Enzymatic Genosensor on Streptavidin-Modified Screen-Printed

Anal. Chem. 2004, 76, 6887-6893

Enzymatic Genosensor on Streptavidin-Modified Screen-Printed Carbon Electrodes David Herna´ndez-Santos, Marı´a Dı´az-Gonza´lez, Marı´a Begon˜a Gonza´lez-Garcı´a, and Agustı´n Costa-Garcı´a*

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

Voltammetric enzyme genosensors on streptavidin-modified screen-printed carbon electrodes (SPCEs) for the detection of virulence nucleic acid determinants of pneumolysin and autolysin genes, exclusively present on the genome of the human pathogen Streptococcus pneumoniae, were described. Alkaline phosphatase (AP) and 3-indoxyl phosphate were used as the enzymatic label and substrate, respectively. The oligonucleotide probes were immobilized on electrochemically pretreated SPCEs through the streptavidin/biotin reaction. The adsorption of streptavidin was performed by deposition of a drop of a streptavidin solution overnight at 4 °C on the surface of the SPCEs. After the hybridization reaction with FITClabeled complementary targets, the enzyme is captured using an anti-FITC antibody conjugated to AP. In nonstringent experimental conditions, these genosensors can detect 0.49 fmol of 20-mer oligonucleotide target and discriminate between a complementary oligo and an oligo with a three-base mismatch. In the presence of 25% formamide in the hybridization buffer, a single-base mismatch on the oligonucleotide target can be detected. Electrochemical devices have received considerable attention in the development of DNA hybridization biosensors.1-3 The high sensitivity of electrochemical transducers, coupled with their compatibility with modern microfabrication and miniaturization technologies, low cost and power requirements, and their independence of sample turbidity, makes such devices excellent candidates for DNA diagnostics. Electrochemical DNA genosensors commonly rely on the conversion of the hybridization event to useful electrical signals. The base-pairing recognition event can be detected via the variation of the current signal of a redox indicator (i.e., cationic metal complexes, daunomycin, electrochemical dye, or methylene blue4-14) that has a different affinity for the resulting target/probe * 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) Wang, J.; Cai, X.; Rivas, G.; Shiraishi, H. Anal. Chim. Acta 1996, 326, 141147. (5) Erdem, A.; Kerman, K.; Meric, B.; Akarca, U. S.; Ozsoz, M. Anal. Chim. Acta 2000, 422, 139-149. (6) Millan, K. M.; Mikkelsen, S. R. Anal. Chem. 1993, 65, 2317-2323. 10.1021/ac048892z CCC: $27.50 Published on Web 11/02/2004

© 2004 American Chemical Society

duplex, as compared to the affinity for the probe alone, from changes in the intrisic nucleic acid electrochemical signals associated with the electroactivity of guanines (nonlabeling detection)9,15-18 or using enzyme tags that can be captured following the hybridization. The enzymes hold great potential for electrical detection of DNA hybridization. Such promise is attributed to the biocatalytic activity of these labels that provides the amplification essential for monitoring very low target levels. This can be accomplished by combining the hybridization step with an electrochemical measurement of the product of the enzymatic reaction. The great potential of enzyme labels for electrical detection of DNA hybridization was demonstrated using horseradish peroxidase (HRP)19-28 and alkaline phosphatase (AP).29-31 The use of enzymes has led to the most sensitive electrochemical detections of DNA,21,32 as well as novel strategies (7) Hashimoto, K.; Ito, K.; Ishimori, Y. Anal. Chim. Acta 1994, 286, 219-224. (8) Wang, J.; Fernandes, J. R.; Kubota, L. T. Anal. Chem. 1998, 70, 36993702. (9) Marrazza, G.; Chianella, I.; Mascini, M. Anal. Chim. Acta 1999, 387, 297307. (10) Marrazza, G.; Chianella, I.; Mascini, M. Biosens. Bioelectron. 1999, 14, 4351. (11) Wang, J.; Rivas, G.; Cai, S. Electroanalysis 1997, 9 (5), 395-398. (12) Hashimoto, K.; Ito, K.; Ishimori, Y. Anal. Chem. 1994, 66, 3830-3833. (13) Takenaka, S.; Uto, Y.; Saita, H.; Yokoyama, M.; Kondo, H.; Wilson, W. D. Chem. Commun. 1998, 1111-1112. (14) Maruyama, K.; Mishima, Y.; Minagawa, K.; Motonaka, J. Anal. Chem. 2002, 74, 3698-3703. (15) Wang, J.; Rivas, G.; Fernandes, J. R.; Lopez-Paz, J. L.; Jiang, M.; Waymire, R. Anal. Chim. Acta 1998, 375, 197-203. (16) Wang, J.; Gru ¨ ndler, P.; Flechsig, G.-U.; Jasinski, M.; Rivas, G.; Sahlin, E.; Lopez-Paz, J. L. Anal. Chem. 2000, 72, 3752-3756. (17) Wang, J.; Kawde, A.-N. Anal. Chim. Acta 2001, 431, 219-224. (18) Lucarelli, F.; Marrazza, G.; Palchetti, I.; Cesaretti, S.; Mascini, M. Anal. Chim. Acta 2002, 469, 93-99. (19) Campbell, C. N.; Gal, D.; Cristler, N.; Banditrat, C.; Heller, A. Anal. Chem. 2002, 74, 158-162. (20) de Lumley-Woodyear, T.; Campbell, C. N.; Heller, A. J. Am. Chem. Soc. 1996, 118, 5504-5505. (21) Zhang, Y.; Kim, H.-H.; Heller, A. Anal. Chem. 2003, 75, 3267-3269. (22) Pividori, M. I.; Merkoci, A.; Alegret, S. Analyst 2001, 126, 1551-1557. (23) Pividori, M. I.; Merkoci, A.; Alegret, S. Biosens. Bioelectron. 2001, 16, 11331142. (24) Pividori, M. I.; Merkoci, A.; Alegret, S. Biosens. Bioelectron. 2003, 19, 473484. (25) Pividori, M. I.; Merkoci, A.; Barbe´, J.; Alegret, S. Electroanalysis 2003, 15 (23-24), 1815-1823. (26) Williams, E.; Pividori, M. I.; Merkoci, A.; Forster, R. J.; Alegret, S. Biosens. Bioelectron. 2003, 19, 165-175. (27) Dequaire, M.; Heller, A. Anal. Chem. 2002, 74, 4370-4377. (28) Zhang, Y.; Kim, H.-H.; Mano, N.; Dequaire, M.; Heller, A. Anal. Bioanal. Chem. 2002, 374, 1050-1055. (29) Bagel, O.; Degrand, C.; Limoges, B.; Joannes, M.; Azek, F.; Brossier, P. Electroanalysis 2000, 12 (18), 1447-1452.

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for the detection of neurodegenerative diseases on the basis of the double surface technique (DST).33 A wide variety of electrodes have been used as support to fabricate genosensor devices, including carbon paste electrodes,4,5,15,16 glassy carbon electrodes,6,20,21,32,34 graphite electrodes,7,17,19,31,33 carbon composites8,22-26 and gold electrodes.12-14 Recently, several genosensor devices have been developed on screen-printed carbon electrodes (SPCEs).9-11,18,27-30,35 The screenprinting microfabrication technology is nowadays well-established for the production of thick-film electrochemical transducers. 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. For the design of a genosensor, the crucial step is the immobilization of single-stranded DNA probes (ssDNA) 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 surfaces4,5,9-11,15-18 or physical adsorption on carbon surfaces or membranes coupled to carbon surfaces7,22-25 and bulk-modification of carbon paste.8 However, in these cases, the 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 polymer-coated surfaces,6,20,21,27,28,34,35 the use of self-assembled monolayers onto gold surfaces,12-14 or the use of avidin/biotin interaction to attach biotinylated probes on the electrode surface,10,19,26 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 an enzymatic genosensor on screen-printed 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 must be performed at a constant temperature above room temperature. Moreover, the electrode surface must be previously electrochemically pretreated at an anodic potential in acidic medium to improve its adsorptive properties. In this way, reproducible, sensitive, and stable sensing phases are obtained.36 This methodology for coating the electrode with streptavidin is very simple, in contrast to other works in which the modification of carbon electrodes with a film of coelectrodeposited avidin and redox polymer19 or a bulk modification of a (30) Wang, J.; Xu, D.; Erdem, A.; Polsky, R.; Salazar, M. A. Talanta 2002, 56, 931-938. (31) Palecek, E.; Kizek, R.; Havran, L.; Billova, S.; Fojta, M. Anal. Chim. Acta 2002, 469, 73-83. (32) Wang, J.; Liu, G.; Jan, M. R. J. Am. Chem. Soc. 2004, 126, 3010-3011. (33) Fojta, M.; Havran, L.; Vojtiskova, M., Palecek, E. J. Am. Chem. Soc. 2004, 126, 6532-6533. (34) Lee, T.-Y.; Shim, Y.-B. Anal. Chem. 2001, 73, 5629-5632. (35) Ruffien, A.; Dequaire, M.; Brossier, P. Chem. Commun. 2003, 912-913. (36) Dı´az-Gonza´lez, M.; Herna´ndez-Santos, D.; Gonza´lez-Garcı´a, M. B.; CostaGarcı´a, A. Talanta, in press.

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carbon-polymer composite electrode with streptavidin26 were carried out. The biotinylated oligo nucleic acid probes used in this work target the pneumolysin (ply), autolysin (lytA), and a cellwall protein (psaA) genes. These targets are randomly labeled with the universal linkage system (ULS).37 This system binds to DNA at the N7 position of guanine, resulting in the attachment of a label molecule to the DNA. The label molecule used in this study was fluorescein (FITC). Electrochemical detection is achieved with an anti-FITC alkaline phosphatase-labeled antibody and using 3-indoxyl phosphate (3-IP) as the electrochemical substrate of AP. Our group has described 3-IP as a suitable electrochemical substrate for AP.38 The resulting enzymatic product is indigo blue, an aromatic heterocycle insoluble in aqueous solutions. Its sulfonation in acidic medium gives rise to indigo carmine (IC), an aqueously soluble compound that shows an electrochemical behavior similar to that of indigo blue. Both 3-IP and IC have already been studied on SPCEs.39,40 Data presented here demonstrate the potential applicability of SPCE genosensors in the diagnosis of a human infectious pulmonary disease. These electrochemical genosensors are stable, sensitive devices for the detection of specific nucleic acid fragments. 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), 3-indoxyl phosphate disodium salt (3-IP), bovine serum albumin fraction V (BSA), streptavidin (molecular weight ) 60 000 g/mol), and rabbit IgG anti-FITC conjugated to alkaline phosphatase (Ab-AP) were purchased from Sigma (Madrid, Spain). 3-Indoxyl phosphate (3-IP, 6 mM) solutions were prepared in 0.1 M Tris, pH 9.8, containing 20 mM MgCl2 and stored in opaque tubes at 4 °C. Working solutions of streptavidin were made in 0.1 M Tris, pH 7.2, buffer. BSA lyophilized powder was reconstituted in ultrapure water. Working solutions of Ab-AP were made in 0.1 M Tris, pH 7.2, containing 2 mM MgCl2 and 1% BSA. Double-labeled poly-T (30-mer, 3′-biotin and 5′-FITC), poly-T (30-mer, 3′-biotin), poly-A (18-mer, 3′-FITC), 3′-biotinylated 30mer oligonucleotide probes with a 10-T spacer in the 3′-end, and the complementary 20-mer labeled targets corresponding to autolysin (lytA), pneumolysin (ply), and the cellwall protein psaA genes, as well as the single-base and three-base-mismatch ply targets were purchased from Isogen Life Science (Maarssen, The Netherlands). ULS-FITC 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 single-base and three-base-mismatch ply oligonucleotides, are shown in Table 1. Concentrated aliquots of all oligonucleotides were prepared in 0.1 M Tris, pH 7.2, and stored (37) Lempers, E. L. M.; Van der Berg, F. M.; Reedijk, J.; Bloemink, M. J. U.S. Patent 5580990, 1996. (38) Ferna´ndez-Sa´nchez, C.; Costa-Garcı´a, A. Electroanalysis 1998, 10 (4), 249255. (39) Dı´az-Gonza´lez, M.; Ferna´ndez-Sa´nchez, C.; Costa-Garcı´a, A. Electroanalysis 2002, 14 (10), 665-670. (40) Dı´az-Gonza´lez, M.; Ferna´ndez-Sa´nchez, C.; Costa-Garcı´a, A. Anal. Sci. 2002, 18, 1209-1213.

Table 1. Oligonucleotide Sequences for Probes and Targets of Pneumolysin, Autolysin, and Cell Wall Protein psaA oligonucleotide

sequence

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

5′-GGT CTT GCC CAA TCG GAA CGT TTT TTT TTT-3′-biotin 5′-CGT TCC GAT TGG GCA AGA CC-3′ 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 1. 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.

at -20 °C. Working solutions of all biotinylated oligonucleotide probes were made in 0.1 M Tris, pH 7.2, buffer, containing 1% BSA. Working solutions of all oligonucleotide targets were diluted in 2xSSC buffer (300 mmol/L sodium chloride + 30 mmol/L sodium citrate), pH 7.2, containing 1% BSA (hybridization solution). In selectivity studies, an amount of 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 using 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. Screenprinted carbon electrodes (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), counter, and silver pseudoreference 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 ringshaped layer further printed around the working area constituted the reservoir of the electrochemical cell, with an actual volume of 50 µL. Methods. Figure 1 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. Then the electrode was washed with 0.1 M Tris buffer, pH 7.2, to remove the excess 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, pH 7.2, buffer containing 1% BSA. The formation of the sensing phase was performed with 40 µL of 3′-biotinylated oligonucleotide probes (0.5 ng/µL) for 15 min. Finally, the electrodes were rinsed with 2xSSC buffer, pH 7.2, containing 1% BSA. Analytical Chemistry, Vol. 76, No. 23, December 1, 2004

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Hybridization Step. Hybridization was performed at room temperature by placing 30 µL of FITC-labeled oligonucleotide target solutions in 2xSSC buffer, pH 7.2, containing 1% BSA, on the surface of the genosensor for 45 min and then rinsing with 0.1 M Tris, pH 7.2, buffer containing 1% BSA. Reaction with Anti-FITC AP Conjugate (Ab-AP). This step was performed by dropping aliquots of 40 µL of Ab-AP solutions (1/100 dilution) on the genosensor device for 60 min. After a washing step with 0.1 M Tris buffer, pH 9.8, containing 1% BSA, the enzymatic reaction and the analytical signal recording were carried out. Enzymatic Reaction and Analytical Signal Recording. The enzymatic reaction was carried out by dropping an aliquot of 30 µL of 6 mM 3-IP on the electrode surface for 20 min. After that, the reaction was stopped by adding 4 µL of fuming sulfuric acid and 10 µL of ultrapure water. In this step, the corresponding indigo product is converted to its parent hydrosoluble compound indigo carmine (IC). The analytical signal was recorded using cyclic voltammetry (CV); the SPCEs were held at a potential of -0.25 V for 25 s, and then a voltammogram was recorded from -0.25 to +0.20 V at a scan rate of 50 mV/s. The anodic peak current was measured in all experiments. Single-Base-Mismatch Detection. The methodology used to detect a single-base mismatch is similar to the one explained above, but an adequate amount of formamide was added to the hybridization solution. RESULTS AND DISCUSSION 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 work36 using biotin conjugated with alkaline phosphatase and 3-indoxyl phosphate (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 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,10 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. Evaluation of the Nonspecific Adsorptions. The direct adsorption of Ab-AP on the electrode surface was evaluated for different dilutions of the antibody. Two parallel experiments were carried out on pretreated streptavidin-coated electrodes. Some electrodes were not blocked with BSA, and others were blocked with a 2% BSA solution for 15 min. Both the modification with the oligonucleotide probe and the hybridization reaction with the complementary target were not carried out in this study. Figure 2 shows two cyclic voltammograms obtained when 1/100 dilution Ab-AP solutions were placed for 90 min on streptavidin-coated SPCEs without BSA (a) and blocked with 2% BSA (b). Enzymatic reaction and analytical signal recording were performed as described in the Experimental Section. The analytical signals obtained when the electrodes are not blocked with BSA increase for decreasing dilutions of Ab-AP (163, 659, and 1204 nA for 6890 Analytical Chemistry, Vol. 76, No. 23, December 1, 2004

Figure 2. Cyclic voltammograms recorded after placing a 1/100 dilution Ab-AP solution for 90 min on a streptavidin-coated SPCE without BSA (a) and blocked with 2% BSA (b), followed by the enzymatic reaction with 3-IP. Scan rate: 50 mV/s.

Figure 3. Effect of double-labeled poly-T oligonucleotide concentration on peak current for ([) a 1/100 dilution Ab-AP (double-labeled poly-T concentrations varying from 0.001 to 10 ng/µL) and for (b) a 1/500 dilution Ab-AP (double-labeled poly-T concentrations varying from 0.1 to 10 ng/µL).

1/10 000, 1/1000, and 1/100 Ab-AP dilution, respectively). When the blocking step with BSA is carried out before the incubation of Ab-AP, the nonspecific adsorption of this antibody is drastically avoided for all dilutions assayed (the analytical signals obtained were 179, 146, and 195 nA for 1/10 000, 1/1000, and 1/100 AbAP dilution, respectively). Different concentrations and accumulation times of BSA have been assayed (data not shown). A 2% BSA solution and an accumulation time of 15 min were enough to avoid the nonspecific adsorption of Ab-AP. Higher concentrations of BSA or accumulation times did not improve the results. The dilution of Ab-AP has been optimized using several concentrations of the double-labeled poly-T oligo for two dilutions of Ab-AP (1/100 and 1/500). Figure 3 displays the results obtained in this study. For a 1/100 dilution of Ab-AP, the peak current increases with the concentration of double-labeled polyT, reaching a current plateau for 1.0 ng/µL of double-labeled poly-T with a current response of 4670 nA. For a 1/500 dilution of Ab-AP, the current plateau is reached for lower concentrations of double-labeled poly-T. However, the peak current does not reach the current values obtained with 1/100 dilution of Ab-AP for none of the concentrations assayed, indicating that the amount of antibody is not enough to saturate the FITC labeled to the strand attached on the electrode surface. Higher concentrations than 1/100 Ab-AP dilution did not increase the analytical signals, so this dilution was chosen for further studies.

Figure 4. Cyclic voltammograms recorded on (b) a pretreated SPCE where a 0.1 ng/µL double-labeled poly-T oligonucleotide solution was placed for 90 min, followed by 15 min of BSA blocking; (9) a streptavidin-coated, BSA-blocked SPCE where a 0.1 ng/µL double-labeled poly-T oligonucleotide solution was placed for 90 min; ([) streptavidin-coated, BSA-blocked, 3′-biotinylated poly-T probe modified SPCEs where a 1 pg/µL 3′-FITC poly-A target solution was incubated for 60 min; and (2) where this incubation was not carried out. In all cases, an incubation of 1/100 dilution Ab-AP for 90 min followed by the enzymatic reaction with 3-IP was carried out. Scan rate: 50 mV/s.

In addition, different solutions of 3′-FITC-labeled poly-A with concentrations varying between 0.05 and 1.0 ng/µL were dropped for 60 min on streptavidin-modified, BSA-blocked SPCEs and then a 1/100 dilution of Ab-AP. The analytical signals did not increase with increasing concentrations of 3′-FITC-labeled poly-A, and the peak currents obtained were similar to those due to the nonspecific adsorption of Ab-AP (data not shown). These results indicate that the main contribution of the nonspecific adsorptions is due to the adsorption of Ab-AP on the electrode surface. Significance of the Attachment of Biotinylated Oligonucleotide Probes through Streptavidin/Biotin Interaction. To test the significance of the streptavidin/biotin interaction in the attachment of oligonucleotide probes on the electrode surface, parallel experiments were carried out using SPCEs modified with streptavidin and SPCEs without streptavidin. Figure 4 displays four cyclic voltammograms obtained for a 1/100 dilution of AbAP solution. The reaction time with Ab-AP was 90 min for all experiments. Enzymatic reaction and analytical signal recording were performed as described in the Experimental Section. The voltammogram with the solid square was recorded using a SPCE modified with streptavidin and blocked with a 2% BSA solution, followed by reaction with 0.1 ng/µL of double-labeled poly-T for 90 min. The voltammogram with the solid circle corresponds to a pretreated SPCE (with neither streptavidin nor BSA) for which a 0.1 ng/µL double-labeled poly-T solution was accumulated for 90 min, followed by blocking with 2% BSA for 15 min to avoid the nonspecific adsorption of Ab-AP. Working solutions used in this study did not contain 1% BSA. When the double-labeled 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, and moreover, in the latter case, the analytical signal was similar to that obtained for the nonspecific adsorption of Ab-AP. Two factors could be responsible for this behavior. One of these is that streptavidin/biotin interaction allows attachment and orientation of the strands of double labeled poly-T on

Figure 5. Effect of 3′-biotinylated ply probe concentration on the genosensor response. Ply probe immobilization time, 90 min; complementary ply target concentration, 5 pg/µL; hybridization time, 60 min; Ab-AP incubation time, 90 min. Data are given as average ( SD (n ) 3).

the electrode surface, whereas the direct adsorption of the oligonucleotide on the electrode surface results in a very poor manner. The other is that the blocking step with BSA, carried out after the direct adsorption of double-labeled poly-T on the electrode surface, hampers the next reaction between FITC and the Ab-AP. Whatever the reason, because a 2% BSA is always needed to avoid the nonspecific adsorptions of Ab-AP, the oligonucleotide cannot be directly adsorbed on the electrode surface. The cyclic voltammograms with the solid diamond and solid triangle of Figure 4 correspond to the analytical signals obtained on streptavidin-coated, BSA-blocked SPCEs for which a 0.1 ng/ µL 3′-biotinylated poly-T solution was incubated for 90 min; the solid diamond voltammogram shows the response after the incubation of 1.0 pg/µL 3′-FITC poly-A (60 min of hybridization time), and the solid triangle voltammogram shows the response when this incubation was not carried out. Both voltammograms show that the hybridization event occurs and that the background signals for the genosensor are similar to those corresponding to the nonspecific adsorption of Ab-AP. Optimization of Genosensor Response. The immobilization of the oligonucleotide probes on the electrode surface occurs through the streptavidin/biotin interaction. Different concentrations of 3′-biotinylated ply probe and immobilization times were assayed. These studies were performed using 60 min of hybridization time and a concentration of the complementary ply target of 5 pg/µL. The reaction time with Ab-AP was 90 min. Figure 5 shows the results obtained for different concentrations of the ply probe with 90 min of immobilization time. The peak current increases with increasing concentrations of the ply probe, reaching a current plateau for 0.1 ng/µL. A saturation concentration of 0.5 ng/µL of the ply probe was chosen for subsequent studies. In the case of the immobilization time, 15 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 10 and 60 min using a ply probe immobilized on the electrode surface. The concentration of the complementary ply target was 5 pg/µL. The reaction time with Ab-AP was 90 min. Figure 6 shows that the peak current increased rapidly with the hybridization time up to 30 min and Analytical Chemistry, Vol. 76, No. 23, December 1, 2004

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Figure 6. Effect of the hybridization time on the genosensor response. Ply probe concentration, 0.5 ng/µL; ply probe immobilization time, 15 min; complementary ply taget concentration, 5 pg/µL; Ab-AP incubation time, 90 min. Data are given as average ( SD (n ) 3).

Figure 7. Ply, lytA, and psaA genosensor responses to the ply (white bars), lytA (gray bars), and psaA (black bars) targets. Concentration of targets, 5 pg/µL.

then more slowly to reach a current plateau at about 45 min. This time was chosen for further studies. The reaction time with Ab-AP was also studied. The optimum time obtained was 60 min, where a current plateau was reached (data not shown). Using the optimized experimental conditions, the response of the genosensor formed with a 3′-biotinylated ply probe (ply genosensor) for different concentrations of the complementary oligonucleotide target was evaluated. A linear relationship between peak current and concentration of complementary ply target was obtained between 0.1 and 5 pg/µL, with a correlation coefficient of 0.9993, according to the following equation.

ip (nA) ) 91 + 220 [ply target] (pg/µL)

Thus, this genosensor can detect 0.1 pg/µL, which is 0.49 fmol of ply target in 30 µL. The ply genosensor, as well as a lytA genosensor (formed with 3′-biotinylated lytA probe) and a psaA genosensor (formed with 3′-biotinylated psaA probe), was tested with complementary and noncomplementary targets (at a concentration of 5 pg/µL for each one). In Figure 7, it can be seen that when the reaction with noncomplementary targets was carried out, the analytical signal decreased drastically for all genosensors. These results show that 6892 Analytical Chemistry, Vol. 76, No. 23, December 1, 2004

Figure 8. 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).

the hybridization reaction with noncomplementary targets does not occur for all 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 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.41 In one of the approaches, DNA hybridization is carried out under low stringent conditions, which enables a formation of hybrid duplexes containing base pairing mismatches; specific structural features of these duplexes affect their electrical and electrochemical behavior, which is used to perform the electrochemical detection of the mismatch.42,43 In another approach, based on the use of highly stringent conditions or peptide nucleic acid (PNA) 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.1,44 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. Three different concentrations (1, 5, and 10 pg/µL) of complementary ply, plymism1, and plymism3 targets were assayed, and three genosensors were used for each concentration. Figure 8 displays the results obtained. For the three concentrations assayed, the analytical signal obtained for the three-base-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 obtained only decrease about 25% with respect to those obtained for the complementary target. Other saline concentrations of the hybridization buffer were tested, but the discrimination between single-base-mismatch ply target and complementary ply target was not improved. (41) Fojta, M. Electroanalysis 2002, 14 (21), 1449-1463. (42) Marques, L. P. J.; Cavaco, I.; Pinheiro, J. P.; Ribeiro, V.; Ferreira, G. N. M. Clin. Chem. Lab. Med. 2003, 41 (4), 475-481. (43) Kerman, K.; Saito, M.; Morita, Y.; Takamura, Y.; Ozsoz, M.; Tamiya, E. Anal. Chem. 2004, 76, 1877-1884. (44) Gooding, J. J. Electroanalysis 2002, 14 (17), 1149-1156.

In both cases, a linear relationship between peak current and concentration of oligonucleotide target was obtained for concentrations between 0.25 and 5 pg/µL according to the following equations:

ip (nA) ) 29 + 175 [ply] (pg/µL) r ) 0.9992 ip (nA) ) 74 + 166 [lytA] (pg/µL) r ) 0.998

Figure 9. Effect of formamide concentration in the hybridization buffer on the genosensor response for the complementary target (ply, white bars) and the single-base-mismatch target (plymism1, gray bars). Targets concentration, 3.5 pg/µL. Data are given as average ( SD (n ) 4).

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

Because of this point, more stringent experimental conditions were tested. Different concentrations of formamide were added to the hybridization buffer. It is well-known that this molecule makes the hybridization reaction more difficult. Figure 9 shows the analytical signals obtained for a concentration of 3.5 pg/µL of both single-base-mismatch ply target and complementary ply target by varying the concentrations of formamide in the hybridization buffer between 5 and 50%. Four ply genosensors were used for each point. The analytical signals decreased with increasing concentrations of formamide in hybridization buffer for both plymism1 and complementary ply targets. For a concentration of 25% of formamide, the analytical signal obtained for plymism1 target is similar to the background signal. Under these more stringent conditions (hybridization buffer containing 25% formamide), calibration plots for both lytA and ply target were obtained using lytA and ply genosensors, respectively.

As expected, the sensitivity decreases under 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 ∼1.2 fmol of complementary target in 30 µL under these more stringent experimental conditions. CONCLUSIONS The formation of sensitive and reproducible enzymatic genosensors on screen-printed carbon electrodes has been achieved by coating the electrode surface with streptavidin and using biotinylated oligonucleotide probes to form the sensing phase. The way the electrodes are coated with streptavidin, at 4 °C overnight, is essential to obtain reproducible genosensors, whereas the use of biotinylated probes is efficient due to the effect of orientation/enhanced adsorption of single strands on the genosensing phase, leaving the probes accessible for the reaction with their complementary targets. The 10-T spacer allows the probe to be more accessible to the oligonucleotide target; however, the use of a nucleotide spacer could cause false positive results with real DNA samples, so the effect of this spacing strategy should be considered and evaluated in the case of PCR product detection. Under nonstringent experimental conditions, these genosensors can detect 0.49 fmol of 20-mer oligonucleotide target 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 2.5 times lower. 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 the ULS labeling of the oligonucleotide targets. Financial support was provided by European Project UE-02-QLK2CT-70963 and by a grant from the Consejerı´a de Educacio´n y Cultura del Principado de Asturias. Received for review July 29, 2004. Accepted September 15, 2004. AC048892Z

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