Simultaneously Amplified Electrochemical and Surface Plasmon

Chem. , 2005, 77 (9), pp 2756–2761 ... The detection limit of this DNA sensor is 10 pM (2 fmol, with signal to noise > 3). ... Analytical Chemistry ...
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Anal. Chem. 2005, 77, 2756-2761

Simultaneously Amplified Electrochemical and Surface Plasmon Optical Detection of DNA Hybridization Based on Ferrocene-Streptavidin Conjugates Jianyun Liu,† Shengjun Tian,† Louis Tiefenauer,‡ Peter Eigil Nielsen,§ and Wolfgang Knoll*,†

Max-Planck-Institute for Polymer Research, Ackermannweg 10, D-55128, Mainz, Germany, Life Sciences Department, Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland, and Center for Biomolecular Recognition, Department for Biochemistry and Genetics, Biochemistry Laboratory B, The Panum Institute, Blegdamsvej 3C, DK-2200, Copenhagen Denmark

A sensitive method based on ferrocene-streptavidin (FcStv) conjugates for the simultaneously amplified electrochemical and surface plasmon optical detection of DNA target hybridization to peptide nucleic acid-modified gold surfaces is reported. The attachment of Fc-Stv to the biotinylated complementary target DNA not only amplified the surface plasmon resonance signal but also enhanced the electrochemical signal due to the many Fc markers per Stv. The ferrocene redox peak current increased with the increase of the target DNA concentration. Consequently, the amount of hybridized target DNA can be estimated by cyclic voltammetry and chronocoulometry. The detection limit of this DNA sensor is 10 pM (2 fmol, with signal to noise > 3). This sensor was also shown to have high selectivity (at the single-base mismatch level) and good reproducibility. The detection of DNA hybridization reactions holds great promise for the rapid clinical diagnosis of genetic diseases. Various techniques have been used for the detection of DNA hybridization, such as optical,1-5 electrochemical,6-14 or piezoelectric transduc* Corresponding author. E-mail: [email protected]. † Max-Planck-Institute for Polymer Research. ‡ Paul Scherrer Institut. § The Panum Institute. (1) (a) Piuuno, P. A. F.; Krull, U. J.; Hodson, R. H. E.; Damha, M. J.; Cohen, H. Anal. Chem. 1995, 67, 2635-2643. (b) Dore, K.; Dubus, S.; Ho, H.-A.; Levesque, I.; Brunette, M.; Corbeil, G.; Boissinot, M.; Boivin, G.; Bergeron, M. G.; Boudreau, D.; Leclerc, M. J. Am. Chem. Soc. 2004, 126, 42404244. (c) Nilsson, K. P. R.; Ingana¨s, O. Nat. Mater. 2003, 2, 419-424. (2) Jenison, R.; Yang, S.; Haeberli, A.; Polisky, B. Nat. Biotechnol. 2001, 19, 62-65. (3) (a) Thiel, A. J.; Frutos, A. G.; Jordan, C. E.; Corn, R. M.; Smith, L. M. Anal. Chem. 1997, 69, 4948-4956. (b) Lee, H. J.; Goodrich, T. T.; Corn, R. M. Anal. Chem. 2001, 73, 5525-5531. (4) (a) Piscevic, D.; Lawall, R.; Veith, M.; Liley, M.; Okahata, Y.; Knoll, W. Appl. Surf. Sci. 1995, 90, 425-436. (b) Liebermann, T.; Knoll, W.; Sluka, P.; Hermann, R. Colloids Surf., A 2000, 169, 337-350. (c) Peterlinz, K. A.; Georgiadis, R.; Herne, T, M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 3401-3402. (5) Lu, J.; Strohsahl, C. M.; Miller, B. L.; Rothberg, L. J. Anal. Chem. 2004, 76, 4416-4420. (6) Palecek, E.; Fojta, M. Anal. Chem. 2001, 73, 74A-83A.

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tion techniques,15-18 with the main interest focused on improving the selectivity and sensitivity of the surface-based DNA hybridization detection systems. Among the techniques used, surface plasmon resonance (SPR) and electrochemical methods have drawn great attention. Conventional SPR has limited applications in ultrasensitive detection due to its inability to measure extremely small changes in refractive indices. However, by using external labels with high molecular weight or with a high refractive index, the sensitivity of SPR has been shown to be greatly enhanced.19-23 (7) (a) Wang, J.; Chem. Eur. J. 1999, 5, 1681-1685. (b) Wang, J. Anal. Chim. Acta 2002, 469, 63-71. (c) Wang, J.; Liu, G.; Jan, M. R. J. Am. Chem. Soc. 2004, 126, 3010-3011. (8) (a) Campbell, C. N.; Gal, D.; Cristler, N.; Banditrat, C.; Heller, A. Anal. Chem. 2002, 74, 158-162. (b) Caruana, D. J.; Heller, A. J. Am. Chem. Soc. 1999, 121, 769-773. (c) Dequaire, M.; Heller, A. Anal. Chem. 2002, 74, 43704377. (d) Zhang, Y.; Kim, H. H.; Mano, N.; Dequaire, M,; Heller, A. Anal. Bioanal. Chem. 2002, 374, 1050-1055. (e) Zhang, Y.; Pothukuchy, A.; Shin, W.; Kim, Y.; Heller, A. Anal. Chem. 2004, 76, 4093-4097. (9) (a) Patolsky, F.; Lichtenstein, A.; Willner, I. Nat. Biotechnol. 2001, 19, 253257. (b) Patolsky, F.; Katz, E.; Bardea, A.; Willner, I. Langmuir 1999, 15, 3703-3706. (c) Patolsky, F.; Lichtenstein, A.; Willner, I. Angew. Chem., Int. Ed. 2000, 39, 940-943. (10) Napler, M. E.; Loomis, C. R.; Sistare, M. F.; Kim, J.; Ecrkhardt, A. E.; Torp, H. H. Bioconjugate Chem. 1997, 8, 906-913. (11) Jelen, F.; Yosypchuk, B.; Kourilove, A.; Novotny, L.; Paleck, E. Anal. Chem. 2002, 74, 4788-4793. (12) Boon, E. M.; Ceres, D. M.; Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2000, 18, 1096-1100. (13) Takennaka, S.; Yamashita, K.; Takagi, M.,; Uto, Y.; Kondo, H. Anal. Chem. 2000, 72, 1334-1341. (14) Yu, C. J.; Wan, Y.; Yowanto, H.; Li, J.; Tao, C.; James, M. D.: Tan, C. L.; Blackburn, G. F.; Meade, J. J. J. Am. Chem. Soc. 2001, 123, 11155-11161. (15) Caruso, F.; Rodda, E.; Furlong, D. N.; Niikura, K.; Okahara, Y. Anal. Chem. 1997, 69, 2043-2049. (16) Bardea, A.; Dagan, A.; Ben-Dov, I.; Amit, B.; Willner, I. Chem. Commun. 1998, 839-840. (17) Patolsky, F.; Lichtenstein, A.; Willner, I. J. Am. Chem. Soc. 2000, 122, 418419. (18) Larsson, C.; Rodahl, M.; Ho ¨o ¨k, F. Anal. Chem. 2003, 75, 5080-5087. (19) He, L; Musick, M. D.; Nicewarner S. R.; Salinas, F. G..; Benkovic, S. J.; Natan, M. J.; Keating, C. D. J. Am. Chem. Soc. 2000, 122, 9071-9077. (20) (a) Lyon, L. A.; Musick, M. D.; Natan, M. J. Anal. Chem. 1998, 70, 51775183. (b) Lyon, L. A.; Pena, D. J.; Natan, M. J. J. Phys. Chem. B 1999, 103, 5826-5831. (21) Wink, T.; van Zuilen, S. J.; Bult, A.; van Bennekom, W. P. Anal. Chem. 1998, 70, 827-832. (22) (a) Kubitschko, S.; Spinke, J.; Bruckner, T.; Pohl, S.; Oranth, N. Anal. Biochem. 1997, 253, 112-122. (b) de Vries, E. F. A.; Schasfoort, R. B. M.; van der Plas, J.; Greve, J. Biosens. Bioelectron. 1994, 9, 509-514. 10.1021/ac048088c CCC: $30.25

© 2005 American Chemical Society Published on Web 03/26/2005

Until now, colloid Au nanoparticles,19 liposomes,21 latex particles,22 and proteins23 have all been used as effective amplification tags. Many other highly sensitive techniques derived from SPR and their (bio-)sensing applications have also been reported recently.24-27 The increased interest in electrochemical methods for hybridization detection arises from the high sensitivity, low cost, and their compatibility with microfabrication technology. One of the most often used approaches for the electrochemical detection of hybridization reactions is based on using enzyme labels in order to generate an amplified signal through different schemes, such as via the production of an electroactive compound,28 via bioelectrocatalytic redox transformations,8b,29 or via the biocatalyzed precipitation of an insoluble product at the electrode as a result of the formation of the double-stranded DNA complexes.9b Although large signal amplification from enzyme labels can be achieved, labeling of an oligonucleotide by an enzyme is not a straightforward route, and once prepared, the activity of the conjugate must be periodically controlled owing to the inherent poor stability of enzymes. Moreover, the quantification of the hybridization reaction by the catalytic signal is also difficult. Streptavidin exhibits strong affinity to biotin (Kd ) 10-13 mol dm-3) and is extremely stable toward thermal denaturation or against chaotropes.35 Moreover, it can be bound to a (sensor) surface by various biotin derivatives for biosensor applications. The amplification of the DNA sensing process by biotinstreptavidin interaction and the transduction of the recognition event by an SPR imaging assay was once reported.23 However, the large size of streptavidin (54 × 58 × 48 Å) 31 blocks heavily the electron transfer and limits its application in electronic transduction schemes for biomolecular recognition reactions. Ferrocene is a very good redox-active molecule, which exhibits excellent reversibility of its redox reaction and offers the versatility of synthesizing various Fc derivatives. Fc has been used as an indicator for gene sequencing by many researchers. Willner’s group reported an amplified bioelectrocatalytic detection of viral DNA by introducing a large number of Fc groups through the replication of Fc-labeled DNA by DNA polymerase.29b Ferrocenylated oligodeoxynucleotides were synthesized and used for (23) Jordon, C. E.; Frutos, A. G.; Thiel, A. J.; Corn, R. M. Anal. Chem. 1997, 69, 4939-4947. (24) (a) Yu, F; Persson, B.; Lofas, S.; Knoll, W. J. Am. Chem. Soc. 2004, 126, 8902-8903. (b) Yu, F.; Tian, S. J.; Yao, D. F.; Knoll, W. Anal. Chem. 2004, 76, 3530-3535. (25) Hutter, E.; Peleni, M-P. J. Phys. Chem. B. 2003, 107, 6497-6499. (26) Malcka, J.; Gryczynski, I.; Gryczynski, Z.; Lakowicz, J. R. Anal. Chem. 2003, 75, 6629-6633. (27) Goodrich, T. T.; Lee, H. J.; Corn, R. M. J. Am. Chem. Soc. 2004, 126, 40864087. (28) Azek, F.; Grosslord, C.; Joammes, M.; Limoges, B.; Brossier, P. Anal. Biochem. 2000, 284, 107-113. (29) (a) de Lumley-Woodyear, T.; Caruana, D. J.; Campell, C. N.; Heller, A. Anal. Chem. 1999, 71, 394-398. (b) Patolsky, F.; Weizmann, Y.; Willner, I. J. Am. Chem. Soc. 2002, 124, 770-772. (c) Zhang, Y.; Kim, H.; Heller, A. Anal. Chem. 2003, 75, 3267-3269. (30) Cosnier, S.; Lepeflec, A. Electrochim. Acta 1999, 44, 1833-1836. (31) Hendrickson, W. A.; Pa¨hler, A.; Smith, J. L.; Satow, Y.; Merritt, E. A.; Phizackerley, R. P. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 2190-2195. (32) Mucic, R. C.; Herrlein, M. K.; Mirkin, C. A.; Letsinger, R. L. Chem. Commun. 1996, 4, 555-558. (33) Ihara, T.; Maruo, Y.; Takenaka, S.; Takagi, M. Nucleic Acids Res. 1996, 24, 4273-4280. (34) Wang, J.; Poisky, R.; Merkoci, A.; Turner, K. L. Langmuir 2003, 19, 989991. (35) Wang, J.; Li, J.; Baca, A. J.; Hu, J.; Zhou, F.; Yan, W.; Pang, D. W. Anal. Chem. 2003, 75, 3841.

the electrochemical probing of DNA hybridization reactions.32,33 Fc-functionalized molecule beads34 or particles35 were also used to tag DNA target, and the binding of Fc molecules was confirmed by chronopotentiometry or cyclic voltammetry. Heller,8c,d Azek28 and Kwak36 used Fc as electrochemical mediators for enzyme catalytic reactions in enzyme-amplified DNA detection. Recently, ferrocene was successfully linked to streptavidin, with the obtained Fc-Stv molecules keeping their bioactivity. Furthermore, electron transfer becomes feasible due to the electron shuttle between Fc and the electrode surface,37 which makes it possible to bind many biotin derivatives to Fc-Stv for electrochemical characterization and detection. Peptide nucleic acid (PNA) represents very good structural mimics of DNA, in which the entire sugar-phosphate backbone is replaced by uncharged N-(2-aminethyl)glycine linkages with nucleobases attached through methylene carbonyl linkages to the peptide backbone. PNA has a high binding affinity to complementary DNA sequences, obeying the Watson-Crick base-pairing rules.38a-e PNA-DNA duplexes are thermally more stable than the corresponding DNA-DNA duplexes.38c Moreover, the stability of PNA-DNA duplexes is almost unaffected by the ionic strength of the medium, in contrast to the behavior of DNA double-helical structures.38d In this report, we used Fc-Stv to bind to biotinylated DNA targets for the detection of their hybridization to PNA probes on an Au electrode surface. Without any other enzyme label, the detection sensitivity can go down to the picomolar level (2 fmol with signal-to-noise > 3) by using square wave voltammetry (SWV). Owing to the large size of streptavidin, we can also detect the hybridization event by SPR at the same time. The quantification of target DNA can be easily obtained from cyclic voltammetry or chronocoulometry. This sensor architecture can also discriminate effectively different DNA sequences (at the single-base mismatch level) and can be regenerated many times for repeated use. EXPERIMENTAL SECTION Chemicals and Reagents. Mercaptohexanol (MCH) was purchased from Sigma. Fecrrocene-streptavidin conjugates were synthesized as reported before.37 All buffer salts and other organic chemicals were obtained from Sigma or Aldrich unless otherwise stated. All chemicals were used as received. The buffer used for the PNA assembly was 100 mM sodium phosphate buffer (PB, pH 7.4). The hybridization buffer and the (36) Kim, E.; Kim, K.; Yang, H.; Kim, Y. T.; Kwak, J. Anal. Chem. 2003, 75, 5665-5672. (37) (a) Padeste, C.; Grubeln, K. A.; Tiefenauer, L. Electrochim. Acta 2003, 48, 761-769. (b) Padeste, C.; Grubelnik, A.; Tiefenauer, L. Biosens. Bioelectron. 2000, 15, 431-438. (c) Padeste, C.; Steiger, B.;Grubelnik, A.; Tiefenauer, L. Biosens. Bioelectron. 2003, 19, 239-247. (d) Padeste, C.; Steiger, B.;Grubelnik, A.; Tiefenauer, L. Biosens. Bioelectron. 2004, 20, 545-552. (38) (a) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991, 254, 1497-1500. (b) Egholm, M.; Buchardt O.; Nielsen, P. E.; Berg R. H. J. Am. Chem. Soc. 1992, 114, 1895-1897. (c) Egholm, M.; buchardt, O.; Christensen, L.; Behrens, C.; Freir, S. M.; Driver, D. A.; Bergh, R. H.; Kim, S. K.; Norden, D.; Nielsen, P. E. Nature (London) 1993, 365, 566-568. (d) Tomac, S.; Sarkar, M.; Ratilainen, T.; Wittung, P.; Nielsen, P. E.; Norden, B.; Graslund, A. J. Am. Chem. Soc. 1996, 118, 5544-5552. (e) Egholm, M.; Behrens, C.; Christensen, L.; Berg, R. H.; Nielsen, P. E.; Buchardt, O. J. Chem. Soc., Chem. Commun. 1993, 9, 800-801. (f) Koch, T. In Peptide Nucleic Acids. Protocols and Applications; Nielsen, P. E., Egholm, M., Eds.; Horizon Scientific Press: Norfolk, 1999; p 21.

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Scheme 1. Amplified Detection of PNA/DNA Hybridization by the Formation of an Electroactive Streptavidin Layer

Table 1. Sequences of PNA Probe and DNA Targets

a

name

sequences

HS-PNAa BT2 BT1 BT15

Cys-[eg1]6TGT-ACA-TCA-CAA-CTA-NH2 5′-biotin-TAG TTG TGA TGT ACA 5′-biotin-TAG TTG TGA CGT ACA 5′-biotin-ATC AAC ACT ACA TGT

Cys(egl)6:

following binding buffer for associating with Fc-Stv consisted of 20 mM phosphate and 0.005% Tween 20 (PB-T, pH 7.4). The dehybridization solution was 50 mM NaOH. Thiolated PNA probes were synthesized as reported before.38e,f All other oligonucleotide targets employed in this work were synthesized by MWG. The sequences of PNA probe and DNA targets are shown in Table 1. Construction of the Fc-Stv Amplified DNA Sensor. Gold electrodes were prepared by evaporation of 2-nm Cr followed by 45-nm Au onto LaSFN9 glass slides. The freshly evaporated electrode was immersed in 100 nM thiolated PNA solution for 10 h, washed with 100 mM PB buffer, and then immersed in 1 mM MCH/water solution for 1 h to exchange the physically adsorbed PNA. The resulting PNA-modified electrodes were hybridized with biotinylated DNA target in PB-T solution. The hybridization step and the following binding steps were carried out in an electrochemical flow cell (200-µL volume) with an inlet and outlet for circulation. Finally, the electrode was rinsed with PB-T buffer for 10 min. Instruments. Electrochemical measurements were performed with an Autolab analyzer (Eco Chemie). The electrode system consists of the above-mentioned modified Au film working electrode, an Ag/AgCl/3 M NaCl reference minielectrode, and a coiled platinum wire counter electrode. The PB-T solution was employed as supporting electrolyte. The electrochemical techniques used are cyclic voltammetry (CV), chronocoulometry (CC), and SWV. The parameters for SWV are as follows: frequency, 200 Hz, step potential 5 mV, amplitude 20 mV. The SPR setup used is based on essential modules of a normal plasmon spectrometer that can be connected with the electrochemical analyzer. A HeNe laser operating at λ ) 632.8 nm passes a chopper, two polarizers for intensing and polarization control, before being reflected from the base of a 90° high index glass prism mounted on a θ-2θ goniometer arrangement. A lens collects the reflected light onto the photodiode. The output signal is fed into a lock-in amplifier. If one monitors the reflectivity at a fixed angle of incidence, any change of the interfacial architecture, 2758

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e.g., induced by adsorption or desorption processes, can be quantified by evaluating the resulting change of the reflected intensity in this kinetic mode of operation.39 RESULTS AND DISCUSSION The sensing configuration is depicted in Scheme 1. The thiolated PNA mixed with MCH was assembled on the Au electrode surface and acts as the sensing interface. Interaction of the PNAs with biotinylated DNA targets results in hybrid duplex formation. Subsequently, 200 nM Fc-Stv was associated with each hybrid by the biotin-streptavidin binding. The intensity of the redox reaction signal indicates the extent of hybridization of the target DNA oligomers. SPR Characterization of the DNA Hybridization Reaction and Amplification by Fc-Stv. At the SPR sensor surface with a submonolayer of thiolated DNA, the hybridization of 12-mer DNA target only led to a very small angular shift of the SPR reflectivity minimum.19 To decrease the steric effect, the thiolated PNA layer in our system is less than a submonolayer; hence, one cannot detect any observable angular shift upon DNA hybridization (Figure 1 solid line). Subsequent exposure of the SPR surface to the solution containing 200 nM Fc-Stv led to a pronounced angle shift (Figure 1 dashed line) due to the interaction of Fc-Stv with the biotin group on the DNA target. The angle shift is ∼0.25°, corresponding to an optical layer thickness of d ) 2.1 nm as obtained from the SPR simulation by assuming a refractive index of n ) 1.45 for the protein layer. This thickness is smaller than the streptavidin layer formed on a thiolated biotin matrix (d ) 3.5 nm),4b which is reasonable, by taking into consideration the submonolayer thiolated PNA matrix and the hybridization efficiency. Moreover, this thickness is comparable with that on a DNA/DNA matrix, corresponding to about half a streptavidin monolayer with a surface coverage of ∼1.5 × 1012 molecules/ cm2.23 The amplifying effect of Fc-Stv for the SPR detection of DNA hybridization can be seen even more clearly in the kinetic measurements. Using a flow cell, kinetic data can be acquired in real time to monitor the progress of all the reactions. Figure 2 shows the kinetic plot after adding different samples by monitoring the SPR reflectivity changes at a fixed incident angle θ ) 55.7°. After exposure of the PNA-modified surface to 1 µM noncomplementary target DNA (BT15), and rinsing with PB-T solution, the injection of Fc-Stv led to only a small increase in reflectivity (a f b), and the following rinsing of the surface with PB-T buffer resulted in a back shift of the reflectivity toward its initial value (b f c), indicating that the sensing surface exhibits virtually no nonspecific adsorption of Fc-Stv. However, after hybridization (39) Knoll, W. Annu. Rev. Phys. Chem. 1998, 49, 569-638.

Figure 1. SPR curves recorded in 20 mM PB-T after PNA immobilization (dotted curve), after hybridization with 500 nM BT2 (solid line), and after Fc-Stv adsorption (200 nM, dashed line). The inset is an expanded view of the SPR minimum part.

Figure 2. SPR kinetic curve measured at a fixed incident angle (θ ) 55.7°): (a) after adsorbing 1 µM BT15 (noncomplementary biotinylated target), then injection of 200 nM Fc-Stv.; (b) rinsing with PB-T buffer; (c) injection in 500 nM BT2 (fully complementary biotinylated target); (d) after rinsing and then injection of 200 nM FcStv; (e) same as (b); (f) 50 mM NaOH; (g-k) the same steps as (be).

with 500 nM complementary DNA target (BT2, c f d), the injection of Fc-Stv under the same conditions (d f e) results in a strong enhancement of reflectivity within the first 5 min, with the binding process being completed within 40 min. Rinsing with PB-T buffer reduces only little of the reflectivity (e f f). Compared to the DNA hybridization step (c f d), binding of Fc-Stv (d f e) exhibits a 10-fold signal enhancement. Electrochemical Characterization of Fc-Stv-Enhanced DNA Hybridization. As described above, Fc-Stv can be easily attached to the DNA strand by the strong biotin-streptavidin interaction. Because of the elasticity of the DNA strands and the long linker between the Fc groups and the Stv (6 glycine linkers),37 the electron transfer between Fc and the underlying electrode can happen rather efficiently. Figure 3 shows the typical CV feature of the surface-bound Fc molecules after the hybridization of complementary BT2 with the PNA probe on the surface and the following binding of Fc-Stv. The CV curves at different scan rates are all perfectly symmetrical, indicating a fully reversible reaction. The peak currents are proportional to the scan rates (Figure 3, inset), confirming a surface-controlled redox process only. The total width at half-height of either the cathodic or the anodic wave is ∼120 mV, larger than the ideal value, 90.6/n mV, for reversible surface responses,40a indicating that there are some

Figure 3. Cyclic voltammograms of Au/PNA/BT2/Fc-Stv measured in PB-T buffer with different scan rates. From inside to outside 0.4, 0.5, 0.6, 0.7, 0.8, 1.0, and 1.2 V/s, respectively. The inset shows the linear relationship between the peak currents and the scan rates.

interactions between the Fc sites. Besides, the random distribution of Fc sites on the film can also produce a broadened wave.40b In the Fc-Stv conjugate, every Stv group possesses at least nine Fc sites.37 Assuming that one biotinylated DNA target can bind one Fc-Stv, and all the Fc sites are electroactive, then the amount of DNA target can be estimated by integrating the area under the cathodic (or anodic) wave in the cyclic voltammogram.41 Using this approach, the calculated value of ΓDNA is 1.1 × 1012 molecules/cm2, which is a little smaller than the value obtained from SPR calculations. However, this is reasonable by taking into consideration that not all the Fc sites are electroactive as assumed. CC provides a more direct method for determining the surface coverage of the adsorbed electroactive reactant independent of the kinetics of the reactants.42-44 Moreover, it is easy to correct for double-layer charging. Shown in Figure 4, curve f, is the chronocoulometric response of the PNA-modified electrode after hybridization with 500 nM complementary DNA BT2 and the following binding of Fc-Stv (200 nM). The potential is stepped up to 0.3 V, which is positive enough to ensure that all reduced ferrocenes on the electrode surface are oxidized. The total charge Q at time t is

Q ) 2nFAc0*(D0t/π)1/2 + nFAΓ0 + Qdl

(1)

with the terms on the right side of the equation representing the contributions from dissolved Fc (here this part is assumed to be 0), adsorbed Fc, and double-layer charging, respectively. As shown in Figure 4, a plot of Q versus t1/2 yields an intercept Q0 given by

Q0 ) nFAΓ0 + Qdl

(2)

The determination of Γ0 requires an independent estimate of Qdl. In our system, the adsorption of the big molecule Fc-Stv will (40) (a) Murray, R. W. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1984; Vol. 13, pp 191-368. (b) Peerce, P. J.; Bard, A. J. J. Electroanal. Chem. 1980, 114, 89. (41) Takada, K.; Diaz, D. J.; Abruna, H. D.; Cuadrado, I.; Casado, C.; Alonso, B.; Moran, M.; Losada, J. J. Am. Chem. Soc. 1997, 119, 10763-10773. (42) Anson, F. C. Anal. Chem. 1966, 38, 54-57. (43) Christie, J. H.; Osteryoung, R. A.; Anson, F. C. J. Electroanal. Chem. 1967, 13, 236. (44) Anson, F. C.; Christie, J. H.; Osteryoung, R. A. J. Electroanal. Chem. 1967, 13, 343.

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Figure 4. Chronocoulometric curves measured in PB-T buffer: (a) Au/PNA surface; (b) after (a) and then hybridization with 500 nM BT2; (c) after (b) and then binding normal Stv; (d-f) after hybridization with 5, 50, and 500 nM BT2 followed by binding with 200 nM FcStv, respectively. The lines represent the fit to the data used to determine the intercept at t ) 0 to obtain Q0 (dotted line) and Qdl (dashed line).

Figure 5. Cyclic voltammograms at a Au/PNA electrode (a) after hybridization with 500 nM BT2; (b) after hybridization with fully mismatched BT15 (1 µM) followed by the binding of 200 nM Fc-Stv; (c) after the hybridization with 500 nM perfectly matched BT2 followed by the binding of 200 nM Fc-Stv. (d) same as (b) but hybridization with single-mismatched BT1 (500 nM); (e) after (c), washing with 50 mM NaOH, and then repeat step (c).

affect Qdl, so we use normal Stv instead of Fc-Stv to get Qdl under the same conditions (curve c in Figure 4). Thus, ΓDNA is calculated to be ∼9.4 × 1011 molecules/cm2, which is similar to the results from CV. In the following experiments, we only compare the change of the intercept Q0 at different target concentrations, instead of giving the absolute Γ0. Curves d-f in Figure 4 correspond to the CC after the PNA probe on the surface hybridized with different concentrations of BT2, followed by the binding of Fc-Stv. The intercept increases from curve d to curve f with the increase of the target concentration, indicating that Γ0 of Fc on the surface is dependent on the target concentration. Figure 5 shows the cyclic voltammograms of the sequenceselective hybridization of various DNA targets and subsequent binding of Fc-Stv on the immobilized PNA probe. In all cases, the surface was washed for at least 10 min before the binding of Fc-Stv to ensure that there is no nonspecifically bound DNA and to differentiate the fully complementary against a single-base mismatched target DNA. In the case of complementary target BT2, a very clear surface peak was observed upon binding of FcStv (curve c). However, for fully mismatched target DNA BT15, there was no redox peak in the same scan range (curve b); only a very small peak appeared in the sensitive SWV (cf. curve g in Figure 6). It indicates that the nonspecific interaction of either target DNA or Fc-Stv is negligible, which was also concluded 2760 Analytical Chemistry, Vol. 77, No. 9, May 1, 2005

Figure 6. (A) SWVs of Au/PNA after hybridization with BT2 at different concentrations (from a to f: 0, 2, 10, 50, 100, and 200 pM, respectively, reaction time 30 min) and the following binding with FcStv (200 nM, 30 min). Curve g is the case for noncomplementary target BT15 (1 µM) as a reference to show the good selectivity and the absence of nonspecific adsorption. Frequency, 200 Hz; amplitude, 20 mV. (B) The relationship between the peak current and the concentration of BT2, with a linear relationship in the range between 10 and 200 pM.

from Figure 2, steps a f c. For the single-base mismatched target DNA (BT1) (curve d, Figure 5), the peak current is only 70% of that of the complementary case. Moreover, the peak current continues to decrease upon further rinsing. However, there is almost no decrease for the complementary ones. Thus, it is apparent that the DNA sensor reported here shows a high specificity, even at the single-base mismatch level. To assess the sensitivity of the sensor, the SWV technique was used.45 In our experiments, the immobilized PNA was allowed to hybridize with the target DNA (BT2) of different concentrations for a fixed time (30 min), followed by association with Fc-Stv. Then the peak current from the Fc on the surface was measured. Shown in Figure 6A are the SWV curves with different target BT2 concentrations. The peak current directly corresponds to the amount of target BT2 hybridized on the surface. Figure 6B shows the plot of the peak current as a function of the target concentration. Here, we can see that the hybridization signal (Ip) increase linearly with the target concentration over the concentration range of 10-200 pM. It is important to note the small baseline signal recorded in the absence of DNA, indicating a very low nonspecific binding. The selectivity of the response was controlled by (45) (a) Osteryoung, J.; O'Dea, J. J. Electroanal. Chem. 1986, 14, 209. (b) Osteryoung, J. Acc. Chem. Res. 1993, 26, 77-83.

replacing the BT2 target with a noncomplementary target DNA (BT15, Figure 6A, curve g). The resulting signal was very close to that obtained in the absence of BT2, even though a much higher concentration for BT15 (1 µM) was used. A detection limit of 10 pM (2 fmol) can be estimated with a signal-to-noise ratio of >3. Regeneration of the Sensor Surface. The reproducibility of a sensor surface is very important for the rapid and accurate detection of analyte molecules. Our sensor surface is readily reusable: by washing the electrode surface with 50 mM NaOH for 5 min and rehybridizing it with target DNA followed by the binding of Fc-Stv, we have successfully recovered up to 90% of the original signal (cf. steps f f k in Figure 2 and curve e in Figure 5). This good reproducibility originates mainly from the covalent connection of thiolated PNA and the stability of Fc-Stv, compared with an enzyme amplification system. CONCLUSIONS We have devised a new DNA sensor by using Fc-Stv conjugate as an amplifier for DNA hybridization detection by SPR in combination with several electrochemical techniques. Using

CV or CC, the amount of the hybridized target DNA can easily be quantified, and also a single-base mismatched oligonucleotide can be clearly discriminated. In contrast to previously reported electrochemical approaches, the reported DNA sensor achieves an impressive sensitivity without sacrificing selectivity or reusability. The DNA sensor thus offers the promise of convenient, reusable detection of DNA at the femtomolar level. Using this architecture, kinetic information of DNA/PNA binding and the differentiation of a single-base mismatch by the difference of binding constants can easily be achieved, and the results will be reported elsewhere. ACKNOWLEDGMENT This work was partly supported by the Deutsche Forschunggemeinschaft (DFG, KN 224/13-1).

Received for review December 27, 2004. Accepted February 25, 2005. AC048088C

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