DNA, Isat the current expected upon complete saturation of binding, K the binding constant, ct the total metal complex concentration, and [DNA] the concentration of CT-DNA. The parameter s is defined as the site size of base pairs with a molecule of osmium complex. The K and s values were obtained by curve fitting to the plot of the Xb value for the concentration of CT-DNA using eq 1. Table 2 summarizes the K and s values calculated by the nonlinear least-squares method. The binding constants of osmium complexes were in the order [Os(DA-bpy)2DPPZ]2+ > [Os(DMbpy)2DPPZ]2+ > [Os(bpy)2DPPZ]2+ > [Os(DC-bpy)2DPPZ]2+ . [Os(bpy)3]3+. The K value for the DPPZ-type osmium complexes are ∼1.0 × 104 times higher than that of [Os(bpy)3]2+. This large difference of K values reflects different binding modes. According to the work of Thorp and co-workers, a binding constant of about >106 M-1 indicates that the DNA-binding mode of the metal complex is by intercalation.34,35 This was confirmed experimentally by the hypochromism effect and red shifting of the DPPZ ligand on adsorption spectra at 372 nm34 and NPV.36 The highest K value (K ) 3.1 × 107 M-1) of the [Os(DA-bpy)2DPPZ]2+ complex suggests that introduction of DA-bpy, which is a hydrophilic group, has a significant influence on the affinity with CT-DNA. Barton and co-workers also reported that the [Rh(MGP)2Phi]2+ (MGP ) 4-(guanidylmethyl)-1,10-phenanthroline; Phi ) phenanthrenequinone diimine) complex, which contains pendant amino functionalities, has a high binding affinity with the DNA sequence due to hydrogen bonding.37,38 The high K value (33) Johnston, D. H.; Thorp, H. H. J. Phys. Chem. 1996, 100, 13837-13843. (34) Welch, T. W.; Corbett, A. H.; Thorp, H. H. J. Phys. Chem. 1995, 99, 9, 11757-11763. (35) Welch, T. W.; Corbett, A. H.; Thorp, H. H. J. Phys. Chem. 1996, 100, 1382913836. (36) Kalsbeck, W. A.; Thorp, H. H. J. Am. Chem. Soc. 1993, 115, 7146-7151. (37) Terbrueggen, R. H.; Johann, T. W.; Barton, J. K. Inorg. Chem. 1998, 37, 6874-6883.

Figure 3. Differential pulse voltammograms of [Os(DA-bpy)2DPPZ]2+ on the (a) dsDNA and (b) ssDNA electrode. Sample in 10 mL of TrisHCl buffer (pH 7.76) at 25 °C. The scan rate, pulse amplitude, and pulse period were 50 mV s-1, 50 mV and 200 ms, respectively. Table 3. Voltammetric Behaviors of Osmium Complexes on the ssDNA- and dsDNA-Modified Electrodesa osmium complex

IssDNAb/ nA

IdsDNAb/ Aa

∆Ic/ nA

EdsDNAd/ mV

[Os(bpy)3]3+ [Os(bpy)2DPPZ]2+ [Os(DA-bpy)2DPPZ]2+ [Os(DM-bpy)2DPPZ]2+ [Os(DC-bpy)2DPPZ]2+

1.22 89 136 102 96

2.82 260 343 320 220

1.60 180 207 218 124

620 701 157 546 460

a Samples were in Tris-HCl buffer (pH 7.76) at 25 °C. The concentration of targeted DNA was 60 ng mL-1. bIssDNA and IdsDNA indicate the anodic current of DPV on the ssDNA and dsDNA electrode, respectively. c∆I is IdsDNA - IssDNA. dEdsDNA are anodic peak potentials (E/mV vs Ag/AgCl) of DPV on dsDNA electrodes.

of the [Os(DA-bpy)2DPPZ]2+ complex may be due to hydrogen bonding between the amino group of the bpy ligand and CT-DNA. DNA-Binding Properties of Osmium Complexes on the Electrode Surface. The voltammetric behaviors of osmium complexes on the ssDNA- or dsDNA-immobilized gold electrode were studied. Figure 3 and Table 3 show the response current (∆I ) IdsDNA - IssDNA) measured using DPV in Tris-HCl buffer (pH 7.76) with dsDNA (after hybridization) and an ssDNAmodified electrode (before hybridization) of the electrode surface. With DPPZ-type osmium complexes, the response anodic current was significantly enhanced on the dsDNA electrode. The ∆I values of DPPZ-type osmium complexes were about 77-136 times higher than that of the [Os(bpy)3]3+ complex. This behavior suggests that DPPZ-type osmium complexes also intercalate between base pairs of dsDNA hybridization on the electrode surface. With the [Os(DA-bpy)2DPPZ]2+ complex, the IssDNA value (136 nA) suggests that the complex has higher nonspecific interaction with ssDNA than the other osmium complexes. Nevertheless, the (38) Terbrueggen, R. H.; Barton, J. K. Biochemistry 1995, 34, 8227-8234.

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Figure 4. Effect of hybridization time. Sample in 10 mL of Tris-HCl buffer (pH 7.76) at 25 °C. The concentration of targeted DNA was 60 ng mL-1.

lowest EdsDNA value and the high sensitivity of the [Os(DAbpy)2DPPZ]2+ complex are advantageous for targeted DNA detection. As a result, the [Os(DA-bpy)2DPPZ]2+ complex was applied to an electrochemical probe because of an applicable redox potential (157 mV vs Ag|AgCl) and a high sensitivity (∆I ) 343 nA) on the electrode surface. Effect of Hybridization Time and Concentration of the [Os(DA-bpy)2DPPZ]2+ Complex. For precise determination of the targeted DNA, highly efficient hybridization on the ssDNAimmobilized electrode was required. The efficiency of hybridization is dependent on the ionic strength and the hybridization time. The effects of ionic strength and temperature have been discussed previously.22,23 Figure 4 shows the anodic peak current of 0.8 mmol dm-3 of the [Os(DA-bpy)2DPPZ]2+ complex at various hybridization times. As hybridization proceeded, the anodic peak current increased due to increase in the local concentration on the dsDNAimmobilized electrode surface by intercalation of the [Os(DAbpy)2DPPZ]2+ complex with dsDNA. The hybridization reaction was completed after 60 min. Figure 5 shows the effect of [Os(DA-bpy)2DPPZ]2+ concentration on the current response of DNA hybridization on the electrode. As can be seen, when the concentration is high enough, a plateau or maximum value of the peak current was obtained. From the results, 0.8 mmol dm-3 [Os(DA-bpy)2DPPZ]2+ complex was chosen as the optimum concentration of an electrochemical probe for determination of the targeted DNA on the ssDNA immobilized electrode. Calibration Curve for the Targeted DNA. Figure 6 shows typical calibration curve of the targeted DNA using DPV with the [Os(DA-bpy)2DPPZ]2+ complex as the electrochemical probe. The hybridization signal (∆I) of [Os(DA-bpy)2DPPZ]2+ increases linearly with concentration in the range from 1.0 pg mL-1 to 0.12 µg mL-1. This plot could be represented by the equation Y ) 0.00324X + 0.00382, where Y is the response current (in ∆A) and X is the targeted DNA concentration (in ng mL-1). The relative standard deviation (RSD) was less than 5.0%. The detection limit was 0.1 pg mL-1 (S/N ) 3.0). This high sensitivity may be 3702 Analytical Chemistry, Vol. 74, No. 15, August 1, 2002

Figure 5. Effect of concentration of [Os(DA-bpy)2DPPZ]2+. Sample in 10 mL of Tris-HCl buffer (pH 7.76) at 25 °C. The concentration of targeted DNA was 60 ng mL-1.

Figure 6. Calibration curve for concentration of targeted DNA. Sample in 10 mL of Tris-HCl buffer (pH 7.76) at 25 °C. Equation: Y ) 0.00324X + 0.00382.

reflected by not only the high binding affinity of the [Os(DAbpy)2DPPZ]2+ complex but also the high redox activity of the osmium complex as a mediator for the enzyme sensor.39 Regeneration of ssDNA and removal of the [Os(DA-bpy)2DPPZ]2+ complex on the electrode surface were performed by rinsing the electrode in hot hybridization buffer (100 °C) for 10 min. The reproducibility was also tested by repetitive hybridization with targeted DNA and intercalation of the [Os(DA-bpy)2DPPZ]2+ complex. As a result, no significant deterioration of the response current was observed in 10 regeneration-hybridization-intercalation cycles. The average response current was 343 ( 3 nA. The RSD foron 10 measurements was 2.1%. Gene Detection. The [Os(DA-bpy)2DPPZ]2+ complex as an electrochemical probe was applied for a specific gene (400-bp yAL3 (39) Motonaka, J.; M Kamizasa, J. Electroanal. Chem. 1994, 373, 75-81.

Table 4. Recognition of Samplesa sample

concn/ng mL-1

∆i/nA

targeted DNA 400-bp yAL3 geneb mDNAc

60 60 60

207 220

a Samples were in Tris-HCl buffer (pH 7.76) at 25 °C. bThe 400-bp y AL3 gene contains a complementary sequence to the immobilized ssDNA on the electrode. cmDNA is noncomplementary to immobilized ssDNA on the electrode. Values are averages for four determinations.

gene) and mDNA detection. The 400-bp yAL3 gene (60 ng mL-1) contained a sequence complementary to the immobilized ssDNA on the electrode and mDNA was not a complementary sequence. These sequences were denatured in a boiling-water bath for 5 min and then rapidly cooled in an ice bath for 5 min. Then the hybridization of 400-bp yAL3 gene-ssDNA and intercalation of the [Os(DA-bpy)2DPPZ]2+ complex were carried out under the above optimum conditions. Table 4 summarizes the response current of the [Os(DA-bpy)2DPPZ]2+ complex interacting with targeted DNA, the yAL3 gene and mDNA, respectively, on the ssDNA-immobilized electrode. On the detection of 400-bp yAL3 gene, the [Os(DA-bpy)2DPPZ]2+ complex also acted as an electrochemical probe with good sensitivity, although the ∆I value of the 400-bp yAL3 gene was slightly higher than that of the targeted DNA. On the other hand, no ∆I value of mDNA was observed when the ssDNA-immobilized electrode reacted with the

mDNA. These results suggest that the 400-bp yAL3 gene can be detected with good selectivity using the [Os(DA-bpy)2DPPZ]2+ complex and the ssDNA immobilized electrode. CONCLUSIONS The metal complex as a DNA probe is desirable to intercalate between base pairs of DNA for precise detection of the targeted DNA, though a large number of DNA-binding molecules, which have high affinity, interact to DNA hybridization without the need for intercalation. In addition, introduction of the bpy ligand with an amino group (DA-bpy) makes it possible to obtain an applicable redox potential and high DNA-binding affinity. The binding constants of osmium complexes were in the order [Os(DAbpy)2DPPZ]2+ > [Os(DM-bpy)2DPPZ]2+ > [Os(bpy)2DPPZ]2+ > [Os(bpy)2DPPZ]2+ > [Os(DC-bpy)2DPPZ]2+ . [Os(bpy)3]3+. The [Os(DA-bpy)2DPPZ]2+ complex has the lowest redox potential (E1/2 ) 147 mV vs Ag/AgCl) due to decrease of the back-donation effect and the highest DNA-binding properties (K ) 3.1 × 107 M-1) by intercalation and hydrogen bonding between the amino group of the bpy ligand and CT-DNA. We succeeded in detecting targeted DNA and the gene using [Os(DA-bpy)2DPPZ]2+ as a novel electrochemical probe on the ssDNA-modified electrode.

Received for review November 5, 2001. Accepted May 1, 2002. AC011148J

Analytical Chemistry, Vol. 74, No. 15, August 1, 2002

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