PNA−DNA Hybridization Study Using Labeled Streptavidin by

CH-5232 Villigen PSI, Switzerland, and Department of Biochemistry B, The Panum Institute, IMBG, Blegdamsvej 3c, DK-2200, Copenhagen N, Denmark. An...
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Anal. Chem. 2006, 78, 470-476

PNA-DNA Hybridization Study Using Labeled Streptavidin by Voltammetry and Surface Plasmon Fluorescence Spectroscopy Jianyun Liu,*,† Louis Tiefenauer,§ Shengjun Tian,† Peter Eigil Nielsen,| and Wolfgang Knoll*,†

Max-Planck-Institute for Polymer Research, Ackermannweg 10, D-55128, Mainz, Germany, Department of Chemistry, Life Sciences Department, Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland, and Department of Biochemistry B, The Panum Institute, IMBG, Blegdamsvej 3c, DK-2200, Copenhagen N, Denmark

Using ferrocene-streptavidin conjugates as amplifiers, we recently have demonstrated the simultaneous detection of DNA hybridization to peptide nucleic acid (PNA)modified gold surfaces at the femtomole level by electrochemical and surface plasmon resonance techniques (Liu, J.; Tian, S.; Tiefenauer, L.; Nielsen, P. E.; Knoll, W. Anal. Chem. 2005, 77, 2756-2761). In this paper, a detailed study of the binding behavior of PNA-DNA is presented by square wave voltammetry and surface plasmon fieldenhanced fluorescence spectroscopy (SPFS). The different binding constants for fully matched and single-mismatched DNA were obtained. The effect of the buffer concentration on the PNA-DNA hybrids was investigated using labeled streptavidin by cyclic voltammetry (CV) and SPFS. At high ionic strength, both the CV and SPFS signals were restrained dramatically, which is most probably due to a conformational change of the short-strand PNA-DNA helices on the surface. We conclude that the combination of electrochemical techniques with SPFS is very useful for the study of short DNA structure transformation.

based on ferrocene (Fc)-streptavidin conjugates for the simultaneously amplified electrochemical and surface plasmon optical detection of DNA target hybridization to the immobilized peptide nucleic acid (PNA).9 The quantification of target DNA can be easily obtained on this sensor platform with good stability and reproducibility. This sensor can also discriminate effectively different DNA sequences and can be regenerated. A comprehensive understanding of interactions between surfaceimmobilized DNA and the free oligonucleotides in solution is the key to realizing genetic diagnostic devices. Some reports have dealt with the nature of surface DNA-DNA interaction.10-13 There is, however, little knowledge about the surface affinity reaction of PNA-DNA.13-16 PNA is a DNA mimic in which the negatively charged sugar-phosphate backbone is substituted with an uncharged pseudopeptide chain.17 PNA binds to the complementary single-stranded target according to the Watson-Crick rules for base pairing, and for most sequences, PNA binds DNA target with high affinity.18,19 This observed high affinity is partially a result of the neutral character of the PNA backbone, which alleviates the standard charge-charge repulsion of duplex formation. More importantly, the mismatch discrimination of PNA is in many cases

Further progress in gene diagnostics requires the development of a simple and sensitive method for DNA analysis. Recently, the electrochemical detection of DNA hybridization has received a great deal of attention due to its high sensitivity, low cost, and compatibility with microfabrication technology. One of the most often used approaches for the electrochemical detection of hybridization reactions is based on using redox labels linked to the hybridized target in order to generate an amplified signal.1-8 In this context, we have recently reported a very sensitive method

(7) Patolsky, F.; Weizmann, Y.; Willner, I. J. Am. Chem. Soc. 2002, 124, 770772. (8) Kim, E.; Kim. K.; Yang, H.; Kim, Y. T.; Kwak, J. Anal. Chem. 2003, 75, 5665-5672. (9) Liu, J.; Tian, S.; Tiefenauer, L.; Nielsen, P. E.; Knoll, W. Anal. Chem. 2005, 77, 2756-2761. (10) Peterson, A. W.; Heaton, R. J.; Georgiadis, R. J. Am. Chem. Soc. 2000, 122, 7837-7838. (11) Peterson, A. W.; Wolf, L. K.; Georgiadis, R. M. J. Am. Chem. Soc. 2002, 124, 14601-14607. (12) Liebermann, T.; Knoll, W.; Sluka, P.; Herrmann, R. Colloid Surf., A 2000, 169, 337-350. (13) Kambhampati, D.; Nielsen, P. E.; Knoll, W. Biosens. Bioelectron. 2001, 16, 1109-1118. (14) Kosaganov, Y. N.; Stetsenko, D. A.; Lubyako, E. N.; Kvitko, N. P.; Lazurkin, Y. S.; Nielsen, P. E. Biochemistry 2000, 39, 11742-11747. (15) Kushon, S. A.; Jordan, J. P.; Seifert, J. L.; Nielsen, H.; Nielsen, P. E.; Armitage, B. A. J. Am. Chem. Soc. 2001, 123, 10805-10813. (16) Jensen, K. K.; Orum, H.; Nielsen, P. E.; Norden, B. Biochemistry 1997, 36, 5072-5077. (17) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991, 254, 1497-1500. (18) Giesen, U.; Kleider, W.; Berding, C.; Geiger, A.; Ørum, H.; Nielsen, P. E. Nucleic Acids Res. 1998, 26, 5004-5006. (19) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norde´n, B.; Nielsen, P. E. Nature 1993, 365, 556-568.

* Corresponding authors. E-mail: [email protected]. Tel: +49 6131 379 160. Fax: +49 6131 379 360. E-mail: [email protected]. † Max-Planck-Institute for Polymer Research. § Paul Scherrer Institut. | The Panum Institute. (1) Caruana, D. J.; Heller, A. J. Am. Chem. Soc. 1999, 121, 769-773. (2) Zhang, Y.; Kim, H.; Heller, A. Anal. Chem. 2003, 75, 3267-3269. (3) Zhang, Y.; Pothukuchy, A.; Shin, W.; Kim, Y.; Heller, A. Anal. Chem. 2004, 76, 4093-4097. (4) Wang, J.; Li, J.; Baca, A. J.; Hu, J.; Zhou, F.; Yan, W.; Pang, D. W. Anal. Chem. 2003, 75, 3941-3945. (5) Wang, J.; Liu, G.; Jan, M. R. J. Am. Chem. Soc. 2004, 126, 3010-3011. (6) 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.

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10.1021/ac051299c CCC: $33.50

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Scheme 1. Detection of PNA-DNA Hybridization by the Formation of a Labeled Streptavidin Layer

better than that of DNA, and so PNA is not only a high-affinity probe for DNA but it also maintains high specificity.18-21 In this report, we investigate the conditions for the electrochemical detection of PNA-DNA hybridization based on the same architecture as before9 and determine the optimal surface coverage for PNA. Based on a Langmuir isotherm, equilibrium binding data were obtained, which will lead to a better understanding of the hybridization processes and will allow for a comparison of surface PNA-DNA hybridization reactions with different nucleobase mismatches. The DNA target sequences that hybridize to the immobilized PNA on the gold surface are labeled with biotin, which binds streptavidin with a very high affinity (Kd ) 10-15 mol dm-3). This complex is extremely stable toward thermal denaturation or against chaotropic ions.4 Therefore, using square wave voltammetry (SWV), the PNA-DNA affinity constants can be obtained from the redox-labeled streptavidin coupled to the duplex. SWV is a very sensitive and fast technique (voltammograms can be obtained in a matter of seconds) among the various electrochemical detection schemes22 and, therefore, very well suited to study DNA hybridization. Using dye-labeled streptavidin as a detection probe, the affinity constants were also obtained by surface plasmon field-enhanced fluorescence spectroscopy (SPFS). Conventionally, DNA forms a plectonemic helix in which both strands are intertwined. However, DNA is highly charged, so that electrostatic repulsion of negatively charged DNA helices opposes folding and especially formation of close contacts between DNA regions. Counterions shield the negative charges of DNA and hence decrease the repulsion between DNA segments. Therefore, any change of the salt conditions influences the geometry of supercoiled DNA. Monte Carlo simulations of supercoiling23-25 have shown that the ion concentration has a strong effect on the (20) Ratilainen, T.; Holmen, A.; Tuite, E.; Haaima, G.; Christensen, L.; Nielsen, P. E.; Norden, B. Biochemistry 1998, 37, 12331-12342. (21) Ratilainen, T.; Holmen, A.; Tuite, E.; Nielsen, P. E.; Norden, B. Biochemistry 2000, 39, 7781-7791. (22) Bard, A. J.; Faulkner, L. R. Electrochemical methods: Fundamentals and applications, 2nd ed.; Wiley: New York, 2001; p 663. (23) Vologodskii, A. V.; Levene, S. D.; Klenin, K. V.; Frank-Kamenetskii, M. D.; Cozzarelli, N. R J. Mol. Biol. 1992, 227, 1224-1243. (24) Tesi, M. C.; Janse van Rensburg, E. J.; Orlandini, E.; Sumners, D. W.; Whittington, S. G. Phys. Rev. E 1994, 49, 868-872, (25) Schlick, T.; Li, B.; Olson, W. K. Biophys. J. 1994, 67, 2146-2166.

conformation of supercoiled DNA. In particular, systematic studies on the effect of the ionic conditions on the geometry of supercoiled DNA were performed.26 However, little is known about a shortstrand duplex on a surface, despite the considerable interest in knowing more about the conformation feature of short-strand DNA duplexes related to their biological function (e.g., biochip design, protein-DNA interactions).27,28 The stability of PNA-DNA duplexes is almost unaffected by the ionic strength of the medium, in contrast to the behavior of DNA-DNA double-helical structures.29 We have employed cyclic voltammetry (CV) in this work in order to monitor how the salt concentration influences the structure of PNA-DNA hybrids fixed on a gold substrate. Because the electron transfer to or from surface-confined Fc molecules is dependent on the structure of the sensor surface architecture and the distance between the Fc sites and the electrode surface, any change of the underlying PNA-DNA duplex structure will be reflected by the redox reaction of the Fc molecules. Finally, with fluorescence-labeled streptavidin as a probe instead of Fc-labeled streptavidin, we can use SPFS,30which gives additional information about the nature of the change of the PNA-DNA hybrid structure as a function of the ionic strength in solution. To our knowledge, no study has examined the role of salt concentration on the conformation of short-strand DNA hybrids at the water-solid interface. These studies should give a better understanding of heterogeneous PNA-DNA hybridization interactions involving thiolated PNA as probe and of the dependence of the configuration of the resulting PNA-DNA duplexes on the ionic strength. It reveals that this electrochemical technique combined with SPFS opens a new way as a supplementary measurement to structural biology, even though no direct spatial information can be given like by the AFM. (26) Bussiek, M.; Mu ¨ cke, N.; Langowski, J. Nucleic Acids Res. 2003, 31, e137. (27) Bates, A. D.; Maxwell, A. DNA Topology. Oxford University Press: Oxford, U.K., 1993. (28) Negri, R.; Costanzo, G..; Buttinelli, M.; Venditti, S.; Dimauro, E. Biophys. Chem. 1994, 50, 169-181. (29) Tomac, S.; Sarkar, M.; Ratilainen, T.; Wittung, P.; Nielsen, P. E.; Norden, B.; Graslund, A. J. Am. Chem. Soc. 1996, 118, 5544-5552. (30) Liebermann, T.; Knoll, W. Colloids Surf., A 2000, 171, 115-130.

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EXPERIMENTAL SECTION Chemicals and Reagents. Mercaptohexanol (MCH) was purchased from Sigma. Two types of ferrocene-labeled streptavidin conjugates (Fc-gly-SAv and Fc-ac-SAv) with different linkers (shown in Scheme 1) were prepared as reported.31-33 Alexa-647labeled streptavidin (Fluor-SAv) (Molecular Probes) was used for SPFS detection. All buffer salts and other organic chemicals were obtained from Sigma or Aldrich unless otherwise stated. All chemicals were used as received. Thiolated PNA probes were synthesized as reported.34,35 The sequences of the PNA probe and the DNA targets were shown in other report.9 The base sequences of three mismatch oligomer (BT3) is 5′-biotin-TAG TTG TCA CGT ACA. Construction of the M-SAv-Linked DNA Sensor. The sensing configuration is depicted in Scheme 1. The assembly of PNA and the hybridization steps can be found in the literature.9 For the binding step of M-SAv during the experiment, two Fclabeled SAvs with different linkers and one fluorescence-labeled SAv (for SPFS detection) were used (shown in Scheme 1). Instruments. Electrochemical measurements were performed with an Autolab analyzer (Eco Chemie, Netherlands). The details about the electrochemical detection are the same as described before.9 The SPFS setup was constructed based on a conventional surface plasmon spectrometer, in combination with the photocounting technique.12,30 The emitted fluorescence light from the sample surface was collected through a minielectrochemical flow cell. A lens (f ) 40 mm; Owis) focused the light through an interference filter (λ ) 670 nm, T ) 69%, LOT) onto a photomultiplier tube (Hamamatsu), which fed the signal to a photon counter (Hewlett-Packard). The whole fluorescence detection unit is mounted to the goniometer rotating together with the prism at the incidence angle θ, while the photodiode detecting the reflected intensity rotates at 2θ, relative to the fixed excitation laser beam. Both signals, the fluorescence from a photocounter and the reflectivity from a lock-in amplifier, were controlled by a computer. RESULTS AND DISCUSSION Detection of Sequence-Selective Hybridization by Fc-glySAv Conjugate with SWV Detection. Scheme 1 shows the stepwise preparation of the DNA sensor. The thiolated PNAs mixed with MCH were assembled on the Au electrode surface and act as the sensing interface. The interaction of the PNAs with biotinylated DNA targets results in hybrid duplex formation. Subsequently, Fc-gly-SAv is attached to the DNA strand by the strong biotin-streptavidin interaction. Due to the elasticity of the DNA strands and the flexibility of Fc moieties on the streptavidin (shown in Scheme 1),32 the electron transfer between the attached Fc moieties and the underlying electrode occurs efficiently, allowing the quantification of target DNA using cyclic voltammetry or chronocoulometry.9 (31) Padeste, C.; Grubeln, K. A.; Tiefenauer, L. Electrochim. Acta 2003, 48, 761769. (32) Padeste, C.; Grubelnik, A.; Tiefenauer, L. Biosens. Bioelectron. 2000, 15, 431-438. (33) Padeste, C.; Steiger, B.; Grubelnik, A.; Tiefenauer, L. Biosens. Bioelectron. 2003, 19, 239-247. (34) Egholm, M.; Behrens, C.; Christensen, L.; Berg, R. H.; Nielsen, P. E.; Buchardt, O. J. Chem. Soc., Chem. Commun. 1993, 9, 800-801. (35) 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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Figure 1. SWVs at a Au/PNA electrode in PB-T buffer (a) after hybridization with 500 nM BT2, (b) after hybridization with 500 nM perfectly matched BT2 followed by the binding of Fc-gly-SAv, (c) after hybridization with 500 nM single-mismatched BT1 followed by the binding of Fc-gly-SAv, (d) same as (c) but two-base-mismatched BT3, (e) after (b), washing with 50 mM NaOH, and (f) after (d) and then repeat step (b).

Figure 2. Peak currents of the binding Fc-gly-SAv after hybridization as a function of exposure time of HS-PNA assembly. The peak currents were detected by SWV with square frequency 100 Hz. HSPNA concentration, 100 nM.

SWV is very sensitive and fast electrochemical method and results can be obtained within seconds. Figure 1 shows the SWV curves after hybridization of three different DNA targets with the immobilized PNA and the subsequent binding of Fc-gly-SAv. In the case of fully complementary target BT2, a sharp peak was observed (curve b). However, for one-single-mismatch DNA, the peak was decreased (curve c), and a much smaller peak signal was found (curve d) for two mismatches. It is apparent that the DNA sensor architecture can discriminate effectively between different DNA sequences. The reproducibility of a sensor surface is very important for the rapid and accurate detection in automatic systems. Our sensor surface shows good performance after regeneration. Washing the electrode surface with 50 mM NaOH for 5 min can remove all hybridized strands (curve e in Figure 1), and after rehybridization with the target DNA followed by binding of Fc-gly-SAv conjugate, the redox response of the Fc moieties has been successfully recovered (curve f in Figure 1). Effect of the PNA Probe Density on Hybridization. There are many reports about the effect of the thiolated DNA probe density on the hybridization efficiency and hybridization kinetics.36-39 Since PNA strands are uncharged, and the big

Figure 3. (A) SWVs in PB-T buffer solution after binding with Fc-gly-SAv following the hybridization with different concentrations of BT2 (from top to bottom: 0, 1, 10, 50, 200, and 500 nM). Frequency, 100 Hz; amplitude, 20 mV. (B) Langmuir isotherm of PNA-DNA hybridization from (A). (C) Linear representation of Langmuir isotherm.

molecule streptavidin binds to the duplex after hybridization, the optimal surface density for this architecture may be different. We varied the exposure time of sensing surface to the thiolated PNA solution in order to achieve an optimal PNA probe density for DNA hybridization as determined by measuring the electrochemical response of bound Fc-gly-SAv. If the PNA concentration is too high (>1 µM), no dependence of the peak currents on the exposure time was found.38 Therefore, a low concentration of the PNA solution (100 nM) was chosen in order to control the density more conveniently. A series of sensing surfaces were prepared by changing the exposure times. Then the same hybridization as well as the subsequent binding of Fc-gly-SAv was performed. The resulting Fc peak currents plotted as a function of exposure time are shown in Figure 2. The peak current increases with longer exposure time, indicating a correlation between the amount of bound Fc-gly-SAv on the surface and the exposure time. The detection signal appears to reach a maximum after 10-h incubation with the PNA solution, with further exposure resulting in no additional increase. Langmuir Isotherm for PNA-DNA Hybridization. The amount of hybridized DNA on the substrate depends on the concentration of DNA target in bulk solution, c0. At low concentrations of DNA, only a fraction of PNA-DNA duplexes will be formed. With increasing target concentration, more and more complexes are formed until a maximum is reached. Assuming that no DNA-DNA interaction occurs because of the low surface density,9 the equilibrium relationship between the bulk solution concentration c0 and the coverage of hybridized DNA molecules can be represented by the simple Langmuir isotherm:

θ ) KAc0/(1 + KAc0)

(1)

where θ is the fractional coverage defined as Γ/Γm (Γm is the DNA maximum coverage), c0 is the bulk DNA target concentration, and KA is the affinity constant. On this sensing architecture shown in Scheme 1, we can assume that one biotinylated DNA target binds one Fc-gly-SAv, (36) Peterson, A. W.; Heaton, R. J.; Georgiadis, R. M. Nucleic Acids Res. 2001, 29, 5164-5168. (37) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916-8920. (38) Steel, A. B.; Levicky, R. L.; Herne, T. M.; Tarlov, M. J. Biophy. J. 2000, 79, 975-981. (39) Levicky, R.; Herne, T. M.; Tarlov, M. J.; Satija, S. K. J. Am. Chem. Soc. 1998, 120, 9787-9792.

and all the Fc sites are electrochemically equivalent and active.31 The coverage Γ of the hybridized DNA will be proportional to the redox current I. Here, for clarity, eq 1 can be expressed as

I ) Imc0/(Kd + c0)

(2)

where Im represents the current of the Fc-streptavidin conjugates for maxium DNA coverage and Kd is the dissociation constants, Kd ) 1/KA. Figure 3A displays a series of SWV scans of Fc-gly-SAv conjugates that were bound to the complementary BT2 following the hybridization with the immobilized PNA. Clearly, with increasing the target concentration, the corresponding redox peak currents increase. If the peak current I is plotted as a function of the bulk concentration c0, a Langmuir isotherm can be obtained, as shown in Figure 3B. Using eq 2 for fitting, the equilibrium binding constants KA is obtained. By replotting the data in a linearized relation of the Langmuir isotherm, i.e., c0/I versus c0 (Figure 3C), KA can be deduced to be 1.5((0.4) × 108 M-1 from the linear regression for fully complementary DNA targets. The KA value for the one-base-mismatch DNA was 5.1 × 107 M-1 (Figure 4). The good agreement of the observed peak currents at increasing target concentrations, c0, with the theoretical fitting based on the simple Langmuir model indicates that this electrochemical method is suitable to follow hybridization reaction processes at interfaces. Moreover, we can differentiate one-base mismatch from completely matched DNA by comparing the affinity constants. However, it is difficult to directly compare the binding constant values obtained here with those reported in the literature because of the differences in the attachment chemistry, probe density, and ionic strength for hybridization. For example, the affinity constant for the fully complementary PNA-DNA duplexes measured with the BIAcore method is only 2.8 × 106 M-1 .16 To confirm the reliability of the SWV method, SPFS was employed as a control method. The affinity constants were determined using SPFS on the same architecture as mentioned above except that the electrochemically active Fc label was replaced by the fluorescent dye Alexa 647 attached to streptavidin. Figure 5A displays a series of angular fluorescence intensity scans taken after the hybridization of targets with increasing concentrations and binding with Fluor-SAv (solid curves). As expected, the fluorescence peak intensity increased gradually by increasAnalytical Chemistry, Vol. 78, No. 2, January 15, 2006

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Figure 4. (A) SWVs in PB-T buffer solution after binding with Fc-gly-SAv following the hybridization with different concentrations of BT1 (from top to bottom: 0, 5, 20, 50, 100, 300. and 600 nM, respectively). Frequency, 100 Hz; amplitude, 20 mV. (B) Langmuir isotherm of PNA-DNA hybridization from (A). (C) Linear representation of Langmuir isotherm.

Figure 5. (A) Fluorescence intensity scans (right axis) measured in PB-T buffer after binding with Fluor-SAv following the hybridization with different concentrations of BT2 (solid lines from bottom to top: 0, 1, 5, 10, 50, 200, and 500 nM, respectively), Dotted and dashed lines are the angular reflectivity scans (left axis) before and after hybridization with 500 nM BT2, respectively, followed by the binding of Fluor-SAv. (B) Langmuir isotherm of the data presented in (A). (C) linearized plot of the data of (B).

ing the target concentration. Additionally, the angular reflectivity scans corresponding to the hybridization with 0 and 500 nM BT2 were also shown here in order to demonstrate the angular shifts induced by the binding of Fluor-SAv to the biotin of the DNA target.9 In a presentation of the SPFS data (Figure 5B and C), according to the Langmuir model the affinity constant KA for the completely matched PNA-DNA duplex was obtained as 2.1 × 108 M-1 and for the one-base mismatch 4.4 × 107 M-1. One can see that the results obtained by SPFS give very similar affinity constants compared to those derived from the SWV 474

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method. They are also comparable to values reported earlier for a different probe attachment chemistry based on biotin-streptavidin.13 Ionic Strength Effect on the Structure of PNA-DNA Hybrids. There are many studies on the effects of the salt concentration on DNA hybridization behavior and the thermal stability of the double-helix.40,41 However, there are few experi(40) Puglisi, J. D.; Tinoco, I., Jr. Methods Enzymol. 1989, 180, 304. (41) Breslauer, K. J. In Protocols for Oligonucleotide Conjugates; Agrawal, S., Ed.; Methods in Molecular Biology 26; Humana Press: Totowa, NJ, 1994; p 347.

Figure 6. Cyclic voltammograms of PNA-DNA hybrids binding with (A) Fc-gly-SAv, (B) Fc-ac-SAv in PB buffer solution with different concentrations (as indicated). Scan rate, 0.8 V/s. (C) Cyclic voltammograms of HS-biotin-modified Au surface binding with Fc-gly-SAv in different concentrations of PB buffers from left to right: 5, 10, 20, 50, 100, 200, and 500 mM, respectively. Other conditions are the same as (A).

ments concerned with the study of the structure of short-strand DNA hybrids. The flexibility of short DNA duplexes at a local scale (a few 10-15 bases) is now regarded as a key issue in understanding many biological processes involving DNA.42The AFM technique has provided direct images of supercoiled DNA structures at different ionic conditions.43 Using an electrochemical method, we tried to get insight into the relationship between the short PNA-DNA hybrid structure and the salt concentration of the buffer. Figure 6A and B show the CV curves of PNA-DNA hybrids conjugated with Fc-gly-SAv and Fc-ac-SAv, respectively. It was found that, with increasing salt concentration, the peak currents decreased dramatically, to the extent that the redox peak almost disappeared in 200 mM PB solution for the PNA-DNA hybrids with Fc-gly-SAv conjugate. The change is not caused by a loss of the target/Fc-streptavidin conjugate, dissociating off the film at high salt concentrations, since the redox peak was fully restored if the salt concentration was changed back to low concentrations (dash-dotted curve of Figure 6A). Moreover, the effect is not due to the Fc-labeled streptavidin alone. If Fc-glySAv is bound directly to a HS-biotin-modified Au surface without the PNA-DNA duplex layer, only a slight increase of the peak current was observed with increasing salt concentration (Figure 6C). The redox potential shifting slightly to positive potentials can probably be attributed to the formation of oxidized ferricenium/ phosphate ion pairs, a change in the liquid junction potentials, the activity coefficients, or both as the electrolyte concentration changed.44 Thus, we believe that the remarkable current difference (42) Zuccheri, G.; Scipioni, A.; Cavaliere, V.; Gargiulo, G.; De Santis, P.; Samori, B. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 3074-3079. (43) Lyubchenko, Y. L.; Shlyakhtenko, L. S. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 496-501.

in Figure 6A is a consequence of some structural transformation of the PNA-DNA duplex and not related to changes of the individual components. Presumably, at low ionic strength, the PNA-DNA duplexes are extended and oriented perpendicular to the electrode surface and become more ordered because of the electrostatic repelling and thus stretching of the DNA strands, facilitating the electron transfer of flexible Fc moieties across the PNA-DNA duplexes. Such an ordered molecular arrangement of DNA may favor long-range electron-transfer reactions along the duplexes as confirmed by the experiments of Heller et al.45 They found that the Faradaic charge transfer occurred along onedimensionally aligned 12-base pair double-stranded DNA helices but not along disordered ones. Oppositely, at high ionic strength, the surface charge of the DNA chains is shielded resulting in a more coiled configuration and partial collapse of the duplexes thereby forming a barrier layer of DNA helices between the Fc moieties and the electrode surface. The collapse of DNA chains not only covers the active domains of the electrode surface but also induces morphological changes of the film, which makes the electrode surface inaccessible for the Fc moieties, resulting in the observed decrease of the redox peak. However, if the more hydrophobic Fc-ac-SAv conjugate is used instead of the hydrophilic Fc-gly-SAv, the current will not disappear completely at high salt concentrations, and even a small increase of the peak height is observed in 500 mM buffer compared to 200 mM buffer (Figure 6B). We assume that the hydrophobic ac linker interacts with the collapsed DNA allowing electron-transfer processes. Anne et al.46 investigated the electron transfer of Fc-labeled oligonucleotide (44) Rowe, G. K.; Creager, S. E. Langmuir 1991, 7, 2307-2312. (45) Hartwich, G.; Caruana, J. D.; Lumley-Woodyear, T.; Wu, Y.; Campbell, C. N.; Heller, A. J. Am. Chem. Soc. 1999, 121, 10803-10812.

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duplexes at varying ionic strength. The usefulness of fluorescence detection to probe hybridization has been demonstrated by our group.47 Shown in Figure 7A are the angular fluorescence intensity scans (solid curves) in different salt concentrations. As expected from the electrochemistry results, the SPFS signal gradually decreased if the salt concentration increased stepwise from 5 to 500 mM (cf. also Figure 7B). Moreover, the signal almost completely recovered (more than 90%) upon changing the salt concentration back again to 5 mM. In addition, the angular reflectivity scan curves (Figure 7A) shifted gradually to higher angles at high ionic strengths due to the refractive index effect of the bulk solution, which simultaneously causes a shift of the fluorescence maximum. These observations confirm that at high ionic strength the duplex structure is collapsed or less stretched because of the efficient screening of the charge of the backbone, and hence, the attached dyes are getting closer to the metal surface. As a result, the emission is partially quenched and the observed fluorescence intensity reduced. In the low ionic strength limit, the duplex chains are stretched and the fluorescence label is separated further away from the surface. These findings are in good agreement with the CV experiment (vide supra).

Figure 7. (A) Angular reflectivity (left axis) and corresponding fluorescence intensity scans (right axis) measured on PNA-DNA hybrids binding with Fluor-SAv in different concentrations of PB buffer (as indicated), solid lines (right axis) from top to bottom: 5, 10, 20, 50, 100, 200, and 500 mM PB buffer, respectively. (B) the plot of normalized fluorescence intensity to PB concentration.

chains end-tethered to gold electrode surfaces and found that the Fc voltammetric signal on the duplexes disappeared at high scan rate (200 V/s) in high ionic strength electrolyte solution but recovered at low scan rates. However, we could not find any Fc voltammetric signal even at a scan rate as low as 20 mV/s in our experiment. These different results may be due to the dissimilarity of the Fc connection. The observed sensitivity of the short-strand duplexes structure to the ionic strength is also consistent with the results found for the supercoiled DNAs.43 Electron-transfer processes can be affected by many factors. To confirm our hypothesis that the structure and orientation of the duplexes are influenced by the ionic strength, SPFS was employed on the same architecture with Fluor-SAv instead of Fclabeled SAv. The fluorescence signal in SPFS detection is sensitive to the separation distance of the dye to the substrates, and the fluorescence will be quenched to some extent if the dye is too close to the Au surface.30 Therefore, fluorescence probes attached to the SAv can effectively monitor structural changes of the underlying PNA-DNA duplexes. SPFS is considered to be a valuable method to investigate global structural changes of (46) Anne, A.; Bouchardon, A.; Moiroux, J. J. Am. Chem. Soc. 2003, 125, 11121113. (47) Thomas Neumann. Ph.D. Thesis, Universita¨t Mainz, Germany, 2001.

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Analytical Chemistry, Vol. 78, No. 2, January 15, 2006

CONCLUSIONS The use of Fc-gly-SAv as an electrochemical probe has allowed us to monitor the sequence-selective PNA-DNA hybridization by the SWV method. A controlled surface density of PNA can be achieved by changing the exposure time of the Au surface to a highly diluted PNA probe solution. The binding behavior has been investigated using SWV as well as SPFS, and the affinity constants of a fully complementary and a single-base-mismatched DNA were obtained with good agreement. This finding confirms that SWV is a valuable method for the study of PNA-DNA hybridization processes at interfaces. We observed that the measured redox current depended on the used electrolyte concentration and assumed some structural reorganization of the immobilized PNA-DNA duplexes by varying the salt concentrations. At high salt concentrations, the redox current is strongly reduced probably because the collapsed helices block the electron transfer. At low salt concentrations, the redox reaction of the Fc moiety attached to the duplexes is facilitated by the stretched helices. SPFS results confirm the structural transformation of the PNA-DNA duplexes. The observed ability of short-strand DNA helices to easily change their structure or packing may be a very important feature related to their biological function. Finally, we put emphasis on the fact that electrochemical and SPFS methods are both very sensitive and well-suited for investigations of nanometer thin layers and provide functional and structural information about the molecular architecture of the sensor surfaces. ACKNOWLEDGMENT This work was partly supported by the Deutsche Forschunggemeinschaft (DFG, KN 224/13-1). Received for review July 21, 2005. Accepted November 3, 2005. AC051299C