Alkaline Phosphatase-Catalyzed Silver Deposition for Electrochemical

Anal. Chem. 2007, 79, 5272-5277

Alkaline Phosphatase-Catalyzed Silver Deposition for Electrochemical Detection Pablo Fanjul-Bolado, 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

Alkaline phosphatase (AP) is one of the most used enzymatic labels for the development of ELISAs, immunosensors, DNA hybridization assays, etc. This enzyme catalyzes the dephosphorylation of a substrate into a detectable product usually quantified by optical or electrochemical measurements. This work is based on a substrate (3-indoxyl phosphate) that produces a compound able to reduce silver ions in solution into a metallic deposit, which is localized where the enzymatic label AP is attached. The deposited silver is electrochemically stripped into solution and measured by anodic stripping voltammetry. Its application to an enzymatic genosensor on streptavidin-modified screen-printed carbon electrodes for the detection of virulence nucleic acid determinants of autolysin gene, exclusively present on the genome of the human pathogen Streptococcus pneumoniae, is described. Compared with the direct voltammetric detection of indigo carmine, the anodic stripping voltammetry of silver ions is 14-fold more sensitive. Very sensitive methods are always required for DNA sensing. In this way, 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 independent of sample turbidity, makes such devices excellent candidates for DNA diagnostics. Different strategies for electrochemical detection have been performed. One of them is based on label-free electrochemical detection, via the intrinsic electrochemical behavior of DNA, through guanine or adenine nucleotides. However, most of the strategies are based on the use of indicators or labels. The first ones are based on the differences in the electrochemical behavior of indicators with double-strand DNA (dsDNA) and single-strand DNA (ssDNA). The indicators for hybridization detection can be anticancer agents, organic dyes, or metal complexes. The latter * To whom correspondence should be addressed. Fax: +34 985 103 125. E-mail: [email protected]. (1) Pividori, M. I.; Merkoçi, 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.

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strategies are the use of labels as metal complexes,4,5 ferrocene,6-8 enzymes,9-18 or gold nanoparticles.19-22 The detection of these nanoparticles can be carried out by measuring the changes in the electrical resistance19 or capacitance20 between electrodes usually after a silver enhancement procedure, which consists of reducing silver using a chemical reducing agent. Although the sensitivity of these methods is at the picomolar level, the main drawback is the high background signals. To overcome the high background signals, another method based on silver electrodeposition catalyzed by colloidal gold can be used.5,23,24 In this case, the background signals are perfectly discriminated from the analytical signal by controlling the deposition potential of silver on the electrode surface. Another possibility is the enzymatic control of metal precipitation that avoids the background that is (4) Hernańdez-Santos, D.; Gonza´lez-Garcıá, M. B.; Costa-Garcıá, A. Anal. Chem. 2005, 77, 2868-2874. (5) De la Escosura-Mun ˜iz, A.; Gonza´lez-Garcıá, M. B.; Costa-Garcıá, A. Biosens. Bioelectron. 2007, 22, 1048-1054. (6) Umek, M.; Lin, S. S.; Chen, Y. P.; Irvine, B.; Paulluconi, G.; Chan, V.; Chong, Y.; Cheung, L.; Vielmetter, J.; Farkas, D. H. Mol. Diagn. 2000, 5, 321328. (7) Fan, C. H.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9134-9137. (8) Liepold, P.; Wieder, H.; Hillebrandt, H.; Friebel, A.; Hartwich, G. Bioelectrochemistry 2005, 67, 143-150. (9) Abad-Valle, P.; Fernańdez-Abedul, M. T.; Costa-Garcıá, A. Biosens. Bioelectron. 2005, 20, 2251-2260. (10) Hernańdez-Santos, D.; Dıáz-Gonza´lez, M.; Gonza´lez-Garcıá, M. B.; CostaGarcıá, A. Anal. Chem. 2004, 76, 6887-6893. (11) Xie, H.; Yu, Y. H.; Xie, F.; Lao, Y. Z.; Gao, Z. Clin. Chem. 2004, 50, 12311233. (12) Azek, F.; Grossiord, C.; Joannes, M.; Limoges, B.; Brossier, P. Anal. Biochem. 2000, 284, 107-113. (13) Dequaire, M.; Heller, A. Anal. Chem. 2002, 74, 4370-4377. (14) Huang, T. J.; Liu, M.; Knight, L. D.; Grody, W. W.; Miller, J. F.; Ho, C. M. Nucleic Acids Res. 2002, 30, e55. (15) Metfies, K.; Huljic, S.; Lange, M.; Medlin, L. K. Biosens. Bioelectron. 2005, 20, 1349-1357. (16) Hwang, G.; Kim, E.; Kwak, J. Anal. Chem. 2005, 77, 579-584. (17) Carpini, G.; Lucarelli, F.; Marrazza, G.; Mascini, M. Biosens. Bioelectron. 2004, 20, 167-175. (18) Lucarelli, F.; Marrazza, G.; Mascini, M. Biosens. Bioelectron. 2005, 20, 20012009. (19) Urban, M.; Mo ¨ller, R.; Fritzsche, W. Rev. Sci. Instrum. 2003, 74, 10771081. (20) Moreno-Hagelsieb, L.; Lobert, P. E.; Pampin, R.; Bourgeois, D.; Remacle, J.; Flandre, D. Sens. Actuators, B 2004, 98, 269-274. (21) Tsai, C. Y.; Chang, T. L.; Chen, C. C.; Ko, F. H.; Chen, P. H. Microelectron. Eng. 2005, 78-79, 546-555. (22) Ruffien, A.; Dequaire, M.; Brossier, P. Chem. Commun. 2003, 912-913. (23) Hernańdez-Santos, D.; Gonza´lez-Garcıá, M. B.; Costa-Garcıá, A. Electrochim. Acta 2000, 46, 607-615. (24) Hernańdez-Santos, D.; Gonza´lez-Garcıá, M. B.; Costa-Garcıá, A. Electroanalysis 2000, 12 (18), 1461-1466. 10.1021/ac070624o CCC: $37.00

© 2007 American Chemical Society Published on Web 06/15/2007

typical in the case of the conventional metal enhancement process of growing nanoparticles. It also leads to a significant increase in sensitivity for electrical detection of DNA chips.25 The methods based on the use of enzymatic labels are without doubt among the most sensitive. Thus, enough sensitivity to avoid PCR amplification has been achieved by use of enzymatic labels.11,12,16 Moreover, because routine laboratories are usually working with enzymatic labels, this might make them the most attractive labels for implementation of DNA diagnosis in such laboratories. The analytical signal normally arises from a redox process of a product of the enzymatic reaction. However, in other cases, the analytical signal is not based on the redox process of the enzymatic product. Thus, Kwak et al.16 used a biometallization process when a nonelectroactive substrate (p-aminophenyl phosphate) was enzymatically converted into the reducing agent p-aminophenol that reduces Ag+ ions leading to deposition of the metal onto electrode surface. In this case, the oxidation peak of deposited Ag was recorded. Mascini et al.18 used the alkaline phosphatase substrate BCIP/NBT that after the enzymatic reaction generates an insoluble and insulating product on the sensing phase that blocks the electrical communication between the electrode surface and the Fe(CN)63-/4- redox pair. In this case, faradaic impedance spectroscopy is finally used to detect the enhanced electron-transfer resistance. This work outlines the use of a new substrate solution that combines an indoxyl compound, 3-indoxyl phosphate (3-IP), and silver ions. An enzymatic reaction mechanism is proposed, and the parameters that affect the enzymatic reaction are studied using the streptavidin-biotin interaction, which is carried out on the surface of a screen-printed carbon electrode (SPCE). Our group has described 3-IP as a suitable electrochemical substrate for alkaline phosphatase (AP).26 The resulting enzymatic product is indigo blue, an aromatic heterocycle insoluble in aqueous solutions. Two strategies can be carried out to detect the product: its sulfonation in acidic medium, giving rise to indigo carmine (IC),27,28 or its solubilization in basic medium and in presence of dithionite salt, giving rise to leucoindigo.29 The main drawback of these methodologies is that, in all cases, it is necessary to add a step for detection after the enzymatic reaction and the use of aggressive agents such concentrated sulfuric acid or sodium dithionite, respectively. The substrate proposed here overcomes these drawbacks and, moreover, improves the sensitivity of the methodology. To demonstrate the better sensitivity obtained with this substrate, an enzymatic genosensor on SPCEs for the identification of nucleic acid determinants exclusively present on the genome of the pathogen Streptococcus pneumoniae has been developed. The different steps of this genosensor have been optimized in a previous work.10 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 (25) Mo ¨ller, R.; Powell, R. D.; Hainfeld, J. F.; Fritzsche, W. Nano Lett. 2005, 77 (2), 579-584. (26) Fernańdez-Sańchez, C.; Costa-Garcıá, A. Electroanalysis 1998, 10 (4), 249255. (27) Dıáz-Gonza´lez, M.; Hernańdez-Santos, D.; Gonza´lez-Garcıá, M. B.; CostaGarcıá, A. Talanta 2005, 65, 565-573. (28) Fanjul-Bolado, P.; Gonza´lez-Garcıá, M. B.; Costa-Garcıá, A. Talanta 2004, 64, 452-457. (29) Fanjul-Bolado, P.; Gonza´lez-Garcıá, M. B.; Costa-Garcıá, A. Anal. Chim. Acta 2005, 534, 231-238.

immobilization of biotinylated oligo probe. The biotinylated oligonucleic acid probe used in this work targets the autolysin (lytA) gene. This target is randomly labeled with the Universal Linkage System (ULS).30 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 substrate proposed here, 3-IP/Ag+. EXPERIMENTAL SECTION Materials. Tris(hydroxymethyl)aminomethane (Tris), 3-indoxyl phosphate disodium salt (3-IP), bovine serum albumin fraction V (BSA), streptavidin (molecular weight, 66 000), biotin conjugated to alkaline phosphatase (B-AP; dimer, four units of B per molecule of AP, molecular weight, 160 000), rabbit IgG antiFITC conjugated to alkaline phosphatase (Ab-AP), and silver nitrate were purchased from Sigma (Madrid, Spain). A mixture solution of 5.6 mM 3-IP and 0.4 mM silver nitrate were prepared daily in 0.1 M Tris-HNO3 pH 9.8 and 20 mM Mg(NO3)2 and stored in opaque tubes at 4 °C. Working solutions of streptavidin were made in 0.1 M TrisHNO3 pH 7.2 buffer. BSA lyophilized powder was reconstituted in ultrapure water. Working solutions of B-AP and Ab-AP were made in 0.1 M Tris-HNO3 pH 7.2 containing 2 mM Mg(NO3)2 and 1% BSA. 3′-Biotinylated 30-mer oligonucleotide probe with a 10-T spacer in the 3′-end and the complementary 20-mer labeled target corresponding to the autolysin (lytA) gene, as well as a noncomplementary target, were purchased from Isogen Life Science (Maarssen, The Netherlands). ULS-FITC labeling of these targets was carried out by Kreatech Biotechnology (Amsterdam, The Netherlands). The sequence of the oligonucleotide probe and its complementary target and noncomplementary target were as follows:

lytA probe: 5′-TTG CAT CAT GCA GGT AGG ACT TTT TTT TTT-3′-biotin lytA target: 5′-GTC CTA CCT GCA TGA TGC AA-3′ noncomplementary target: 5′-GAT TTG CCT GGT TTG GCA AG-3′ Concentrated aliquots of all oligonucleotides were prepared in 0.1 M Tris-HNO3 pH 7.2 and stored at -20 °C. Working solutions of all biotinylated oligonucleotide probes were made in 0.1 M Tris-HNO3 pH 7.2 buffer, containing 1% BSA. Working solutions of all oligonucleotide targets were diluted in 2× SSC buffer (300 mmol/L sodium nitrate-30 mmol/L sodium citrate) pH 7.2, containing 1% BSA (hybridization solution). Ultrapure water obtained with a Milli-Q plus 185 from Millipore Ibe´rica S.A. (Madrid, Spain) was used for all solutions. Nitrocellulose membranes were purchased from Pierce to carry out the qualitative assays. Apparatus and Electrodes. Cyclic voltammetric experiments were performed with an Eco Chemie Autolab PGSTAT 12 potentiostat interfaced to a AMD K-6 266-MHz computer system (30) Lempers, E. L. M.; Van der Berg, F. M.; Reedijk, J.; Bloemink, M. J. US patent 5580990, 1996.

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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. The procedure to perform the reaction between streptavidin and B-AP on SPCEs has been previously optimized27 and it comprises the following steps: Streptavidin Coating of SPCEs. An electrode pretreatment was carried out before each voltammetric experiment with the aim of improving the sensitivity and repeatability of the results. A 40-µL sample of 0.1 M H2SO4 was dropped on the SPCEs, and an anodic current of +5.0 µA was applied for 2 min. Then, the electrodes were washed using 0.1 M Tris-HNO3 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-HNO3 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-HNO3 pH 7.2 buffer containing 1% BSA. Reaction with B-AP. An aliquot of 40 µL of B-AP (different concentrations) was dropped on the streptavidin-modified electrode for 1 h. Then, the electrode was washed with 0.1 M TrisHNO3 buffer pH 9.8 to remove the excess of protein Enzymatic Reaction and Metallic Silver Deposition. This step was developed with an aliquot of 35 µL of a mixture of 5.6 mM 3-IP and 0.4 mM silver nitrate solutions for 20 min, protected from light. Detection Step. The SPCE was held at -0.20 V for 5 s, and a cyclic voltammogram was recorded (in the same enzymatic reaction medium) from -0.20 to 0.50 V at a scan rate of 50 mV/s. Design of the Genosensor Device. The electrode pretreatment was carried out following the procedure mentioned above. The formation of the sensing phase was optimized in a previous work.10 It was performed with 40 µL of 3′-biotinylated oligonucleotide probe (0.5 ng/µL) for 15 min. Then, the electrodes were rinsed with 2× SSC buffer pH 7.2 containing 1% BSA. After that, the hybridization was performed at room temperature placing 30 µL of FITC-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-HNO3 pH 7.2 buffer containing 1% BSA. Then, a reaction with Ab-AP was performed 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-HNO3 buffer pH 9.8, containing 1% BSA, the enzymatic reaction and metallic silver deposition and the detection step were carried out as mentioned above. Qualitative Assay on Nitrocellulose Membranes. Four assays were carried out on these membranes. In the first assay, 5274


three aliquots of 2 µL of 1.25 × 10-5 M of B-AP solution were dropped on the membrane and left there until dry. After that, three aliquots of 2 µL of 5.6 mM 3-IP were dropped. In the second assay, the same procedure was carried out, but after the drops of B-AP were dried, three aliquots of 2 µL of a mixture of 5.6 mM 3-IP and 0.4 mM silver nitrate solutions were dropped. The third assay was carried out as in the second assay but without 3-IP in the mixture. The last assay was performed without B-AP. After 5 min of enzymatic reaction, a photograph of all assays was taken. RESULTS AND DISCUSSION Alkaline Phosphatase-Catalyzed Silver Deposition Mechanism. 3-IP is basically formed by an indolic ring substituted in position 3 by a phosphate group. In the presence of alkaline phosphatase, this substrate is hydrolyzed in position 3, giving an indoxyl intermediate. It suffers from a keto-enol tautomerism and in presence of atmospheric oxygen is oxidized to produce an indigo blue. This product is characterized by its insolubility in aqueous solution and an intense blue color. Sometimes for the development of assays on membranes, indoxyl substrates are combined with other compounds as tetrazolium salts that enhance the reaction rate and the color of the enzymatic product. In this way, after the generation of the indoxyl intermediate, that acts as a reducing agent, another product, a formazan derivate, is co-deposited with indigo blue, resulting in an enhancement of blue color (Figure 1A). Through the simultaneously generation of two colored products, the sensitivity of the assay is improved. By using the same reaction mechanism, we propose the combination of silver ions with the indoxyl substrate. By this way, we will achieve a simultaneous deposition of indigo and metallic silver (Figure 1B). A qualitative assay was carried out on nitrocellulose membranes to demonstrate the reaction mechanism we have proposed, using 3-IP as substrate and AP as enzyme. The results are shown in Figure 2. In column A, a blue indigo product is generated and visually detected when B-AP is mixed with 3-IP. When silver ions are added to the substrate solution, an intensification of the product color is observed due to the simultaneous precipitation of metallic silver (column B). Thus, silver ions act as enhancers of the enzymatic reaction. When the assay is developed in the absence of the indoxyl substrate (column C) or in the absence of AP (column D), generation of any colored product is not observed. Then, the conclusion to this qualitative assay is that the enzymatic substrate produces a compound able to reduce silver ions in solution into a metallic deposit, which is located where the enzyme is attached. Some potential applications for the reaction mechanism proposed are as follows: immunohistochemistry, immunocytochemistry, tests performed on membranes, nanotechnology, and biosensors. Electrochemical Detection of Silver Deposition Catalyzed by Alkaline Phosphatase. For this study, the reaction between streptavidin and biotin conjugated with alkaline phosphatase (BAP) was performed on the surface of SPCEs. The experimental conditions used to modify the electrode surface with streptavidin by physical adsorption and the reaction with B-AP have been studied in detail in a previous work,27 using 3-IP as the electrochemical substrate. In order to avoid repeatability problems associated with the use of streptavidin, previously reported by

Figure 1. Enzymatic reaction in the presence of alkaline phosphatase using 3-indoxyl phosphate and (A) nitro blue tetrazolium salt or (B) silver ions.

Figure 2. Qualitative assays on nitrocellulose membranes. Column A: 45 µg of B-AP + 5.6 mM 3-IP. Column B: 45 µg of B-AP + 5.6 mM 3-IP + 0.4 mM silver nitrate. Column C: 45 µg of B-AP + 0.4 mM silver nitrate. Column D: 5.6 mM 3-IP + 0.4 mM silver nitrate. Enzymatic reaction time, 5 min.

other authors,31 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. When the substrate 3-IP/Ag+ is used to perform the enzymatic reaction, metallic silver is deposited on the electrode surface in

the presence of B-AP. After the enzymatic reaction, the metallic silver deposited on the electrode surface is oxidized when anodic stripping is carried out from -0.20 to +0.50 V, obtaining a stripping peak of silver at ∼ +0.18 V (Figure 3, curve a). In the absence of B-AP, no metallic silver is deposited on the electrode surface, and consequently, no stripping peak is observed (Figure 3, curve b). The height of the stripping peak of silver depends on the concentration of B-AP. Moreover, another anodic process can be observed in both voltammograms. This process corresponds to the oxidation of 3-IP that in basic medium occurs at ∼ +0.5 V. The parameters that affect the enzymatic reaction have been optimized. The initial potential of the anodic stripping scan is the first important parameter to optimize. In Figure 4, three linear voltammograms corresponding to background signals (without B-AP) obtained for 3 mM 3-IP and 0.2 mM silver nitrate mixture are shown. The initial potential is different in each linear voltammogram. It can be observed that if this initial potential is too negative, an electrochemical reduction of silver ions is produced on the electrode surface, and consequently, a stripping peak is recorded. This electrochemical reduction interferes with the enzyme-catalyzed silver deposition. Therefore, to avoid this interference, the initial potential was fixed at -0.2 V (linear voltammogram c in Figure 4), where there is no analytical signal due to the electrochemical reduction of silver ions in solution. (31) Marrazza, G.; Chianella, I.; Mascini, M. Biosens. Bioelectron. 1999, 14, 4351.


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Figure 3. Cyclic voltammograms obtained for 4.5 × 10-11 M B-AP (curve a) and in the absence of B-AP (curve b). Enzymatic reaction time, 20 min; 3-IP concentration, 5.6 mM; silver ion concentration, 0.4 mM; scan rate, 50 mV/s. The rest of the experimental conditions are in the text.

Figure 5. Cyclic voltammograms recorded for a concentration of silver nitrate in substrate solution of (a) 1.2 and (b) 0.4 mM. B-AP concentration, 4.5 × 10-11M; 3-IP concentration, 3.0 mM; enzymatic reaction time, 20 min; scan rate, 50 mV/s.

Figure 6. Effect of the concentration of 3-IP in the substrate solution on the biosensor response for a B-AP concentration of 9.0 × 10-12 M. Silver concentration, 0.4 mM; enzymatic reaction time, 20 min.

Figure 4. Linear voltammograms obtained in the absence of B-AP for a mixture of 3.0 mM 3-IP and 0.2 mM silver nitrate solution from -0.4 (a), -0.3 (b), and -0.2 (c) to +0.2 V. Scan rate, 50 mV/s.

The concentration of the two main components of the substrate solution, 3-IP and silver ions, has also been optimized, using the interaction between streptavidin and B-AP. Figure 5 shows two cyclic voltammograms obtained for a concentration of B-AP of 4.5 × 10-11 M. In the case of silver ion concentration, a double stripping peak was detected when silver concentrations higher than 0.4 mM were used in the substrate solution. Thus, in curve a of Figure 5, a double stripping peak is obtained for a concentration of 1.2 mM. This behavior is because the concentration of silver ions is so high that the electrochemical reduction of silver occurs at the initial potential of -0.20 V. However, when this concentration was 0.4 mM, only one narrow peak was obtained. Comparing Figure 5a with Figures 3 and 4, it can be observed that the potential of the first peak (at +0.06 V) is similar to that obtained in Figure 4, whereas the potential of the second peak (at +0.18 V) of Figure 5a is similar to that obtained in the voltammogram of Figure 3. The first one corresponds to silver electrochemically deposited, and the second one corresponds to the silver enzymatically deposited. The different oxidation potentials could be because the silver enzymatically generated is codeposited with insoluble indigo blue, which can make the oxidation of this silver difficult, whereas the silver electrochemically generated is deposited on the electrode surface without any compound that can hamper its oxidation. To ensure that the electrochemical reduction of silver does not occur on the electrode surface, a concentration of 0.4 mM was chosen for further studies. Figure 6 displays the effect of the concentration of 3-IP on the analytical signal for a fixed concentration of 9.0 × 10-12 M B-AP. 5276


The best analytical signals were obtained for concentrations between 3 and 9 mM 3-IP. For concentrations higher than 9 mM, the oxidation wave of 3-IP increases and begins at a lower potential, overlapping the stripping peak of silver and, consequently, making its measurement difficult. A concentration of 5.6 mM 3-IP was chosen for further studies. Other parameters, such pH and magnesium ion concentration of the substrate solution, that affect the enzymatic reaction were evaluated. The higher analytical signals were obtained for a pH of 9.8 and a concentration of magnesium ions of 20 mM (data not shown). The enzymatic reaction time was also studied. The results are shown in Figure 7. The analytical signal increases with enzymatic reaction time whereas the background signals appear only at times higher than 30 min. Under the above-mentioned optimized experimental conditions, the relative standard deviation of five parallel experiments was 7.0% with a mean peak current of 1.5 µA for a 9.0 × 10-13 M B-AP, when an enzymatic reaction time of 20 min was used. Moreover, the dose-CV response curve recorded under the above experimental conditions, fits -the following equation:

ip (µA) ) 7 × 1011 [B - AP] (M) + 0.11; r ) 0.999;

n)6

A linear range from 4.5 × 10-13 to 9.0 × 10-11 M was obtained. Moreover, the sensitivity of the assay could be improved by increasing the enzymatic reaction time. For these experimental conditions and in this range of concentrations of B-AP, no background signals (without streptavidin on the electrode surface) are obtained. Comparing these results with those obtained in a previous work,27 where the analytical signal was obtained through the measure of a reversible electrode process of a sulfonic derivate of indigo blue (called indigo carmine) by square wave voltamme-

Figure 7. Effect of the enzymatic reaction time on the analytical (gray line) and background (black line) signals. B-AP concentration, 2.0 × 10-11 M; 3-IP concentration, 5.6 mM; silver ion concentration, 0.4 mM.

try, the sensitivity of the biosensor is improved ∼3.5-fold in terms of slope using the enzymatic silver deposition, despite the fact that square wave voltammetry is an electrochemical technique usually more sensitive than cyclic voltammetry. Moreover, the reproducibility observed for lower concentrations of B-AP using SWV is improved considerably. Genosensor Response. Once the procedure was optimized, an enzymatic genosensor for the identification of a nucleic acid determinant exclusively present on the genome of the pathogen S. pneumoniae was developed. This DNA sensor has been described and optimized by our research group in a previous work.10 In this work, for the electrochemical detection step, 3-IP was used as substrate and then sulfuric acid was added to generate an electroactive compound termed indigo carmine, which is quantified by cyclic voltammetry. In this case, by combining the 3-IP with silver ions, the metallic silver deposited on the electrode surface is detected directly without the need of any more steps to obtain the analytical signal. Thus, the use of sulfuric acid is avoided. Using the optimized experimental conditions, the response of the genosensor formed with 3′-biotinylated lytA probe for different concentrations of the complementary oligonucleotide target has been evaluated. In Figure 8 is shown the calibration plot (Figure 8A) and the voltammograms corresponding to each concentration as well as the voltammogram corresponding to the noncomplementary target for the highest concentration assayed (Figure 8B). A linear relationship between peak current and concentration of complementary lytA target is obtained between 7 and 700 fg/µL with a correlation coefficient of 0.9995, according to the following equation:

ip (µA) ) 0.55 + 0.064 [lytA target] (fg/µL) The reproducibility of the analytical signal for the concentrations of complementary target assayed are shown with error bars in Figure 8A. It is composed between 4 and 10 in terms of percent RSD. Also, comparing linear ranges obtained for target autolysin through both methodologies, the sensitivity of the assay is improved by at least 1 order of magnitude. Thus, this genosensor can detect 7 fg/µL, which is 35 amol of lytA target in 30 µL, ∼14-fold lower than that obtained when the enzymatic reaction was carried out only with 3-IP.10 Also, the use of 3-IP as the enzymatic substrate allows a better control of the silver deposition versus the use of another substrate such as p-aminophenyl phosphate that is more unstable16 and produces higher background signals.

Figure 8. (A) lytA genosensor responses for different concentrations of complementary target. Data are given as average ( SD (n ) 3). (B) Cylic voltammograms corresponding to the background (700 fg/ µl of noncomplementary target) and to each concentration of complementary target of the linear calibration curve.

Moreover, the hybridization reaction with noncomplementary target does not occur for all concentrations assayed (see the voltammogram in Figure 8B for the highest concentration of noncomplementary target assayed, 700 fg/µL). This fact shows that nonspecific adsorptions are not observed. Regarding the selectivity of the genosensor, this system has been studied in a previous work10 and this is able to discriminate one base mismatched strands. CONCLUSIONS In this work, we demonstrate the advantages of the combination of 3-indoxyl phosphate and silver ions as a substrate of alkaline phosphatase. After the generation of the indoxyl intermediate, which acts as a reducing agent, another product, metallic silver, is codeposited with indigo blue, and consequently, an intensification of the product color is observed due tothe simultaneous precipitation of metallic silver. Some potential applications to use the reaction mechanism proposed here are immunohistochemistry, immunocytochemistry, tests performed on membranes, nanotechnology, and biosensors. As an example of the latter case, a genosensor device for the detection of S. pneumoniae has been developed. Thus, the silver enzymatically deposited on the electrode surface can be detected through the anodic peak of the silver when an anodic stripping scan is carried out. This methodology is more sensitive than the direct detection of IC, being able to detect 35 amol in 30 µL. ACKNOWLEDGMENT This work has been supported by project FC-04-PC-04-11. Received for review March 29, 2007. Accepted May 14, 2007. AC070624O Analytical Chemistry, Vol. 79, No. 14, July 15, 2007

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Alkaline Phosphatase-Catalyzed Silver Deposition for Electrochemical

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