Genosensor Based on a Platinum(II) Complex as Electrocatalytic Label

Anal. Chem. 2005, 77, 2868-2874

Genosensor Based on a Platinum(II) Complex as Electrocatalytic Label David Hernańdez-Santos, Marıá Begonã Gonza´lez-Garcıá, and Agustıń Costa-Garcıá*

Departamento de Quı´mica Fı´sica y Analı´tica, Facultad de Quı´mica, Universidad de Oviedo. 33006 Oviedo, Asturias, Spain

Voltammetric genosensors on streptavidin-modified screenprinted carbon electrodes (SPCEs) for the detection of virulence nucleic acid determinants of pneumolysin (ply) and autolysin (lytA) genes, exclusively present on the genome of the human pathogen Streptococcus pneumoniae, were described. The oligonucleotide probes were immobilized on electrochemically pretreated SPCEs through the streptavidin/biotin reaction. After that, the hybridization reaction was carried out with labeled complementary targets on the electrode surface. The ply and lytA targets were labeled using the universal linkage system, which consists of the use of a platinum(II) complex that acts as coupling agent between targets and a, usually fluorescent, molecule label. In this case, the platinum(II) complex acts as a label itself because the analytical signal is achieved by measuring chronoamperometrically the current generated by the hydrogen evolution catalyzed by platinum. In nonstringent experimental conditions, these genosensors can detect 24.5 fmol of 20-mer oligonucleotide target and discriminate between a complementary oligo and an oligo with a three-base mismatch. In presence of 25% formamide in the hybridization buffer, a single-base mismatch on the oligonucleotide target can be detected. Electrochemical assays of nucleic acids have received considerable attention in connection with the detection of DNA hybridization.1-4 The high sensitivity of such assays coupled to their compatibility with modern microfabrication technologies, low cost, minimal power requirements, and independence of sample turbidity or optical pathway makes them excellent candidates for DNA diagnostics. In addition, electrochemistry offers innovative routes for interfacing the nucleic acid recognition system with a signal-generating element and for amplifying electrical signals. In most of these electrochemical DNA assays, the hybridization reaction and detection is carried out on the electrode surface (genosensor devices).5-33 However, in some cases, the electrode * To whom correspondence should be addressed. Fax: +34 985 103 125. E-mail: [email protected]. (1) Pividori, M. I.; Merkocy, A.; Alegret, S. Biosens. Bioelectron. 2000, 15, 291303. (2) Wang, J. Anal. Chim. Acta 2002, 469, 63-71. (3) Lucarelli, F.; Marrazza, G.; Turner, A. P. F.; Mascini, M. Biosens. Bioelectron. 2004, 19, 515-530. (4) Gooding, J. J. Electroanalysis 2002, 14 (17), 1149-1156. (5) Wang, J.; Rivas, G.; Cai, S. Electroanalysis 1997, 9 (5), 395-398.

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only acts as a detector of the hybridization event,34-45 which occurs in a separate step, either because it takes place in a microwell34,36 or because the hybridization reaction occurs on the surface of magnetic beads, which are separated from the hybridization solution and then redissolved.37-39 In other cases two different surfaces are used, one of them to carry out the hybridization event and the other one to carry out the detection (double surface technique).40 (6) Wang, J.; Cai, X.; Rivas, G.; Shiraishi, H. Anal. Chim. Acta 1996, 326, 141147. (7) Millan, K. M.; Mikkelsen, S. R. Anal. Chem. 1993, 65, 2317-2323. (8) Wang, J.; Fernandes J. R.; Kubota, L. T. Anal. Chem. 1998, 70, 36993702. (9) Maruyama, K.; Mishima, Y.; Minagawa, K.; Motonaka, J. Anal. Chem. 2002, 74, 3698-3703. (10) Marrazza, G.; Chianella, I.; Mascini, M. Anal. Chim. Acta 1999, 387, 297307. (11) Marrazza, G.; Chianella, I.; Mascini, M. Biosens. Bioelectron. 1999, 14, 4351. (12) Hashimoto, K.; Ito, K.; Ishimori, Y. Anal. Chim. Acta 1994, 286, 219-224. (13) Wang, J.; Kawde, A.-N. Anal. Chim. Acta 2001, 431, 219-224. (14) Hashimoto, K.; Ito, K.; Ishimori, Y. Anal. Chem. 1994, 66, 3830-3833. (15) Kara, P.; Meric, B.; Zeytinoglu, A.; Ozsoz, M. Anal. Chim. Acta 2004, 518, 69-76. (16) Takenaka, S.; Uto, Y.; Saita, H.; Yokoyama, M.; Kondo, H.; Wilson, W. D. Chem. Commun. 1998, 1111-1112. (17) Lucarelli, F.; Marrazza, G.; Palchetti, I.; Cesaretti, S.; Mascini, M. Anal. Chim. Acta 2002, 469, 93-99. (18) Wang, J.; Rivas, G.; Fernandes, J. R.; Lopez-Paz, J. L.; Jiang, M.; Waymire, R. Anal. Chim. Acta 1998, 375, 197-203. (19) Wang, J.; Gru ¨ ndler, P.; Flechsig, G.-U.; Jasinski, M.; Rivas, G.; Sahlin, E.; Lopez-Paz, J. L. Anal. Chem. 2000, 72, 3752-3756. (20) Erdem, A.; Kerman, K.; Meric, B.; Akarca, U. S.; Ozsoz, M. Anal. Chim. Acta 2000, 422, 139-149. (21) Babkina, S. S.; Ulakhovich, N. A.; Medyantseva, E. P.; Zyavkina, Y. I. J. Anal. Chem. 1999, 54 (11), 1206-1211. (22) Babkina, S. S.; Ulakhovich, N. A.; Zyavkina, Y. I. Anal. Chim. Acta 2004, 502, 23-30. (23) Ihara, T.; Nakayama, M.; Murata, M.; Nakano, K.; Maeda, M. Chem. Commun. 1997, 17, 1609-1610. (24) Zhu, N.; Cai, H.; He, P.; Fang, Y. Anal. Chim. Acta 2003, 481, 181-189. (25) Dequaire, M.; Heller, A. Anal. Chem. 2002, 74, 4370-4377. (26) Zhang, Y.; Kim, H.-H.; Mano, N.; Dequaire, M.; Heller, A. Anal. Bioanal. Chem. 2002, 374, 1050-1055. (27) de Lumley-Woodyear, T.; Campbell, C. N.; Heller, A. J. Am. Chem. Soc. 1996, 118, 5504-5505. (28) Zhang, Y.; Kim, H.-H.; Heller, A. Anal. Chem. 2003, 75, 3267-3269. (29) Campbell, C. N.; Gal, D.; Cristler, N.; Banditrat, C.; Heller, A. Anal. Chem. 2002, 74, 158-162. (30) Williams, E.; Pividori, M. I.; Merkoci, A.; Forster, R. J.; Alegret, S. Biosens. Bioelectron. 2003, 19, 165-175. (31) Pividori, M. I.; Merkoci, A.; Alegret, S. Biosens. Bioelectron. 2003, 19, 473484. (32) Pividori, M. I.; Merkoci, A.; Barbe´, J.; Alegret, S. Electroanalysis 2003, 15 (23-24), 1815-1823. (33) Hernańdez-Santos, D.; Dıáz-Gonza´lez, M.; Gonza´lez-Garcıá, M. B.; CostaGarcıá, A. Anal. Chem. 2004, 76, 6887-6893. 10.1021/ac048091w CCC: $30.25

© 2005 American Chemical Society Published on Web 03/31/2005

In all these DNA assays, the hybridization event is converted to useful electrical signals. The base-pairing recognition event can be detected using unlabeled DNA in different ways. One is the variation of current signal of a redox indicator (i.e., cationic metal complexes,5-9 daunomycin,10-13 electrochemical dye,14 meldola blue,15 or naphthalene with two ferrocenes16) that has a different affinity for the resulting target/probe duplex, as compared to the affinity for the probe alone; another strategy is to measure the changes in the intrinsic nucleic acid electrochemical signals associated with the electroactivity of guanine or adenine.17-19 In other cases, the hybridization reaction is monitored through a molecule that binds to guanines of the DNA strands, using its electroactivity (i.e., methylene blue)20 or using a catalytic property of this molecule (i.e., platinum complexes21,22). Other approaches use labeled DNA with labels such as metal particles,34-38 ferrocene,23 Co(bpy)3+-silica nanoparticles,24 Osbipy complex,40,45 or enzyme tags such as horseradish peroxidase25-32,41,42 or alkaline phosphatase33,43-45 that can be captured following the hybridization. These kinds of labels have led to the most sensitive electrochemical detection of DNA. A wide variety of electrodes have been used as detectors or supports/detectors (genosensors) in the electrochemical DNA assays, including carbon paste electrodes,6,18-20 glassy carbon electrodes,7,24,27,28 graphite electrodes,12,13,15,29,40,45 carbon composites,8,30-32,42 gold electrodes,9,14,16,23 mercury electrodes, or Hg film on silver electrodes.21,22 Moreover, screen-printing microfabrication technology has been used and different DNA assays have been carried out on screen-printed carbon electrodes (SPCEs), as either genosensors5,10,11,17,25,26,33 or detectors.34-38,43,44 This technology allows the mass production of reproducible yet inexpensive and mechanically robust strip solid electrodes. Other important features that these electrodes exhibit are related to the miniaturization of the corresponding device along with their ease of handling and manipulation in a disposable manner. In the case of genosensor devices, the crucial step is the immobilization of single-stranded DNA (ssDNA) probes onto the electrode surface. The immobilization method will determine the sensitivity and reproducibility of the genosensor. General strategies for the immobilization of ssDNA probes on solid surfaces include adsorption at controlled potential onto pretreated carbonbased surfaces,5,6,10,11,14,18-21 or physical adsorption on carbon surfaces or membranes coupled to carbon surfaces,13,25,32,33,42,43 and bulk modification of carbon paste.8 However, in these cases, the (34) Authier, L.; Grossiord, C.; Brossier, P. Anal. Chem. 2001, 73, 4450-4456. (35) Wang, J.; Xu, D.; Kawde, A.-N.; Polsky, R. Anal. Chem. 2001, 73, 55765581. (36) Ruffien, A.; Dequaire, M.; Brossier, P. Chem. Commun. 2003, 912-913. (37) Wang, J.; Polsky, R.; Xu, D. Langmuir 2001, 17, 5739-5741. (38) Wang, J.; Xu, D.; Polsky, R. J. Am. Chem. Soc. 2002, 124, 4208-4209. (39) Wang, J.; Liu, G.; Merkoci, A. Anal. Chim. Acta 2003, 482, 149-155. (40) Palecek, E.; Kizek, R.; Havran, L.; Billova, S.; Fojta, M. Anal. Chim. Acta 2002, 469, 73-83. (41) Pividori, M. I.; Merkoci, A.; Alegret, S. Analyst 2001, 126, 1551-1557. (42) Pividori, M. I.; Merkoci, A.; Alegret, S. Biosens. Bioelectron. 2001, 16, 11331142. (43) Wang, J.; Xu, D.; Erdem, A.; Polsky, R.; Salazar, M. A. Talanta 2002, 56, 931-938. (44) Bagel, O.; Degrand, C.; Limoges, B.; Joannes, M.; Azek, F.; Brossier, P. Electroanalysis 2000, 12 (18), 1447-1452. (45) Fojta, M.; Havran, L.; Vojtiskova, M.; Palecek, E. J. Am. Chem. Soc. 2004, 126, 6532-6533.

immobilized ssDNA probes are not totally accessible for hybridization, resulting in poor hybridization efficiency. Other systems, such as covalent attachment to activated surfaces or to polymercoated surfaces,7,26-29 the use of self-assembled monolayers onto gold surfaces,9,15,17,24 or the use of avidin/biotin interaction to attach biotinylated probes on the electrode surface,11,30,31 allow one to obtain a sensing phase with more strands of DNA than by direct adsorption on the electrode. Moreover, the ssDNA probes are oriented in the genosensing phase, leaving the probes accessible for the reaction with their complementary targets. This work outlines the development of a genosensor on screenprinted carbon electrodes for the identification of nucleic acid determinants exclusively present on the genome of the pathogen Streptococcus pneumoniae. Orientation of the strands in the sensing phase is achieved by modifying the surface of the electrode with streptavidin by physical adsorption followed by the immobilization of biotinylated oligo probes. The physical adsorption of streptavidin has been performed as described in a previous work46 at a constant temperature below the room temperature. Moreover, the electrode surface must be previously electrochemically pretreated at an anodic current in acidic medium to improve its adsorptive properties. The biotinylated oligo nucleic acid probes used in this work target the pneumolysin (ply) and autolysin (lytA) genes. These targets are randomly labeled with the universal linkage system (ULS).47 This labeling system consists of the use of a platinum(II) complex that acts as a coupling agent between DNA strands and a, usually fluorescent, label molecule. This platinum complex is a monofunctional derivate of cisplatin (a potent anticancer agent used in the treatment of a variety of tumors) that binds to DNA at the N7 position of guanine with release of one Cl- ion per molecule of the complex. There are some works in the literature21,22,48,49 where this binding between platinum complexes and DNA strands is studied and used to determine platinum complexes and denature DNA or autoantibodies against DNA. Our group has already used this kind of labeling to attach fluorescein to ply and lytA targets.33 The electrochemical detection was achieved with an anti-fluorescein alkaline phosphatase (AP)labeled antibody and using 3-indoxyl phosphate (3-IP) as electrochemical substrate of AP. However, although these genosensors were stable and sensitive devices for the detection of specific nucleic acid fragments, the need for two additional steps to obtain the analytical signal resulted in a large time-consuming analysis. This fact can be avoided using the analytical signal obtained from the platinum(II) complex, through its catalytic properties toward hydrogen evolution. This catalytic property of platinum complexes has been used by some authors21,22 to determine cisplatin or other complexes but using mercury films on silver electrodes. In the presence of platinum on the electrode surface and fixing an adequate potential in acidic medium, the protons are catalytically reduced to hydrogen. The current generated by this catalytic reduction can be measured and increases with platinum concentration and consequently with labeled target concentration. Data presented here demonstrate the potential applicability of SPCE (46) Dıáz-Gonza´lez, M.; Hernańdez-Santos, D.; Gonza´lez-Garcıá, M. B.; CostaGarcıá, A. Talanta 2005, 65, 565-573. (47) Lempers, E. L. M.; Van der Berg, F. M.; Reedijk, J.; Bloemink, M. J. U.S. Patent 5,580,990, 1996. (48) Yan, F.; Sadik, O. A. J. Am. Chem. Soc. 2001, 123, 11335-11340. (49) K’Owino, I. O.; Agarwal, R.; Sadik, O. A. Langmuir 2003, 19, 3444-4350.

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Figure 1. Chemical structure of the monofunctional square-planar platinum(II) complex used in the ULS with (a) a BOC group and (b) a fluorescein label.

genosensors in diagnosis of a human infectious pulmonary disease. Moreover, these devices allow the detection of a singlebase mismatch on the targets if adequate experimental conditions are used. EXPERIMENTAL SECTION Materials. Tris(hydroxymethyl)aminomethane (Tris), bovine serum albumin fraction V (BSA), and streptavidin (molecular weight 60 000 g/mol) were purchased from Sigma (Madrid, Spain). (NH4)2[PtCl4] complex was purchased from Riedel-de Hae¨n (Germany) whereas BOC-ULS, another platinum complex, (Figure 1a) was kindly provided by Kreatech Biotechnology (Amsterdam, The Netherlands). Working solutions of streptavidin were made in 0.1 M Tris buffer, pH 7.2. BSA lyophilized powder was reconstituted in ultrapure water. The solutions of the two platinum complexes were prepared in an adequate concentration of HCl. 3′-Biotinylated 30-mer oligonucleotide probes with a 10-T spacer in the 3′-end and the complementary 20-mer labeled targets corresponding to lytA and ply genes, as well as the single-base and three-base mismatch ply targets were purchased from Isogen Life Science (Maarssen, The Netherlands). ULS-fluorescein (Figure 1b) labeling of these targets was carried out by Kreatech Biotechnology (Amsterdam, The Netherlands). This system binds to DNA at the N7 position of guanine resulting in the attachment of a label molecule to the DNA. The sequences of the oligonucleotide probes and their complementary targets as well as the singlebase and three-base mismatch ply oligonucleotides are shown in Table 1. Concentrated aliquots of all oligonucleotides were prepared in 0.1 M Tris buffer, pH 7.2, and stored at -20 °C. Working solutions of all biotinylated oligonucleotide probes were made in 0.1 M Tris buffer, pH 7.2, containing 1% of BSA. Working solutions of all oligonucleotide targets were diluted in 2 × SSC buffer (300 mmol/L sodium chloride + 30 mmol/L sodium citrate), pH 7.2, containing 1% BSA (hybridization solution). In selectivity studies, a 25% formamide was added to the hybridization solution.

Ultrapure water obtained with a Milli-Q plus 185 from Millipore Ibe´rica S. A. (Madrid, Spain) was used for all solutions. Apparatus and Electrodes. Cyclic voltammetric experiments were performed with an ECO Chemie µAutolab Type II potentiostat interfaced to a Pentium 166 computer system and controlled by the Autolab GPES software version 4.8 for Windows 98. All measurements were carried out at room temperature. SPCEs were purchased from Alderon Biosciences Inc. (Durham, NC), together with an edge connector. The Alderon Biosciences electrodes incorporate a conventional three-electrode configuration, which comprises a disk-shaped working electrode (4-mm diameter) and counter and silver pseudo-reference electrodes printed on polycarbonate substrates (4.5 × 1.5 cm). Both working and counter electrodes were made of heat-cured carbon composite inks. An insulating layer was printed over the electrode system, leaving uncovered a working electrode area of 7 × 5 mm and the electric contacts. A ring-shaped layer further printed around the working area constituted the reservoir of the electrochemical cell, with an actual volume of 50 µL. Methods. Figure 2 shows a scheme of the analytical procedure. Immobilization of Oligonucleotide Probes onto the Electrode Surface. An electrode pretreatment was carried out before each voltammetric experiment with the aim of improving the sensitivity and repeatability of the results. A 50-µL portion of 0.1 M H2SO4 was dropped on the SPCEs and an anodic current of +3.0 µA was applied for 2 min; then the electrodes were washed using 0.1 M Tris buffer, pH 7.2. The adsorption of streptavidin onto the electrode surface was performed leaving an aliquot of 10 µL of a 1 × 10-5 M streptavidin solution overnight at 4 °C. The electrode was washed with 0.1 M Tris buffer, pH 7.2, to remove the excess of protein. Free surface sites were blocked placing a drop of 40 µL of a 2% (w/v) solution of BSA for 15 min followed by a washing step with 0.1 M Tris buffer, pH 7.2, containing 1% of BSA. The formation of the sensing phase was performed with 40 µL of 3′-biotinylated oligonucleotide probes (0.5 ng/µL) for 30 min. Finally, the electrodes were rinsed with 2 × SSC buffer, pH 7.2, containing 1% BSA. Hybridization Step. Hybridization was performed at room temperature placing 30 µL of ULS-fluorescein-labeled oligonucleotide target solutions in 2 × SSC buffer, pH 7.2, containing 1% BSA, on the surface of the genosensor for 45 min and then rinsing with 0.1 M Tris buffer, pH 7.2, containing 1% BSA. Analytical Signal Recording. A 50-µL portion of a 0.2 M HCl solution was dropped on the electrode surface, and the electrode was held at a potential of +1.35 V for 1 min. Then, the chronoamperometric detection was performed at -1.40 V, recording the electric current generated for 5 min. Single-Base Mismatch Detection. The methodology used to detect a single-base mismatch is similar to the one explained

Table 1. Oligonucleotide Sequences for Probes and Targets of Pneumolysin and Autolysin

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oligonucleotide

sequence

lytA probe lytA target ply trobe ply target single-base mismatch ply target (plymism1) three-base mismatch ply target (plymism3)

5′-TTG CAT CAT GCA GGT AGG ACT TTT TTT TTT-3′-biotin 5′-GTC CTA CCT GCA TGA TGC AA-3′ 5′-CTT GCC AAA CCA GGC AAA TCT TTT TTT TTT-3′-biotin 5′-GAT TTG CCT GGT TTG GCA AG-3′ 5′-GAT TTG CCT GGT TTG TCA AG-3′ 5′-GAT TTG CCT GCT TTG GAA CG-3′


Figure 2. Schematic representation of the analytical procedure followed for the construction of the genosensor and the detection of a complementary target and a single-base mismatch target.

above, but a 25% formamide was included in the hybridization buffer. RESULTS AND DISCUSSION Analytical Signal Based on the Catalytic Hydrogen Evolution. The oligonucleotide targets used in this work are labeled using the ULS. This kind of labeling consists of the use of a squareplanar complex of platinum(II) which acts as a coupling agent. The label is attached to the Pt structure using the complex shown in Figure 1a (BOC-ULS). One of the ends of this complex finishes in a BOC group (tert-butoxycarbonyl group) that is substituted by a molecule (the label, such as fluorescein (Figure 1b), digoxigenin, biotin, etc.50). The complex “platinum/label”, so obtained, is monofunctional; that is, the other end of the complex finishes in one Cl, through which the attachment of the complex to the oligonucleotide takes place. The Cl is substituted by the N7 position of the guanines of the oligonucleotides in a very simple labeling reaction that takes no more than 35 min. Our group has already used this kind of labeling in a previous work33 where the ply and lytA targets were labeled with ULSfluorescein. In this case, the detection of the fluorescein label was carried out indirectly, using an antibody anti-fluorescein conjugated to alkaline phosphatase followed by an enzymatic reaction using 3-IP as electrochemical substrate. This methodology resulted in a sensitive and selective approach. However, it is well known that platinum catalyzes the hydrogen evolution. This fact, due to the presence of platinum(II) in the label complex, can be used to obtain an analytical signal in a simple manner. Figure 3 displays three cyclic voltammograms recorded (50) Heetebrij, R. J.; Talman, E. G.; Velzen, M. A.v.; van Gijlswijk, R. P. M.; Snoeijers, S. S.; Schalk, M.; Wiegant, J.; Rijke, F. vd.; Kerkhoven, R. M.; Raap, A. K.; Tanke, H. J.; Reedijk, J.; Houthoff, H.-J. ChemBioChem 2003, 4 (7), 573-583.

Figure 3. Cyclic voltammograms recorded from +1.35 to -1.40 V after placing an aliquot of 50 µL of (a) 0.1 M HCl solution, (b) 0.1 M HCl solution containing 4.8 × 10-6 M BOC-ULS, and (c) 0.1 M HCl solution containing 4.0 × 10-4 M PtCl42- on the electrode surface. Scan rate, 50 mV/s. Inset, voltammogram (a).

from +1.35 to -1.40 V at a scan rate of 50 mV/s, obtained for background (0.1 M HCl solution, cyclic voltammogram a and inset of figure), for 4.8 × 10-6 M BOC-ULS complex in 0.1 M HCl solution (b) and for 4.0 × 10-4 M PtCl42- in 0.1 M HCl solution (c). In the cyclic voltammogram obtained for background it can be observed that the reduction of protons of the medium occurs at very negative potentials, but to a lower extent than in the presence of any platinum complex on the electrode surface (on SPCEs the oxygen reduction at very negative potentials is not of relevant importance, so the background signals are not affected). In the presence of a platinum complex, this current is generated by the platinum complex reduction itself, but also, and to a higher extent, by the reduction of protons to hydrogen, which is catalyzed by the platinum complex (it is known that Pt is also a catalyst for the oxygen reduction so, at -1.40 V, oxygen reduction could also contribute to the current measured but to a lower extent, because Analytical Chemistry, Vol. 77, No. 9, May 1, 2005

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Figure 4. Chronoamperograms recorded using (a) 0.2 M HCl (background signal) and (b) 1.0 × 10-7 M BOC-ULS in 0.2 M HCl (analytical signal). Electrodeposition potential, -1.40 V.

the protons concentration is much higher than that of oxygen). Moreover, similar behavior is observed for both platinum complexes; the higher current registered for PtCl42- complex compared to the one obtained for BOC-ULS complex is due to the higher concentration assayed for the first complex. The current generated increases with increasing concentrations of the platinum complex. Thus, if an adequate potential is fixed, the intensity registered during the electrodeposition step informs about the presence or absence of platinum on the electrode surface. Figure 4 shows two chronoamperograms recorded during 5 min when the electrode was held at a fixed potential of -1.40 V (electrodeposition potential); chronoamperogram a was recorded when a 50-µL portion of 0.2 M HCl was placed on the electrode (background signal), and chronoamperogram b corresponds to the analytical signal obtained when 1.0 × 10-7 M BOC-ULS in 0.2 M HCl was used. All parameters that affect the platinum detection were studied. The electrodeposition step must be performed in an acidic medium because the analytical signal depends on the protons concentration. Thus, when other media such as NaOH or NaCl were employed, similar currents were obtained in the presence and in absence of platinum, being impossible its detection. Moreover, HCl was chosen as the most suitable acidic medium among other ones (data not shown). The electrodeposition potential and HCl concentration were optimized. Analytical signals for a concentration of 1.0 × 10-8 M BOC-ULS and background signals, obtained for all combinations of the electrodeposition potentials tested (-1.00, -1.20, and -1.40 V) and the HCl concentrations tested (0.05, 0.1, and 0.2 M), are shown in Figure 5. The best result was obtained for an electodeposition potential of -1.40 V and a concentration of 0.2 M HCl. In these conditions, 0.5 pmol of platinum in 50 µL can be detected with a signal-to-background ratio S/B ) 2. Higher electrodeposition potentials and HCl concentrations did not improve the results. Streptavidin-Coated Electrodes. The experimental conditions used to modify the electrode surface with streptavidin by physical adsorption have been studied in detail in a previous work,46 using biotin conjugated with alkaline phosphatase and 3-IP as electrochemical substrate. The electrode surface must be pretreated by applying an anodic constant current (+3.0 µA) in 0.1 M H2SO4 for 2 min to improve its adsorptive properties. The use of this electrochemical pretreatment resulted in an increase 2872 Analytical Chemistry, Vol. 77, No. 9, May 1, 2005

Figure 5. Effect of the electrodeposition potential and the HCl concentration on the background signals (gray bars) and analytical signals (white bars). BOC-ULS concentration, 1.0 × 10-8 M.

in the hydrophilicity of the transducer, allowing the adsorption of streptavidin through hydrophilic and electrostatic attraction. To avoid repeatability problems associated with the use of streptavidin, previously reported by other authors,11 the adsorption of streptavidin on the electrode must be performed at 4 °C overnight. Doing this, repeatable signals are obtained and the streptavidin-coated SPCEs are stable for months if they are stored at 4 °C. Moreover, the significance of the attachment of biotinylated oligonucleotide probes through the streptavidin/biotin interaction has been tested in a previous work.33 When a doublelabeled (biotin and fluorescein) poly-T was attached to the electrode surface through the streptavidin/biotin interaction, the peak currents were much higher than those obtained when it was accumulated on the electrode surface by physical adsorption. This fact means that streptavidin/biotin interaction allows it to attach and orient the oligonucleotide strands on the electrode surface whereas the direct adsorption of the oligonucleotide on the electrode surface leads to poor results. Optimization of Genosensor Response. As the immobilization of the oligonucleotide probes on the electrode surface occurs through the streptavidin/biotin interaction, different concentrations of 3′-biotinylated lytA probe and immobilization times were assayed. These studies were performed using 60 min of hybridization time and a concentration of the complementary lytA target of 50 pg/µL. Figure 6 shows the results obtained for different concentrations of lytA probe with 30 min of immobilization time. The peak current increases with increasing concentrations of lytA probe, reaching a current plateau for 0.5 ng/µL. This saturation concentration of lytA probe is chosen for subsequent studies. In the case of the immobilization time, 30 min is enough to reach a saturation of the electrode surface (data not shown). The influence of the hybridization time on the genosensor response was studied between 5 and 60 min using lytA probe immobilized on the electrode surface. The concentration of complementary lytA target was 25 pg/µL. Figure 7 shows that the peak current increases with the hybridization time, reaching a current plateau at ∼45 min. This time is chosen for further studies. At this point, the electrodeposition potential was again studied using the lytA genosensor, obtaining results similar to those obtained with BOC-ULS complex (E ) -1.40 V) (data not shown).

Figure 6. Effect of the 3′-biotinylated lytA probe concentration on the genosensor response. LytA probe immobilization time, 30 min; complementary lytA target concentration, 50 pg/µL; hybridization time, 60 min.

Figure 7. Effect of the hybridization time on the genosensor response. LytA probe concentration, 0.5 ng/µL; lytA probe immobilization time, 30 min; complementary lytA target concentration, 25 pg/ µL.

Figure 8. Ply and lytA genosensor responses to the ply (gray bars) and lytA (white bars) targets. Concentration of targets, 100 pg/µL.

Using the optimized experimental conditions, the lytA genosensor as well as a ply genosensor (formed with 3′-biotinylated ply probe) were tested with complementary and noncomplementary targets (in a concentration of 100 pg/µL each one). In Figure 8, it can be seen that when the reaction with noncomplementary targets was carried out, the analytical signal decreased drastically for both genosensors. These results show that the hybridization reaction with noncomplementary targets does not occur for both genosensors. Base-Mismatch Detection. It is known that numerous human diseases are associated with specific changes in normal DNA base sequences. For this reason, detecting a single-base mismatch in

Figure 9. Ply genosensor response to the complementary target (ply, white bars), the single-base mismatch target (plymism1, gray bars), and the three-base mismatch target (plymism3, black bars) for different concentrations. Data are given as average (SD (n ) 3).

a DNA sequence is of increasing interest. The way the detection of mutations using electrochemical genosensors is accomplished can be divided in two broad classes.51 In one of the approaches, DNA hybridization is carried out under low stringent conditions, which enables a formation of hybrid duplexes containing the basepairing mismatches; specific structural features of these duplexes affect their electrical and electrochemical behavior, which is used to perform the electrochemical detection of the mismatch.52,53 In another approach, based on the use of highly stringent conditions or peptide nucleic acid probes, only target DNA with a perfect complementarity to the immobilized probe forms the hybrid duplex while mutated forms of the target do not; formation of the hybrid can be detected by the different electrochemical detection strategies.1,4 This is the approach used in the present work. The ply genosensor was used for detecting oligonucleotide sequences containing a single- or three-base mismatch. Figure 9 displays the results obtained for three different concentrations (10, 25, and 100 pg/µL) of complementary ply, plymism1, and plymism3 targets assayed on ply genosensors. For the three concentrations assayed, the analytical signal obtained for the threebase mismatch oligonucleotide sequence is almost the background signal, indicating that three-base mismatch ply targets can be perfectly discriminated from the complementary ply target. For the single-base mismatch oligonucleotide sequence, the analytical signals were ∼40% lower than those obtained for the complementary target. Moreover, in these experimental conditions, a linear relationship between registered current and the logarithm of the concentration of ply target is obtained for concentrations between 5 and 100 pg/µL, according to the following equation:

i5min (µA) ) 510 log{[ply] (pg/µL)} - 22;

r ) 0.997 (n ) 5)

These genosensors can detect 5 pg/µL (24.5 fmol in 30 µL) of complementary ply target. The limit of detection, calculated as the oligonucleotide concentration yielding a signal three times the standard deviation of the blank response divided by the slope (51) Fojta, M. Electroanalysis 2002, 14 (21), 1449-1463. (52) Marques, L. P. J.; Cavaco, I.; Pinheiro, J. P.; Ribeiro, V.; Ferreira, G. N. M. Clin. Chem. Lab. Med. 2003, 41 (4), 475-481. (53) Kerman, K.; Saito, M.; Morita, Y.; Takamura, Y.; Ozsoz, M.; Tamiya, E. Anal. Chem. 2004, 76, 1877-1884.


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Figure 10. Ply genosensor responses for different concentrations of complementary target (ply, white bars) and the single-base mismatch target (plymisms1, gray bars) when 25% formamide is included in the hybridization buffer. Data are given as average ( SD (n ) 3).

of the calibration curve, is 4 pg/µL. Although the chronoamperometric detection of the current generated by catalytic hydrogen evolution due to the presence of platinum on the electrode surface, used to obtain the analytical signal, allows one to discriminate between the complementary oligo and the oligo with a three-base mismatch, the sensitivity of this method is 50-fold lower than that obtained with an enzymatic detection optimized in a previous work.33 To improve the selectivity of the ply genosensor, more stringent experimental conditions were tested. A concentration of 25% formamide, which had been optimized in a previous work,33 was added to the hybridization buffer. It is well known that this molecule makes difficult the hybridization reaction. In these more stringent conditions, a calibration plot for ply target was obtained using the ply genosensor. A linear relationship between peak current and the logarithm of the concentration of oligonucleotide target is obtained for concentrations between 50 and 1000 pg/ mL, according to the following equation:

i5min (µA) ) 360 log{[ply] (pg/µL)} - 275; r ) 0.991 (n ) 5) The limit of detection, calculated as described above, is 50 pg/ µL. As expected, the sensitivity decreases in these experimental conditions but the detection of a single-base mismatch on an oligonucleotide sequence can be performed for any concentration assayed (Figure 10). Despite this loss of sensitivity, the genosensors can detect ∼245 fmol of complementary target in 30 µL in these more stringent experimental conditions. However, the sensitivity obtained with this methodology is ∼2 orders of magnitude lower than that obtained with enzymatic detection.33 CONCLUSIONS The formation of genosensors on screen-printed carbon electrodes is achieved by coating the electrode surface with streptavidin and using biotinylated oligonucleotide probes to form the sensing phase. The oligonucleotide targets are labeled using the ULS. This kind of labeling consists of the use of a squareplanar complex of platinum(II), which acts as a coupling agent

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between DNA and a label molecule. The presence of platinum(II) can be used to obtain an analytical signal in a simple manner, based on the catalytic properties of this metal on hydrogen evolution. The current registered at a fixed potential of -1.40 V in acidic medium is due to the reduction of protons to hydrogen catalyzed by platinum attached to the electrode surface through oligonucleotide targets, which have been previously hybridized. Using this chronoamperometric detection, under nonstringent experimental conditions these genosensors can detect 24.5 fmol of a 20-mer oligonucleotide target in 30 µL and discriminate between a complementary oligo and an oligo with a three-base mismatch. If a single-base mismatch on the oligo target needs to be detected, 25% formamide must be included in the hybridization buffer, although in this case the sensitivity of the genosensor is 10 times lower. Although the sensitivity of this method is 50- (under nonstringent conditions) and 200-fold (using 25% formamide in the hybridization solution) lower than that obtained with an enzymatic method previously published, the analysis time is considerably shorter, because the analytical signal is achieved directly from the platinum complex whereas in the previous work two additional steps were necessary to obtain the analytical signal: the reaction with antibody anti-fluorescein and the enzymatic reaction. Thus, the overall analysis time of this chronoamperometric method is about the half than that resulting from the enzymatic method. All the experiments showed in this work have been carried out with single-stranded oligonucleotides. In a future application, the detection of PCR products coming from amplification of real DNA samples will be tested. In this case, previous steps for denaturing (heating or chemical treatment) the amplicons in order to obtain single-stranded DNA and the use of adequate stringent conditions to allow efficient hybridization of targets with immobilized probes instead of hybridization with complementary strands in solution will be necessary. All this, together with he fact that PCR products are longer DNA strands than the oligonucleotides used in this work, will probably affect negatively to the sensitivity of the genosensor in terms of limits of detection of amplified DNA. However, other important data such as the amount of DNA (before amplification) able to be detected after PCR using these sensors will be evaluated and compared to others reported in the literature. ACKNOWLEDGMENT The authors thank Dr. Willem B. Van Leeuwen (Erasmus Medical Centre, Rotterdam, The Netherlands) for providing some of the oligonucleotides and for his valuable comments during the course of the research, as well as Dr. Rob van Gijlswijk and Dr. Jack Veuskens (Kreatech Biotechnology, Amsterdam, The Netherlands) for providing the BOC-ULS complex and for the ULS labeling of the oligonucleotide targets. Financial support was provided by European project UE-02-QLK2-CT-70963 and by a grant from the Consejerıá de Educacioń y Cultura del Principado de Asturias. Received for review December 26, 2004. Accepted February 18, 2005. AC048091W

Genosensor Based on a Platinum(II) Complex as Electrocatalytic Label

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