Charge Transfer through Modified Peptide Nucleic Acids - Langmuir

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Charge Transfer through Modified Peptide Nucleic Acids Emil Wierzbinski,† Arnie de Leon,‡ Kathryn L. Davis,† Silvia Bezer,‡ Matthaü s A. Wolak,§ Matthew J. Kofke,† Rudy Schlaf,§ Catalina Achim,*,‡ and David H. Waldeck*,† †

Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States § Department of Electrical Engineering, University of South Florida, Tampa, Florida 33620, United States ‡

S Supporting Information *

ABSTRACT: We studied the charge transfer properties of bipyridine-modified peptide nucleic acid (PNA) in the absence and presence of Zn(II). Characterization of the PNA in solution showed that Zn(II) interacts with the bipyridine ligands, but the stability of the duplexes was not affected significantly by the binding of Zn(II). The charge transfer properties of these molecules were examined by electrochemistry for self-assembled monolayers of ferrocene-terminated PNAs and by conductive probe atomic force microscopy for cysteine-terminated PNAs. Both electrochemical and single molecular studies showed that the bipyridine modification and Zn(II) binding do not affect significantly the charge transfer of the PNA duplexes.

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backbone is based on N-(2-aminoethyl)glycine,11 which is neutral, the PNA binds to complementary nucleic acids such as DNA and RNA with high affinity. Neutral PNAs are also better candidates for surface self-assembly than nucleic acids with a charged backbone because of the reduced electrostatic repulsion between the nucleic acid strands or duplexes. For these reasons, PNA has been used for electrochemical investigations of charge transfer in single-stranded (ss) and ds nucleic acid films.12 Herein, we report the effect of zinc binding to the bipyridinemodified PNA duplexes on the charge transfer of the PNA. We chose Zn(II) because it is an electrochemically inactive metal ion, thus allowing us to exclusively probe the effect of the perturbation of the bipyridine by coordination, without the metal ion providing new hopping centers within the conductance pathway. The sequences of the five PNA duplexes we studied here are the same except for one central base pair position (Chart 1). The sequences are labeled by the identity of the variable, central base pair/ligand. The spectroscopic studies we report here showed that Zn(II) binds to the bipyridines in the ligand-modified PNA but does not affect the thermal stability of the modified PNA. We and others have previously shown that well-packed self-assembled monolayers (SAMs) of PNA can be formed on gold surfaces.13 Previous studies of

INTRODUCTION DNA and its synthetic analogues have great potential as scaffolds for nanostructures.1 They are considered also as candidates for active elements in nanoelectronic devices.1a,2 Their promise arises in part from the ability to encode information in their sequence that determines the formation of well-defined, supramolecular structures by Watson−Crick hybridization. Experimental studies of double-stranded (ds) DNA by break junction,3 scanning tunneling microscopy,4 and conductive probe atomic force microscopy (CP-AFM)5 indicated a dependence of their charge transfer properties on their nucleobase sequence. Additional control over the structure, and implicitly the electronic properties, of the DNA duplexes is possible by changing the hydrogen-bond basepairing motif with alternate pairing interactions, such as hydrophobic interactions6 or site-specific metal coordination.7 Formation of metal complexes within the nucleic acid duplexes is particularly appealing for altering the electronic properties of nucleic acids because of the electronic and magnetic properties of the metal ions. Recently, Liu et al. showed by singlemolecule conductance experiments that the molecular conductance of a DNA duplex containing three pairs of hydroxypyridone ligands increased when the duplex was exposed to Cu(II),8 although the conductance of a duplex containing one pair of ligands in the presence of Cu(II) was similar to that of a DNA duplex with no ligand. We have demonstrated that it is possible to synthesize metalcontaining peptide nucleic acid (PNA) duplexes using bipyridine and terpyridine9 and 8-hydroxyquinoline10 ligands that are programmed into the PNA sequence. Because the PNA © 2012 American Chemical Society

Special Issue: Bioinspired Assemblies and Interfaces Received: November 11, 2011 Revised: January 3, 2012 Published: January 4, 2012 1971

dx.doi.org/10.1021/la204445u | Langmuir 2012, 28, 1971−1981

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Chart 1. The Sequences of PNA Duplexesa

ization of the oligomers was performed by MALDI-ToF mass spectrometry on an Applied Biosystems Voyager Biospectrometry Workstation with Delayed Extraction and an α-cyano-4-hydroxycinnamic acid matrix (10 mg/mL in 1:1 water:acetonitrile, 0.1% trifluoroacetic acid (TFA)). (Information about the masses of studied oligomers is given in the Supporting Information, Table S1.) PNA stock solutions were prepared in deionized water and were stored at −18 °C. The PNA solutions for UV and circular dichroism (CD) spectroscopy were in pH 7.0 10 mM phosphate buffer (hereafter called buffer). The concentration of these solutions was determined by UV absorption at 95 °C using the sum of the published extinction coefficients at 260 nm; ε260 of the constituent PNA monomers were taken to be 8600 M−1 cm−1 for T, 6600 M−1 cm−1 for C, 13700 M−1 cm−1 for A, and 11700 M−1 cm−1 for G.15c The extinction coefficient for the bipyridine monomer (ε260 = 9770 M−1 cm−1 at pH 7) was previously determined.16 The PNA solutions for electrochemistry and conducting AFM experiments were in a 1:1 v/v mixture of buffer and acetonitrile. To verify that the PNA duplexes exist under the different conditions (solvent/temperature) required for sample preparation for the different experiments described in this paper, the melting temperature of Bpy:Bpy was also measured in a “universal” buffer of boric acid, phosphoric acid, and acetic acid adjusted with sodium hydroxide to pH 7.0 or pH 4.1 as well as in the pH 7.0 buffer mixed in a 1:1 v/v ratio with acetonitrile. Under all of these conditions, the duplex showed cooperative melting. The melting temperatures were 50, 48, and 42 °C, respectively. Melting Temperature. Melting temperature experiments were performed in 10-mm path length quartz cells on a Varian Cary 300 spectrophotometer equipped with a programmable temperature block. UV melting curves were recorded in the temperature range of 15−95 °C. The rate of both cooling and heating was 1 °C/min. Prior to the measurement of the melting profiles, the solutions were kept at 15 °C for at least 5 min. The melting temperature Tm was taken at the inflection point of the Boltzmann sigmoidal fit function, which assumes a two-state model. Spectrophotometric Titration. UV−visible spectrophotometric titrations were performed at room temperature in a 1-mL optical path cuvette. Stock solutions of Zn(II) in deionized water were prepared from Zn(NO3)2. One milliliter of a 5 μM solution of Bpy:Bpy duplex in buffer was titrated with a 0.2 mM Zn(II) solution. UV absorption spectra were recorded after each addition of 2 μL of the Zn(II) solution. CD Spectroscopy. CD spectra were measured for 10 μM of the PNA duplex in buffer. CD measurements were conducted on a JASCO J-715 spectropolarimeter equipped with a thermoelectrically controlled, single-cell holder. CD spectra were collected using the following parameters: 1 nm bandwidth, 1 s response time, 100 nm/ min scan speed, 20 mdeg sensitivity, and 8 scan accumulation. Photoemission Spectroscopy. All experiments were performed in a commercial (SPECS Nano Analysis GmbH, Berlin, Germany) ultrahigh vacuum (UHV) multichamber system. This system consists of a fast entry lock with an attached Plexiglas glovebox, which enables a direct sample transfer from the glovebox to vacuum. The system also contains two preparation chambers and an analysis chamber equipped with X-ray photoemission spectroscopy (XPS), which has a base pressure of approximately 2 × 10−10 mbar. During sample preparation, the glovebox was flushed and filled with 99.995% purity N2 and kept under a slight overpressure to suppress sample contamination from the ambient environment. In order to reduce residual carbohydrate and H2O contamination, a diaphragm pump was used in series with filters containing active carbon and Drierite drying agent to circulate the N2 atmosphere. Sample Preparation. The substrates, thin films of Au (100 nm thick) deposited on glass slides with an approximately 20 nm thick Ti adhesion layer, were obtained from EMF Corp. They were cut into 0.8 cm × 1 cm pieces and mounted on stainless steel sample holders. Electrical contact between the Au layer and the sample holder was ensured through direct contact with the mounting screws. The Au surface was sputtered with Ar+ ions subsequently after transferring the sample into a vacuum in order to clean the substrate

a All sequences were derived from the 10-bp PNA duplex TCACT-XGATG:CATC-Y-AGTGA, where X:Y = A:T, A:C, Bpy:Bpy, Bk:Bk, and Bk:Bpy; Bk indicates a PNA monomer that does not have a ligand or nucleobase attached to the backbone. In the text, duplexes are identified by the name of the central pair of monomers, i.e., A:T, A:C, etc. bThe parenthesis at the end of each strand identifies the substituent at that end of that strand for electrochemistry/single molecule conductance experiments. For electrochemical studies, duplexes contained a strand with a C-end cysteine (Cys), which allows assembly of the molecules on the gold surface, and an N-en ferrocene (Fc), which functioned as a redox reporter, and another strand that had a C-end Lysine. For single-molecule conductance measurements, both strands of the duplex had a C-end cysteine.

SAMs of DNA showed that the charge transfer resistance of the SAMs decreased upon incubation with Zn(II) only if the pH was higher than 8, which was attributed to the formation of MDNA.25 In the current studies, we have created SAMs of PNA only at pH lower or equal to 7 to ensure preferential coordination of Zn(II) to bipyridine rather than to the nucleobases. Charge transport through the nonmodified and bipyridinemodified PNA duplexes, in the absence and presence of Zn(II), was studied by electrochemistry and single-molecule conductance. Cyclic voltammetry allowed us to determine the charge transfer rate constant for SAMs of duplex PNA that contained a ferrocene redox reporter group tethered to the PNA at the SAM/solution interface. CP-AFM, an approach first proposed by Lindsay and co-workers, allowed us to measure the single molecule conductance.14 To the best of our knowledge, no combined study of charge transfer and conductance on unmodified or metal-containing DNA has been reported to date. The electrochemical and single molecule conductance results reported here show that Zn(II) binding to bipyridine in the ligand-modified PNA duplexes does not affect the charge transport properties of the PNA.

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EXPERIMENTAL SECTION

Solid-Phase PNA Synthesis. PNA oligomers were synthesized by typical solid phase peptide synthesis methods using the Boc-protection strategy.15 PNA monomers were purchased from Applied Biosystems and used without further purification. Ferrocene carboxylic acid was purchased from Sigma Aldrich. The bipyridine PNA monomer was synthesized by published methods.16 After cleavage, PNA was precipitated using ethyl ether and was purified by reverse-phase high-performance liquid chromatography (HPLC) using a C18 silica column on a Waters 600 Controller and Pump. Absorbance was measured with a Waters 2996 Photodiode Array Detector. Character1972


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Figure 1. (A) Schematic representation of the sample preparation for single-molecule conductance measurements. Two complementary ss-PNAs, each containing a terminal cysteine, were incubated separately. One of the strands was incubated with a solution of GNPs (see blue border panels), and the other one with the gold substrate (see yellow border panels). The hybridization of the GNPs incubated with PNA to the PNA strands attached to the surface led to GNP-modified PNA duplexes located in a SAM of ss-PNA. (B) An AFM image of the gold substrate decorated with GNPs (bright objects). The height of the nanoparticles measured on cross section (red line in bottom panel), is close to the nominal diameter of the GNPs of about 5 nm. The apparent diameter in the AFM image is somewhat larger because of the finite size of the AFM tip. A stepwise procedure based on that of Maskus and Abruña18 was used to form ds-PNA SAMs containing metal−ligand complexes. This procedure involves the formation of metal-free PNA SAMs on the electrode before exposure of the PNA to zinc to avoid its binding to the C-terminal cysteine. First, the ds-PNA SAM was formed on the surface of the gold ball electrode via immersion in a 20 μM ds-PNA solution in 1:1 v/v acetonitrile/buffer at 27 °C for 28−40 h. Following incubation with the PNA, the electrodes were thoroughly rinsed with deionized water and then stored in deionized water until use. Prior to metal exposure, electrodes were characterized by cyclic voltammetry. For the formation of the PNA/metal complexes, electrodes were exposed to 0.5 M Zn(NO3)2 for 1 min, after which the electrode was rinsed with deionized water and characterized again using cyclic voltammetry. Following characterization, the electrode was returned to the Zn(NO3)2 solution. This metal exposure, rinsing, and characterization procedure was repeated at total Zn(NO3)2 exposure times of 1 min, 2−3 h, and 18−24 h. Electrochemical Measurements of Charge Transfer Rate Constant. Measurements were performed in a three-electrode setup in a supporting electrolyte solution of 1 M NaClO4 (pH ≈ 6−7), with a Ag/AgCl (1 M KCl) reference electrode, a platinum wire counter electrode, and a PNA-modified gold ball electrode. Scan rates (v) ranged from 1 mV/s to 100 mV/s in a potential window of 0.0 to 0.8 V. Where applicable, k0 values were obtained by plotting the anodic and cathodic peak separation (Ep − E0) versus the normalized scan rate (v/k0) and fitting the data by a curve based on Marcus theory,19 using a reorganization energy (λ) of 0.8 eV.19a Surface coverages Γ were estimated from integration of the charge under the Faradaic current peaks at a scan rate of 5 mV/s. Single-Molecule Conductance Measurements. All measurements were performed using the CP-AFM technique with an Agilent 5500 system equipped with an environmental chamber. The AFM head was placed in a homemade, acoustically isolated Faraday cage and placed on an active antivibrational system (TableStable) located on an optical table. Conductance measurements were performed using an approach developed by Lindsay and co-workers.14 In this approach, molecules of interest are immobilized on gold substrates, and are diluted within an insulating matrix. The studied molecules are covalently bonded on one end to a gold substrate and on the other

surface from ambient atmospheric contamination. A SPECS IQE 11/ 35 ion source was used; this source produces Ar+ ions with a kinetic energy of 5 keV at an emission current of 10 mA and an Ar pressure of ∼4 × 10−5 mbar. After characterization of the sputtered Au surface, the substrate was moved from the analysis chamber into the glovebox. Solutions of 20 μM A:T, Bk:Bpy, and Bpy:Bpy in deionized water were prepared before each photoemission spectroscopy experiment. Sputtered Au samples were incubated in each of the solutions for 40 h at an approximate temperature of 27 °C. After the incubation was completed, each sample was removed from its solution and rinsed with deionized water. The residual solvent on the surface was evaporated by placing the samples in the nitrogen flow inside the glovebox. Each sample was then separately moved back into the load lock and transferred into the analysis chamber for XPS analysis. Next, the samples were moved back into the glovebox and incubated in a 0.5 M solution of Zn(NO3)2 overnight. The next day, the samples were removed from the solution and thoroughly rinsed with ethanol. Residual solvent was again evaporated by placing the samples in the nitrogen flow of the glovebox. The samples were then transferred back into the analysis chamber for XPS analysis. Photoemission Spectroscopy Measurements. Characterization of the samples by photoemission spectroscopy before and after each incubation step was performed using a SPECS XR 50 X-ray gun. The Mg Kα (hν = 1235.6 eV, 20 mA emission current) X-ray emission line was used for standard core-level XPS, and the analysis of the photoelectrons was performed using a SPECS Phoibos 100 hemispherical analyzer. The spectrometer was calibrated to yield the standard Cu 2p3/2 line at 932.66 eV and the Cu 3p3/2 line at 75.13 eV. The photoemission spectra were analyzed using Igor Pro software (WaveMetrics, Inc.), and the core-level emission features were fitted using Gaussian−Lorentzian profiles in order to determine peak positions and full widths at half-maximum (fwhm's).17 Electrochemical Measurements. Charge transfer properties of ferrocene-terminated PNA SAMs on gold ball electrodes were studied by cyclic voltammetry. Electrode characterization was performed using a CH Instruments 618B or 430A electrochemical analyzer. Preparation of the Electrodes. Gold ball electrodes were prepared and annealed in a manner similar to that described in earlier reports.12 1973


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end to a gold nanoparticle (GNP). The electrical circuit is closed by touching the nanoparticle with a gold-coated AFM tip. Sample Preparation. To produce the sample in which single dsPNA molecules attributed with GNPs are embedded in the ss-PNA monolayer, we followed the procedure developed for DNA by Naaman and co-workers.5b In this procedure, batches of the thiolated complementary single strands of the DNA are incubated separately: one batch with the gold substrate and the other in a solution containing GNPs. Subsequently, the batches are combined so that they hybridize on the surface to form a DNA duplex. In the presented experiments, freshly annealed gold-on-mica substrates (Agilent) were incubated with a 20 μM solution of ss-PNA in 1:1 v/v acetonitrile/ buffer for 12 h to form an ss-PNA monolayer. In a separate step, the complementary PNA strands were incubated for 48 h with commercially available gold colloid (Ted Pella) that contained citrate-stabilized GNPs of about 5 nm diameter. The concentrations of the PNA solutions and the gold colloid were adjusted to provide 10fold excess of the nanoparticles over the PNA strands. Finally, the gold substrate covered with an ss-PNA monolayer was incubated for 48 h with the solution containing GNP-premodified complementary PNA strands. All incubations were performed at room temperature. A schematic diagram of this procedure is given in Figure 1A. Zn(II) was incorporated into the Bpy:Bpy duplexes by an additional overnight incubation in a 0.5 M aqueous solution of Zn(NO3)2. Samples were rinsed with ethanol and dried under a stream of argon gas prior to experiments. Figure 1B shows the morphology of a sample substrate decorated with GNPs that are attached to ds-PNA molecules diluted within an ss-PNA SAM. Conductance Measurements. Experiments were performed using gold-coated AFM cantilevers with a nominal spring constant of 0.2 N/ m (PPP-ContAu, Nanosensors). Precise values of the spring constants were determined using a thermal oscillation technique.20 Typically, three to five cantilevers were used to collect sufficient data for a single PNA sequence. To limit the adhesion force between the AFM tip and the SAMs, all experiments were performed in bicyclohexyl under argon atmosphere. To monitor the topography, the samples were scanned with a force of about 3 to 4 nN applied to the AFM tip. After collecting topographical images of the substrate surface, individual GNPs were targeted with the AFM tip, and the force was increased to 7−14 nN for conductance measurements. No influence of the applied force on the measured conductance was observed in this range of applied forces. Smaller forces resulted in poor contact between the AFM tip and the nanoparticle. Higher forces were not applied because they could cause damage to the SAMs or displacement of the GNPs. About 20 to 50 current−voltage curves were collected on each single nanoparticle. Typically, a few tens of the nanoparticles were targeted within a single sample. This procedure was performed on at least three independently prepared samples, resulting in the collection of a total number of about 10 000 current−voltage curves for each type of PNA duplex. Conductance results were plotted in the form of normalized histograms. Conductance values are expressed in units of the quantum conductance G0 = 2e2/h or 7.748 × 10−5 S.

Figure 2. (A) UV melting curves are shown for 5 μM solutions of the Bpy:Bpy (blue), Bk:Bpy (red), and Bk:Bk (black) PNA duplexes; (B) UV melting curves are shown for 5 μM solutions of the Bpy:Bpy PNA duplex in the absence (black) and presence of 1 (red) or 4 (blue) equiv of Zn(II).

caused a decrease in the cooperativity and hyperchromicity of the melting of the duplex (Figure 2B, Table 1). Table 1. Melting Temperatures Tm of PNA Duplexes Tm / oC

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RESULTS AND DISCUSSION Thermal Stability of the Duplexes. Thermal denaturation curves were measured for both nonmodified and ligandmodified PNA duplexes in the absence and in the presence of Zn(II). The melting temperature of the nonmodified PNA was 66 °C, whereas the melting temperature of the modified PNAs was between 48 and 50 °C (Figure 2A). This decrease in melting temperature is similar to the change in the thermal stability of PNA duplexes of similar length that is induced by a central base pair mismatch.9,16 Addition of 1 equiv of Zn(II) to the Bpy:Bpy and Bk:Bpy PNA duplexes did not affect their thermal stability but caused a decrease in the hyperchromicity change associated with the melting of the duplex. The addition of 4 or more equivalents of Zn(II) destabilized the duplex and

sequence

0 Zn

+ 1eq Zn(II)

+ 4eq Zn(II)

A:T A:C Bpy:Bpy Bk:Bk Bk:Bpy

66 52 50 48 49

49 48 49

45 44 45

We have previously studied the interaction of Co(II), Ni(II), and Cu(II) with Bpy:Bpy and showed that there is a correlation between the stability constants of the complexes formed by bipyridine and metal ions and the increase of the melting temperature of the Bpy:Bpy duplex.16 The current results on the interaction of Zn(II) with Bpy:Bpy are in agreement with those observed previously for Cu(II), in that study the binding of Cu(II) and Zn(II) to the PNA does not affect the thermal stability of the ligand-modified duplex, although UV spectroscopy (and electron paramagnetic resonance (EPR) spectroscopy in the case of Cu(II)) showed that the metal ions bind to the Bpy:Bpy PNA. 1974


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Spectrophotometric Titration of the Bipyridine-Containing PNAs with Zn(II). Titration of the preannealed Bpy:Bpy PNA duplex with Zn(II) at low temperature caused the appearance of a band at 300−320 nm, which arises from the π−π* transition of the coordinated bipyridine (Figure 3). A

Figure 4. CD spectra are shown for 10 μM solutions of the Bpy:Bpy− (panel A) and Bk:Bpy−PNA (panel B) duplexes in the absence of Zn(II) (black line) and in the presence of 1 equiv of Zn(II) (red line) or 4 equiv of Zn(II) (blue line).

introduced in the PNA duplexes. The data do not allow us to establish the coordination mode of the Zn(II) to bipyridine, which could be intraduplex, with the formation of Zn(II) complexes with two bipyridine ligands from the same duplex, or interduplex, with Zn(II) coordinating bipyridines from different duplexes.21 Solvent or nucleobases could complete the coordination sphere of Zn(II) in both cases. Binding of the Zn(II) to the PNA SAMs. To ascertain whether Zn(II) binds to the PNA duplexes within SAMs, SAMs of nonmodified and bipyridine-modified PNAs on gold surfaces were studied by XPS in the absence and presence of Zn(II) exposure. Figure 5 shows the measured spectral features for C 1s, N 1s, O 1s, Zn 2p, S 2p, and Au 4f regions of the photoemission spectra of SAMs of A:T, Bk:Bpy, and Bpy:Bpy, before and after incubation with a solution of Zn(NO3)2. Using the attenuation of the Au 4f spectrum, an estimate of the film thickness can be made. The peak intensities of the core level emission at 84 eV were fitted by the exponential function

Figure 3. (A) UV spectra are shown for the titration of a 5 μM solution of Bpy:Bpy PNA with Zn(II). (B) UV−visible spectra are shown for Bpy:Bpy PNA in the presence of 4 equiv of Zn(II) at 22 °C (black) and 95 °C (red).

similar band was observed in the 22 °C spectrum of the Bpy:Bpy PNA annealed in the presence of Zn(II) (Figure 2B). These spectral changes indicate that Zn(II) coordinates to the bipyridines in Bpy:Bpy PNA in both cases. However, titration curves measured at 314 and 260 nm did not show a clear inflection point. This observation indicates that the binding of Zn(II) to the bipyridine ligands is weak (see Supporting Information, Figure S1A). The CD spectra of Bpy:Bpy and Bk:Bpy PNA duplexes, in the absence and the presence of Zn(II), exhibited a biphasic exciton coupling pattern at 254 nm, which is characteristic of a left-handed helix (Figure 4).11 The intensity of this spectral feature decreased when Zn(II) was added to the solutions of bipyridine-modified PNA, which indicates that Zn(II) affected the structure of the duplex. The spectra of the Bpy:Bpy and Bk:Bpy PNA duplexes exhibited a negative peak at 288 nm, which can be attributed to the bipyridine ligands. Taken together these spectral changes indicate that Zn(II) binds selectively but weakly to the bipyridine ligands

I = I0 exp( − d /α)

(1)

in which I is the measured intensity of the covered substrate, I0 is the intensity of the uncovered substrate, d is the thickness of the thin film, and α is the mean free path of the emitted electrons, which was taken to be 36 Å.22 On the basis of the I/I0 intensity ratios determined from the peak intensity of the corelevel emission at 84 eV, average film layer thicknesses were 1975


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Figure 5. The panels show core-level XPS spectra for C 1s, N 1s, O 1s, S 2p, Zn 2p, and Au 4f on sputtered Au and after incubation in solutions containing A:T, Bk:Bpy, and Bpy:Bpy sequences, as indicated. The spectra were taken before and after incubation with 0.5 M Zn(NO3)2.

films23 and indicate that Zn(II) binds to the PNAs during incubation. The stoichiometric C/Zn ratios were calculated by integration of the areas of the C1s and Zn2p3/2 emission lines and taking into account the different ionization cross sections, according to Scofield.24 The C/Zn ratios were 64:1, 40:1, and 65:1 for SAMs of nonmodified A:T PNA, bipyridine-containing Bk:Bpy PNA, and Bpy:Bpy PNA, respectively, which implies that four to five zinc ions are present for every PNA duplex, irrespective of the presence in the PNA of bipyridine. This nonspecific binding of Zn(II) to the PNA is not surprising, given the large excess of Zn(II) present during incubation. Electrochemical Measurements. Table 3 summarizes the rate constant and surface coverage for the four PNA duplexes: A:T, A:C, Bpy:Bpy, and Bk:Bpy. All PNAs have similar surface coverage, which indicates that modification of the PNA duplexes with bipyridine does not influence the PNA assembly and packing on the gold surface. The surface coverage reported here is in good agreement with that reported earlier for SAMs of 10-bp PNA duplexes composed exclusively of AT base pairs.12c Voltammogram sets and plots of (Ep − E0) versus normalized scan rate (log(v/k0)) for each of these PNAs are shown in Figure 7. The solid curves in Figure 7C,D are the simulations based on the Marcus model and assume a

determined to be 32 Å for the A:T SAM, 27 Å for the A:T monolayer incubated in the Zn(NO3)2 solution, 20 Å for the Bk:Bpy SAM, 22 Å for the Bk:Bpy monolayer incubated in the Zn(NO3)2 solution, 26 Å for the Bpy:Bpy SAM, and 25 Å after Zn(NO3)2 incubation of the Bpy:Bpy SAM. It should be noted that this thickness estimate is based on the assumption that the layer is of homogeneous thickness. The XPS data indicate that the cysteine binds to the Au and the duplex is presented to the vacuum. As the sulfur atom in the C-terminus of the double PNA strand is expected to bind with the Au surface, it should be buried by the molecules, leading to a suppression of the S 2p signal. Indeed, no S 2p emissions were detected. The C 1s emission features of all three samples display three peaks located at binding energies around 285, 286, and 288 eV. After incubation with Zn(II), these features do not change significantly, either in relative intensity or energy. This result indicates that Zn(II) does not significantly affect the oxidation state of the carbons. Figure 6 shows the fitted Zn 2p3/2 emission features for A:T, Bk:Bpy, and Bpy:Bpy SAMs after incubation with Zn(NO3)2. The fits of the emissions yielded Zn 2p3/2 binding energies of 1022.4 eV for the A:T monolayer, 1022.44 eV for the Bk:Bpy monolayer, and 1022.51 eV for the Bpy:Bpy. These values are in very good agreement with the values measured on ZnO thin 1976


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for the ferrocene reporter group occurs near 0.45 V vs Ag/AgCl (1 M KCl). For the mismatch duplex A:C, the ferrocene E0 was the same; however the rate constant k0 was 0.05 ± 0.02 s−1, which is a factor of 4−5 smaller than that measured for the fully matched A:T PNA duplex. Thus, a mismatch in the nucleobase sequence significantly slows charge transfer, but does not block it. The standard electrochemical rate constant for the Bpy:Bpy PNA duplex is nearly identical to that measured for the nonmodified A:T PNA duplex. By contrast, the rate constant for the Bk:Bpy PNA duplex is almost half of the rate constant for the A:T and Bpy:Bpy PNA duplexes (Table 2 and Figure Table 2. Electrochemical Charge Transfer Rate Constants k0 and Surface Coverages Γ for ds-PNA SAMs sequence A:T A:C Bpy:Bpy Bk:Bpy

k0 / s−1 0.23 0.05 0.25 0.13

± ± ± ±

0.10 0.02 0.12 0.03

Γ / pmol cm−2 42 52 52 39

± ± ± ±

15 33 22 19

8). This difference suggests that the π stacking interactions and/or the duplex structure are affected significantly by the abasic site, but that the bipyridines have a minimal effect on the structure and electronic coupling. Figure 8 shows data that explores how the electrochemistry depends on the time of exposure to Zn(II). Figure 8A shows the cyclic voltammograms for SAMs of Bpy:Bpy PNA duplexes for a single electrode following 1 min, 2.5 h, and 19.5 h incubation with a solution of Zn(NO3)2. Figure 8B shows the dependence of the peak position on the scan rate, (Ep − E0) versus log(v/k0), for the same electrode as a function of exposure time. In order to see the change in k0 over time, each (Ep − E0) plot in panel B has been normalized to the k0 value

Figure 6. These spectra correspond to Zn 2p3/2 emission features for A:T, Bk:Bpy, and Bpy:Bpy SAMs after incubation with Zn(NO3)2 and are fit to a Gaussian line shape.

reorganization energy of 0.8 eV for the ferrocene redox couple. The electrochemical properties of the SAMs of A:T and A:C duplexes are similar to those of other PNA SAMs previously examined by cyclic voltammetry.12 In particular, the scan rate dependence of the voltammetry for the A:T duplex gives a standard electrochemical rate constant k0 of 0.22 s−1, and the E0

Figure 7. Cyclic voltammograms are shown at 5 (blue), 10 (red), 15 (green), and 20 (violet) mV/s for a SAM composed of the fully complementary A:T (A) or the mismatched A:C (B) PNA duplex are shown in the top panel. Plots of peak position relative to the formal potential (Ep − E0) as a function of the normalized scan rate (v/k0) for A:T and A:C sequences are shown in panels C and D, respectively. The anodic and cathodic peak positions are shown by filled and open symbols, respectively. Scan rates of 3−50 mV/s for the A:T PNA were normalized to k0 = 0.22 s−1 and scan rates of 1−20 mV/s for the A:C PNA were normalized to k0 = 0.062 s−1 (solid lines). 1977


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Figure 8. Voltammograms (A) and relative peak potential (Ep − E0) versus log of the normalized scan rate log(v/k0) plots (B) are shown for one electrode coated with a SAM composed of Bpy:Bpy duplexes, following exposure to 0.5 M Zn(NO3)2 for 0 min (blue), 1 min (red), 2.5 h (violet), and 19.5 h (green). The anodic and cathodic peak positions are represented by filled and open symbols, respectively. Panel A shows cyclic voltammograms taken at 5 mV/s at each Zn(NO3)2 exposure time; panel B shows plots of (Ep − E0) versus log(v/k0) for the same electrode that was used to obtain the data shown in (A). The scan rate ranged from 3 to 75 mV/s and was normalized to the k0 value of 0.25 s−1, which was determined prior to Zn(NO3)2 exposure (solid black curve).

prior to Zn(NO3)2 exposure. Incubation with Zn(II) does not perturb the redox potential of ferrocene significantly and does not change the surface coverage of active redox centers. Aside from a decrease in the large background current at the positive edge of the potential window (∼0.5 to 0.7 V) after Zn(NO3)2 exposure, no significant change in the appearance of the voltammograms occurs over the time of Zn(NO3)2 exposure. The variations of the k0 values measured for a single electrode are not much larger than the calculated errors. The average value of k0 for the Bpy:Bpy PNA duplex does not change upon exposure to Zn(II) (Table 3). The fact that charge transfer Table 3. Summary of Electrode Properties for Films of Bpy:Bpy Sequence Exposed to 0.5 M Zn(NO3)2 for Varying Times exposure time 0 1 min 1−3 h 18−21 h

k0 / s−1 0.25 0.15 0.17 0.35

± ± ± ±

0.12 0.02 0.08 0.03

Γ /pmol cm−2 52 55 87 43

± ± ± ±

22 13 29 16

through the Bpy:Bpy PNA is the same in the absence and presence of Zn(II) means that either the bipyridine is not involved at all in charge transfer or that the change induced in Bpy:Bpy PNA by Zn(II) has no effect on the charge transfer. Because one bipyridine substitution, i.e., Bk:Bpy sequence, does significantly affect the charge transfer rate constant (Table 2), the latter explanation is the more likely one. Single-Molecule Conductance. The molecular conductance was measured by a procedure that was originally developed by Lindsay and co-workers for alkanedithiols.14 The molecules of interest, i.e., nonmodified and bipyridinemodified PNA duplexes, were immobilized on gold substrates in an insulating matrix of ss-PNAs. The PNA duplexes were covalently bonded on one end to the gold substrate and on the other end to a GNP. The electrical circuit was closed by touching the nanoparticle with a gold-coated, AFM tip. A schematic representation of this experimental approach is shown in Figure 9A. Multiple measurements of current−voltage curves were performed, and the conductance of each PNA

Figure 9. (A) Cartoon representation of the experimental system for single-molecule conductance measurements. (B) Sample current− voltage characteristics recorded for the A:T sequence. Red curve was recorded on the ss-PNA matrix.

duplex was determined from the near-zero bias slopes of these curves (typically from −0.1 to +0.1 V). Figure 9B shows several current−voltage scans recorded for A:T PNA duplexes and the characteristic curve (the red curve) recorded for the insulating ss-PNA matrix. Differences in the slope of individual current− voltage curves recorded for duplexes with the same sequence can originate from variations in the geometry and/or contact configurations of the junction.12d,26 1978


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Figure 10. Conductance distributions constructed from the analysis of near-zero bias current−voltage characteristics are shown for A:T (A), A:C (B), and Bpy:Bpy (C) duplexes and for Bpy:Bpy duplexes after incubation with Zn(II) (D). For easier comparison, panels B and D show the distributions for A:T (black line) and Bpy:Bpy (blue line) PNA duplexes, respectively.

faster charge transfer through the A:T SAM than that measured for the A:C SAMs. The Bpy:Bpy duplexes had a broad peak centered at 1.4 × 10−3 G0, which is similar to that measured for A:T duplexes (Figure 10C). The conductance distributions of the Bpy:Bpy PNA duplex become somewhat broader, but the average conductance does not change notably after incubation with Zn(II) (Figure 10D). These observations are in agreement with the results of the electrochemical measurements, which showed that the rate constant for charge transfer for Bpy:Bpy PNA duplexes was not affected by the incubation with Zn(II) and was similar to that determined for SAMs of the nonmodified A:T PNA. The fact that neither the charge transfer rate constant nor the conductance of a PNA duplex containing one pair of Bpy ligands is affected by Zn(II) coordination is consistent with the recent report by Liu et al., who showed that the electrical conductance of a DNA duplex that contained one pair of hydroxypyridone ligands was not affected by Cu(II) coordination.8 By contrast, the electrical conductance of a single DNA

Conductance distributions for the A:T, A:C, and Bpy:Bpy sequences are presented in Figure 10. In all cases, the distributions show a broad range of conductance values, with a majority of the data below 3 × 10−3 G0. The high number of counts at low conductance (

Charge Transfer through Modified Peptide Nucleic Acids - Langmuir

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