Electronic Structure of Self-Assembled Peptide Nucleic Acid Thin

Metal Complexes as Alternative Base Pairs or Triplets in Natural and Synthetic Nucleic Acid Structures. Arnie De , Jing Kong , Catalina Achim. 2013,33...
0 downloads 0 Views 3MB Size
ARTICLE pubs.acs.org/JPCC

Electronic Structure of Self-Assembled Peptide Nucleic Acid Thin Films Matth€aus A. Wolak,† Alexander Balaeff,‡ Sebastian Gutmann,§ Harry J. Helmrich,|| Ruan Vosloo,† Martin M. Beerbom,† Emil Wierzbinski,^ David H. Waldeck,^ Silvia Bezer,# Catalina Achim,# David N. Beratan,‡ and Rudy Schlaf*,† †

Department of Electrical Engineering, University of South Florida, Tampa, Florida 33620, United States Department of Chemistry, Duke University, Durham, North Carolina 27708, United States § Department of Chemistry, University of South Florida, Tampa, Florida 33620, United States Department of Chemical Engineering, University of South Florida, Tampa, Florida 33620, United States ^ Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States # Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States

)



bS Supporting Information ABSTRACT: The electronic structure of self-assembled monolayers (SAMs) of peptide nucleic acid (PNA) formed on Au substrates was investigated. Cys-appended PNA 7-mers of thymine (Cys-T7) were incubated on Au substrates in a nitrogen glovebox attached to a photoemission spectrometer. Ultraviolet and X-ray photoemission spectroscopy (UPS and XPS) measurements on the resulting SAMs revealed the hole injection barrier at the interface and the interface dipole. Electronic structure calculations based on molecular dynamics sampling of the PNA structure yielded the HOMOLUMO gap and the electronic density of states for PNA. Combined with the UPS data, the theoretical calculation enabled estimation of the charge injection barriers at the interface, as well as the assignment of individual UP spectral features to specific molecular orbitals. Interestingly, the dipole moment of the PNA backbone is predicted to polarize PNA MOs, shifting the preferred HOMO localization toward the C-terminus of PNA. Control measurements on Cys-appended, abasic PNA backbone 7-mers (Cys-Bckb7) allowed the identification of the emissions related to the PNA backbone in the UP spectra. The orbital line-up at the interface between the Au substrate and the Cys-PNA indicates a significant interface dipole resulting in the alignment of the Au Fermi level near the center of the PNA HOMOLUMO gap. This alignment causes large charge injection barriers for both holes and electrons, and thus impedes charge transfer from Au into the Cys-PNA SAM.

’ INTRODUCTION This paper explores the electronic structure of interfaces between self-assembled monolayers (SAMs) of peptide nucleic acid (PNA) and the Au substrate on which the monolayer forms. PNA SAMs are of interest for applications in molecular recognition, biosensing, and molecular electronics.15 PNA is a synthetic analogue of DNA with an electrically neutral backbone usually based on N-aminoethyl glycine (Scheme 1), and as such, it withstands hydrolysis by nucleases.6,7 PNA forms Watson Crick duplexes with itself or with other nucleic acids. Both PNA homoduplexes and PNA heteroduplexes with DNA or RNA have thermal stability higher than that of DNA and RNA homoduplexes.6,7 These properties make PNA a promising alternative for DNA in nano- and bio-technology applications in which hybridization and chemical robustness are key requirements. Recently, rate constants of charge transfer through SAMs of single-strand (ss) PNA with a cysteine moiety at the C-terminus and a ferrocene redox probe at the N-terminus were measured by cyclic voltammetry, as a function of PNA length and r 2011 American Chemical Society

sequence.811 These measurements showed that the charge transfer rate and mechanism depend strongly on the electronic structure of the PNA strands, which is defined by the PNA sequence, and is strongly influenced by PNA thermal fluctuations. The charge may either tunnel between the Au substrate and the redox probe or hop via a temporarily occupied state on the nucleobase bridge. In this context, it is particularly interesting to investigate the frontier orbital line-up at the AuPNA interface, i.e., to determine the electron and hole charge injection barriers, as well as the electronic interface dipole induced by localized charge transfer between PNA film and substrate. The influence of charge injection barriers on conductivity at electrode contacts is well-known in molecular systems. Experiments have shown that the magnitude of charge injection barriers strongly influences the conductivity values of single molecules12 and thin molecular films.13 Measured conductivities can vary by Received: February 17, 2011 Revised: July 1, 2011 Published: July 06, 2011 17123

dx.doi.org/10.1021/jp201602j | J. Phys. Chem. C 2011, 115, 17123–17135

The Journal of Physical Chemistry C Scheme 1. Diagram Representation of 7-mer PNA That Is (a) a Thymine Oligomer (Cys-T7) or (b) Abasic (Cys-Bckb7)

ARTICLE

Here, photoemission spectroscopy was applied to study SAMs of single-stranded, cysteine-modified, thymine PNA 7-mers (Cys-T7, Scheme 1) and of the abasic 7-mer PNA backbone (Cys-Bckb7, Scheme 1) formed on Au substrates. The SAMs were prepared in a N2 filled glovebox directly attached to the photoemission spectroscopy chamber. Photoemission spectra were obtained for a control sample incubated in deionized water as well. The electronic structure of both T7 and Bckb7 was theoretically calculated on the basis of the molecular ensembles generated by molecular dynamics (MD) simulations. Comparison of the photoemission spectra of Cys-T7 and Cys-Bckb7 with the theoretical spectra revealed the molecular electronic structure underlying specific spectral features. The measured and calculated DOS are in good agreement for the highest lying valence states (at low binding energy) and differ somewhat for the lower-lying states (at higher binding energy). The difference is attributed to the broadening effect from the PNAPNA and PNAwater interaction. The charge injection barriers and the interface dipole potentials between the PNA and the Au substrate were determined using the experimental data and the computed HOMOLUMO gaps. The results reveal substantial charge injection barriers of 2.81 eV for Cys-T7 and 3.23 eV for CysBckb7, relative to the Au Fermi level.

’ EXPERIMENTAL AND COMPUTATIONAL METHODS

several orders of magnitude, depending on the alignment between the energy levels of the electrode and those of the adsorbed molecules. The energy level alignment is strongly influenced by the interfacial dipole potential, which is determined by the physicochemical interactions between the adsorbed molecules and the electrode surface. The interface dipole needs to be distinguished from additional dipole potentials that can arise within molecular layers due to dipoles intrinsic to the molecules.14 The interface dipole potential and the hole injection barrier at an interface can be directly measured by photoemission spectroscopy applied in combination with density of states (DOS) calculations.1522 This technique is complementary to currentbased methods, which measure charge transport properties of molecular systems but provide only indirect access to the systems’ electronic properties. The photoemission spectroscopy technique originated in investigations of the band line-up at inorganic semiconductor interfaces.15 In recent decades, the technique was extended to molecular materials1619 and, more recently, to macromolecular interfaces composed of semiconducting polymers, nanoparticles, and biomolecules.2022 The high surface sensitivity of photoemission spectroscopy, resulting from the short escape length of photoemitted electrons (∼1050 Å23), makes the technique very suitable for investigating thin molecular films and interfaces. The films are typically produced by clean in-vacuum electrospray deposition of macromolecules from solution onto the surface of the electrode. A photoemission spectroscopy experiment compares the electronic structure of the electrode A before and after the deposition of the macromolecular material B. The differences between the electronic structure of A and A + B yields the hole injection barrier at the interface and the electronic interface dipole.

PNA Synthesis. PNA oligomers were obtained using solid phase peptide synthesis methods with a Boc protection strategy.24 MBHA resin (Peptides International, Louisville, KY) with a loading of 0.18 mequiv/g was down-loaded using Boc-L-Cys(4-MeOBzl)-OH (NovaBiochem/Merck Biosciences, Switzerland) to an estimated loading of 0.040.06 mequiv/g. 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium (HATU) was used in the coupling of cysteine to the resin. Thereafter, depending on sequence, Boc-T-OH or N-(2-Boc-aminoethyl)-N-(methyl)-glycine were coupled using O-Benzotriazole-N,N,N0 ,N0 -tetramethyl-uronium-hexafluorophosphate (HBTU),3-oxide (HCTU) (Peptides International) as a coupling agent. Oligomers were cleaved from the resin using trifluoroacetic acid (TFA) and trifluoromethanesulfonic acid (TFMSA), precipitated in cold ethyl ether, and dried under nitrogen. The solid products were dissolved in water and purified by reverse-phase HPLC using a solvent gradient, from 0 to 50% acetonitrile in water over 40 min on a Waters Delta 600 pump with a 2996 photodiode-array detector (Milford, MA). PNA oligomers were characterized by MALDI-TOF mass spectrometry on an Applied Biosystems Voyager-DE STR Workstation, in reflection mode, using an R-cyano-4-hydroxycinnamic acid matrix and a laser intensity of 1000 instrument units. The measured/calculated mass ratios of Cys-T7 and Cys-Bckb7 were 1984.01 Da/1985.34 Da and 1116.01 Da/1117.57 Da, respectively. PNA stock solutions were prepared in deionized water. The concentration of the solutions of Cys-T7 was determined by UVvis spectrophotometry assuming ε260 = 8,600 cm1M1 for T. Solutions of approximate concentrations of Cys-Bckb7 were prepared by weight. Sample Preparation. All experiments were performed in a commercial (SPECS Nano Analysis GmbH, Berlin, Germany) ultrahigh vacuum (UHV) multichamber system, which consists of a fast entry lock, two preparation chambers, and an analysis chamber equipped with X-ray and ultraviolet photoemission spectroscopy (XPS, UPS). The base pressure of this system is approximately 2  1010 mbar. A Plexiglas glovebox was 17124

dx.doi.org/10.1021/jp201602j |J. Phys. Chem. C 2011, 115, 17123–17135

The Journal of Physical Chemistry C attached to the load lock of the vacuum system, enabling a direct sample transfer from the glovebox to the vacuum. 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 during sample preparation. A diaphragm pump was used in series with filters containing active carbon and Drierite drying agent to circulate the N2 atmosphere and further reduce residual carbohydrate and H2O contamination. 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. (Ithaca, NY). The substrates were cut into 1 cm  1 cm pieces and mounted on sample holders. Electrical contact of the Au layer was ensured through direct contact with the mounting screws. After transfer into the UHV, the Au surface was sputtered with Ar+ ions in order to clean the substrate surface from ambient atmospheric contamination. The SPECS IQE 11/35 ion source produced Ar+ ions with a kinetic energy of 5 keV, an emission current of 10 mA, and an Ar pressure of ∼4  105 mbar. 20 μM Cys-T7 and Cys-Bckb7 solutions in deionized water were prepared shortly before each photoemission spectroscopy experiment. After characterization of the sputtered Au surface, the substrate was moved from the analysis chamber into the glovebox. The sample was incubated in the solution for 24 h at an approximate temperature of 37 C to accelerate SAM formation. After the sample was removed from the solution, it was rinsed with a 50% acetonitrile/50% deionized (DI) water solution, pure DI water, sodium perchlorate, and finally with DI water again. This sequence was used since preliminary experiments showed that rinsing with DI water alone resulted in a monolayer covered by excessive PNA molecules on the surface, which did not bond to the Au substrate and created charging artifacts during the UPS measurements. Using the same solvents to rinse PNA monolayers prepared for ellipsometry experiments resulted in the removal of excess molecules deposited on top of the SAMs.9 The residual solvent on the surface was removed by bringing one corner of the sample into contact with a lab tissue (Kim Wipes). The remaining solvent was then evaporated by placing the sample in the nitrogen flow inside the glovebox. After this process, the sample was moved back into the load lock and transferred into the analysis chamber. Ellipsometric Measurements of PNA Film Thickness. Samples for thickness measurements were prepared using the incubation and rinsing procedures described above. Prior to the measurements, films were dried in a stream of argon. A Gaertner L-117 Null Ellipsometer was used to measure the thickness of the PNA films. The refractive index of the SAMs was assumed to be n = 1.6, as it was reported in earlier studies on PNA assemblies.8,9 Photoemission Spectroscopy Measurements. Characterization of the sample by photoemission spectroscopy before and after deposition was performed using a SPECS UVS 10/35 ultraviolet source and a SPECS XR 50 X-ray gun. All UPS measurements were carried out with He I (hν = 21.22 eV) radiation. The Mg KR (hν = 1235.6 eV, 20 mA emission current) X-ray emission line was used for standard core level XPS and low intensity XPS (LIXPS) work function measurements. LIXPS allows direct detection of typical photoemission spectroscopy artifacts such as charging and photochemical surface modification.25,26 A 15 V sample bias was applied during UPS characterization and LIXPS work function measurements to separate the sample and analyzer spectral cutoffs and to increase the secondary electron yield. Analysis of the photoelectrons was performed

ARTICLE

with 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 2p3/2 line at 75.13 eV. The analysis of photoemission spectra was carried out using Igor Pro software (WaveMetrics, Inc.). Work function values were determined from the intersection of the fitting lines to the secondary edge with the baseline of the spectra.2729 0.1 eV was added to these cutoff values to account for the analyzer broadening.28 Inelastic background removal of UPS valence bands/ HOMO spectra was done by subtraction of the integrated spectrum adjusted for the background intensity.27 Core level emission features were fitted with GaussianLorentzian profiles to determine peak positions and full widths at half-maximum (fwhm).29 Molecular Dynamics Simulations. Molecular structure fluctuations are known to have a strong effect on the molecular charge transfer rate and mechanism.8,10,11 Therefore, MD was employed to generate structural ensembles of Cys-T7 and CysBckb7 for subsequent electronic structure analysis. The simulated system consisted of a single-stranded T7 PNA molecule (without the Cys cap) solvated in a box of water molecules. Ideally, the model system would have consisted of a PNA SAM bound to a Au surface and covered with a water layer.30,31 Such a simulation would better account for PNAPNA interactions in the monolayer and would directly generate the molecular conformations adopted by PNA in the monolayer. However, the computational cost of such a simulation would be prohibitive because of the large size of the system (∼105 atoms) and the expectedly long equilibration time for the PNA SAM structure (in the range of 102 ns or longer). Besides, the initial structure of such a model system would be unclear due to uncertainties in the PNA surface density and distribution pattern, and the force field parameters for PNAAu interaction are nonexistent. Presumably, PNA adopts a straight helical structure in the SAM, judging by the ellipsometry measurements of the SAM thickness.8 However, the helical parameters of the PNA in SAM and the amount of their fluctuations are unknown. This uncertainty was addressed by using PNA structural ensembles from two very different simulations (Table 1). Simulation A8 involved a single-stranded T7 PNA twisted into a right-handed helix with the structural parameters derived from the crystal structure by Rasmussen et al.32 Simulation B10 involved a double-stranded PNA duplex T7:A7 twisted into a left-handed helix with the structural parameters derived from the NMR structure by He et al.33 The PNA backbone in simulation A was harmonically restrained to the helical conformation because of the instability of such conformation in a simulated single-stranded PNA.8 In simulation B, the helical conformation was stable due to Watson Crick bonds between the two PNA strands. Further differences between simulations A and B included the force field, the simulated system size, and the simulation time. The parameters for both simulations are summarized in Table 1, and the computational protocol is explained in the Supporting Information. Despite the many differences between the simulations, the molecular orbitals and DOS computed for the T7 PNA structures extracted from each simulation were found to be in very good agreement. Electronic Structure Calculations. Atomic-resolution structures of the T7 PNA strand were extracted from the MD snapshots saved during simulations A and B. For each snapshot, the molecular orbitals (MOs) were computed using single-point, self-consistent field calculations with the semiempirical INDO/s 17125

dx.doi.org/10.1021/jp201602j |J. Phys. Chem. C 2011, 115, 17123–17135

The Journal of Physical Chemistry C

ARTICLE

Table 1. Parameters for MD Simulations simulated system

simulation A:8

simulation B:10

ss-T7 with constrained backbone

T7:A7 duplex

initial structure

right-handed helix built by

left-handed helix built based on

modifying the X-ray structure

the average helical parameters

of Rasmussen et al.32 (PDB code 1PUP)

of the NMR structure by He et al.33 (PDB code 2K4G)

force field

modified8,33,47,64 AMBER36

modified65 CHARMm66

water model

TIP3P

TIP3P

system size (with the solvent box)

70 Å  70 Å  70 Å,

60 Å  60 Å  60 Å, 20 157 atoms

30 519 atoms ensemble

NPT (298 K/1 atm)

time step

0.5 fs

0.5 fs

PNA H atoms free to move periodic boundary conditions

yes yes

yes yes

full electrostatics (PPPME)

yes

yes

equilibration time

1 ns

2.5 ns

production run

0.5 ns (500 snapshots saved)

2 ns (2000 snapshots saved)

method implemented in the CNDO program.34 The INDO/s method is used for the analysis of DNA and PNA electronic structure8,10,11,3537 and has been shown to produce orbital energies and couplings comparable to those found with ab initio quantum mechanical (QM) methods such as CASPT2 and CASSCF.37 CNDO calculates the MOs as linear combinations of atomic orbitals (AOs). The MO energies are computed with respect to the vacuum, and the energies of filled MOs approximate ionization energies for the molecule. The spatial electron densities of MOs are described in terms of ensemble-average Mulliken populations of the MOs on the PNA atoms. In both simulations A and B, the CNDO computations were performed for three subsystems: nucleobases only (capped with H atoms), backbone only (capped with H atoms), and complete PNA (including both bases and backbone). Including the backbone in the computations proved to have an important effect on the base-localized MOs and their energies, as discussed below. The water molecules were not included in the CNDO computations and affected the QM results only indirectly by affecting the PNA shape in the MD simulations. The MOs of the bare backbone, used to model the emission spectra of the Cys-Bckb7 system, were calculated for the structural ensemble of the PNA backbone generated in the T7 PNA simulation. It is unclear how well such a structural ensemble matches that of abasic Bckb7 in the SAM. Therefore, the present calculations are better viewed as dissecting the base and backbone contribution to the electron density of the PNA molecule, rather than faithfully reproducing the differences between CysT7 and Cys-Bckb7. For each system, the energies of all the MOs of each snapshot of the MD ensemble are pooled together and used to calculate the electronic DOS. The DOS is defined for a given energy E as the number of MOs having the energy between E  dE and E + dE, normalized by the number of snapshots. The DOS plots in this manuscript employ dE = 0.2 eV; varying dE from 0.1 to 0.5 eV did not change the DOS features substantially.

’ RESULTS Photoemission Spectroscopy. XPS measurements were performed before and after the incubation of the Au substrate

NPT (298 K/1 atm)

in deionized water or in aqueous solutions of Cys-T7 or CysBckb7. Figure 1 shows the C 1s, N 1s, O 1s, S 2p, and Au 4f core level emission lines of the sputtered substrate before (bottom spectra) and after each of the incubations (top three spectra on each plot). The spectra measured for the clean substrate show only weak O 1s and C 1s emissions at around 530.5 eV and 285.5 eV, which are attributed to residual contamination of the sample holder assembly. The Au 4f signal was attenuated after each incubation because of partial absorption of the photoelectrons emitted from the Au substrate by the molecular overlayers. None of the films showed S 2p emissions despite the presence of thiol groups in the cysteine moieties, which is attributed to the S-down orientation of the PNA molecules and the low photoionization cross section of S atoms.38 After each incubation, changes were observed in the C 1s, N 1s, and O 1s spectral emission ranges, which is indicative of changes in surface stoichiometry. The adsorbed H2O layer showed a single O 1s peak at around 532.0 eV, as well as a C 1s peak at 284.5 eV. The peaks indicate hydrocarbon contamination in the water, likely originating from the ambient atmosphere. The C 1s emissions for the Cys-T7 and Cys-Bckb7 SAMs consist of at least three distinct emission lines related to the peptide backbone and the thymine bases. Comparison of our results to earlier XPS measurements of cysteine monolayers on Au39 and uracil bases on highly oriented pyrolytic graphite (HOPG)40,41 suggest the following assignment. The high-binding energy emissions at about 288 eV are related to the CdO bonds found in the PNA backbone and thymine bases. The central component at around 286 eV corresponds mostly to the C—N bonds. The main component at about 285 eV is related to C—C bonds in the CysBckb7 and additional CdC bonds in the thymine bases of CysT7. Accordingly, the 285 eV line has a higher intensity in the CysT7 spectrum than in the Cys-Bckb7 spectrum. The O 1s spectra of both surfaces can be fit by two emission lines at approximately 532 and 533 eV to account for the large fwhm. Since both the Cys-T7 and Cys-Bckb7 molecules contain only CdO bonds, it is likely that the 533 eV component arises from the coadsorbed water. This conclusion agrees well with the location of the O 1s line measured for the sample incubated in pure H2O (Figure 1), and with values given in the literature.42 17126

dx.doi.org/10.1021/jp201602j |J. Phys. Chem. C 2011, 115, 17123–17135

The Journal of Physical Chemistry C

ARTICLE

Figure 1. C 1s, N 1s, O 1s, S 2p, and Au 4f core level XP spectra measured on sputtered Au and after incubation in deionized water, in Cys-Bckb7 solution, and in Cys-T7 solution, as indicated.

The N 1s line is also split in two components. The main component is a strong emission line at about 400 eV, which can be related to NH and NC nitrogen bonding configurations. The weak component at about 401 eV likely accounts for the amine groups located at the N-terminus of the PNA and, to a lesser degree, the cysteine group.43 Figure 2 shows the UP spectra measured in conjunction with the XPS characterization. The center graph shows the full spectra before and after each of the three incubations of the substrate. The top edges of the spectra located directly below the Au Fermi energy (the 0 eV point) correspond to the valence bands/highest occupied MOs (Figure 2, right panel). The secondary edges of the spectra, located between 16 eV and 18 eV, are defined by the work function of the sample (Figure 2, left panel). Each incubation produced intense spectral lines corresponding to the frontier orbital states of the deposited molecules, while strongly attenuating the Au emissions 5 eV below the Fermi level. The secondary cutoff shifted to higher binding energy after each of the incubations, indicating a decrease in the Au surface work function due to the interaction between the surface and the molecular films. The work functions after each incubation are listed in Table 2. Modeled Spectra. Two MD simulations were performed to obtain the structural ensembles of T7 and Bckb7 PNA, as described in the Computational Methods section. The helical conformation of the T7 PNA strand was expectedly stable during each simulation yet different between the two simulations. The ensemble-average root-mean-square deviation (rmsd) among the T7 structures was 0.87 Å for simulation A, 1.27 Å for simulation B, and 1.93 Å for cross-comparison between ensembles A and B (see the Supporting Information for more details). It was necessary to use different structural ensembles due to the uncertainty about the ss PNA conformation within the SAM. Next, QM calculations were performed in order to obtain the electronic structure of each T7/Bckb7 molecule in the MD ensembles. A typical practice in DNA/PNA electron transfer theory is to include only the nucleobases in QM computations of the electronic energies and couplings, because the frontier orbitals of nucleic acids are localized predominantly on the

nucleobases.8,4450 However, the experimental UP spectra include contributions from both the nucleobases and the backbone of the PNA molecule. Therefore, the QM computations for the T7 molecule were performed for both the nucleobase stack only and for the whole molecule, including the PNA backbone. The DOS resulting from both computations were compared to the Cys-T7 UP spectra. The QM computations were also performed for the T7 backbone with no bases; the resulting DOS was compared to the Cys-Bckb7 UP spectra. The electronic DOS computed for ensembles A and B are shown in Figure 3 and Figure S4 of the Supporting Information. Despite the significant difference in PNA helix structure between the two simulations, the computed DOS are remarkably similar. The bases-only DOS show the most similarity: both the number of DOS peaks and their energies match very closely between the two simulations. The backbone-only DOS are the most different, with the DOS for system A shifted up in energy by approximately 1 eV with respect to system B. Consequently, the complete PNA DOS in system A is shifted by approximately 1 eV upward from the complete PNA DOS in system B. Including the PNA backbone proved to have a significant effect on the bases-only MO energies, DOS, and the electron density distribution. The DOS of the complete PNA not only combines the nucleobase-localized and backbone-localized electronic states but also has the DOS peaks of the nucleobase states broadened due to the basebackbone interactions (Figure 3). The broadening results not only from an increased thermal noise due to the backbone fluctuations but mainly from the significant dipole moment of the backbone, produced by aligned peptide groups. The electric field of the backbone dipole shifts the energies of the electronic states localized on C-terminal nucleobases up and the energies of the states localized on the N-terminal nucleobases down (cf. Figure 8). On the other hand, the mixing between the bases and the backbone orbitals appears to be limited; e.g., HOMO to HOMO-2 of the complete PNA molecule are localized mainly on the thymine bases and have no presence on the backbone (Figure 4). Due to the static broadening, the complete PNA molecule has a higher average HOMO energy than the bases-only system. 17127

dx.doi.org/10.1021/jp201602j |J. Phys. Chem. C 2011, 115, 17123–17135

The Journal of Physical Chemistry C

ARTICLE

Figure 2. UP spectra measured before and after incubation in deionized water, in Cys-Bckb7 solution, and in Cys-T7 solution, as indicated. The X axis is calibrated such that 0 eV corresponds to the Au Fermi energy. Full spectra are shown in the center. The normalized secondary edge, i.e., the high binding energy spectral cutoff, and the conduction bands/HOMO emission range after removal of the inelastic background are shown on the left and right, respectively.

Table 2. Emission Lines and Work Functions (eV) for the Au Substrates Incubated with Water, Cys-T7, and Cys-Bckb7 clean substrate

water

O1s 530.6

532

N1s

C1s work function

285.4 5.4

284.6 4.63

For example, the average HOMO energy in simulation B is 8.80 ( 0.10 eV for bases only and 8.31 ( 0.16 eV for the entire PNA segment. The electron density of the HOMO shifts toward the C-terminal bases where the electrostatic potential of the backbone dipole is the highest (Figure 4), whereas the HOMO of the bases-only system is more evenly spread over the nucleobase stack (cf. Figure S3, Supporting Information). In contrast, the populations of HOMO-4, HOMO-5, and HOMO-6 shift toward the N-terminal bases (cf. Figure 4 and Figure S3, Supporting Information). A similar effect has been recently predicted for proteins where the dipole moments of alpha-helices were shown to localize hole states on the N-termini of the helices.51 It should be noted that the aforesaid shifts in the density of the PNA MOs reflect not so much changes in MO geometry as changes in the MO energy ordering. In fact, the upper MOs in the majority of PNA snapshots are predominantly localized on only one or two neighboring bases in both the nucleobases-only

Cys-T7

Cys-Bckb7

assignment

531.8

531.5

O in peptide bond

533

532.4

O in water

400

400

N in NH2 and peptide bond

400.9

400.6

N in C—N

285.1

284.9

C in C—C and CdC

286.4

286.4

C in C—H and C—N

288.4

288.3

C in CdO bonds

4.32

3.84

system and the bases-and-backbone system. However, the MO energy fluctuations (caused mainly by the fluctuations of the chemical bond lengths47) keep reshuffling the energy ordering of the localized MOs. For example, the same MO may have the highest energy and count as a HOMO in one MD snapshot but have a second highest energy and count as a HOMO-1 in another snapshot. From that perspective, ensemble-average delocalization of the HOMO in the bases-only system (Figure S3, Supporting Information) means that several base-localized MOs have an approximately equal chance to have an energy at the top of the MO energy order. In the bases-and-backbone system, the electric field of the backbone pushes the energies of the MOs localized near the C-terminus up, causing the localization of the ensemble-average HOMO population on the C-terminus (Figure 4). Certainly, there are a number of MD snapshots where MOs are truly delocalized among several nucleobases and/or the 17128

dx.doi.org/10.1021/jp201602j |J. Phys. Chem. C 2011, 115, 17123–17135

The Journal of Physical Chemistry C backbone. Overall, MO energy and geometry fluctuations in the T7 PNA are complex and their detailed analysis is beyond the scope of this paper. Due to the small mixing between the backbone and nucleobase orbitals, the DOS of the complete PNA effectively represents the superposition of the backbone and the nucleobase DOS, each broadened and/or shifted due to the electric field of the other. Thus, the frontier peak of the backbone DOS overlaps with the second peak of the nucleobase DOS, resulting in a high broad peak around 10.5 eV (Figure 3). The height of the DOS peak at 9.0 ( 0.5 eV, corresponding to the nucleobase frontier orbitals, is reduced due to the static broadening of the peak, especially in

Figure 3. DOS spectra computed for the two simulated T7 structures: (A) simulation A, (B) simulation B (see Computational Methods). Only the parts of the spectra roughly 10 eV below the top edge (cf. Figures 2 and 7) are shown. The three curves in each plot show the DOS computed for the complete PNA strand (solid black line), only the T nucleobases (solid blue line), and only the PNA backbone (dashed green line). The energy values are computed with respect to the vacuum rather than to the Au Fermi level (cf. Figure 7).

ARTICLE

simulation B (Figure 3B). It sounds paradoxical that the DOS of the whole molecule including both the bases and the backbone could be lower than the DOS of the bases only. However, the effect is clear if one considers that the top filled orbitals of the seven bases combine into a single DOS peak in the bases-only system but become spread in energy when the backbone is added to the system, lowering the DOS in the middle of the peak (cf. Figure 8). The added density of backbone states is too low at 9 eV to compensate for the lowering of the bases’ peak. Similar lowering of the DOS is seen at other energies; e.g., the DOS of the whole molecule is lower than the DOS of the backbone only at ∼9.5 eV (simulation A) and ∼13 eV (simulation B). The analysis of atomic contributions to the DOS peak at 9 eV shows that the electron density of HOMO through HOMO-6, contributing to that peak, arises predominantly from the π orbitals of the nucleobases and backbone peptide groups near the C-terminus (Figure 5a). On the bases, the

Figure 5. Ensemble-average per-atom contributions to the peaks of the computed DOS curve of the whole PNA (simulation B). The atoms are shown as spheres colored according to the atom chemical type (C, cyan; O, red; N, blue). The radius of each sphere is proportional to the square root of the ensemble-average MO population on that atom, summed for all the MOs that contributed to a given DOS peak. (a) The first (low) DOS peak at 9 ( 0.5 eV: the contributing MOs reside mostly on the nucleobases and the C-terminus of the PNA backbone. (a-1) The upper part of the first peak (8.7 ( 0.2 eV) is mostly localized on the two C-terminal T bases. (a-2) The middle of the first peak (9 ( 0.1 eV) is distributed between T2T6 and the C-terminal part of the backbone. (b) The second (high) DOS peak at 10.5 ( 0.5 eV comprises MOs from both the bases and the backbone, attributed for the most part to the π electrons of the nucleobases and the peptide groups of the backbone.

Figure 4. Ensemble-average Mulliken populations of the top MOs of a complete PNA (data from simulation B only). The chemical bonds in the T7 PNA structure are shown as blue lines, and the atoms are shown as spheres. The radius of each sphere is proportional to the square root of the average MO population on that atom. The PNA structure shown is the initial structure of the PNA in simulation B; the actual MO Mulliken populations in that structure may differ from the ensemble-average populations shown. 17129

dx.doi.org/10.1021/jp201602j |J. Phys. Chem. C 2011, 115, 17123–17135

The Journal of Physical Chemistry C

ARTICLE

’ DISCUSSION

Figure 6. GaussianLorentzian fits to C 1s, N 1s, and O 1s emission peaks measured on the Cys-T7 (left) and Cys-Bckb7 (right) films.

orbitals are localized mostly on N1, C5, and C6 atoms, with some contribution from O2 and O4. The high DOS peak at 10.5 eV consists of contributions from about 50 MOs that are also composed mainly of base and backbone π orbitals (Figure 5b). The contributing base orbitals are localized mainly on N3, O2, and O4 (Figure 5b).

A. Interface Morphology. Figure 6 shows Gaussian Lorenzian fits to the C 1s, N 1s, and O 1s emission lines measured on the Cys-T7 SAM (left) and the Cys-Bckb7 SAM (right). Table 2 summarizes the energies of these lines. The O 1s emission feature for Cys-T7 consists of two lines at 531.8 eV and 533 eV, while the two lines in the Cys-Bckb7 emission are at 531.6 eV and 532.4 eV. The more intense line is attributed to the O atoms in the peptide bonds of the PNA, whereas the weaker line is attributed to the residual water present in the PNA layer.42 The latter peak is higher in the Cys-T7 spectrum than in the Cys-Bckb7 spectrum, indicating a larger amount of water present in the Cys-T7 system. Possible explanations include lower PNA surface density in the Cys-T7 film (see below) and/or water interaction with the T bases whose polar moieties offer more hydrogen-bonding sites in addition to those found in the backbone.52 The variation in the position of the H2O-related peak (533 eV for Cys-T7 vs 532.4 eV for Cys-Bckb7) may either result from uncertainties of the fitting process or indicate differences in charge and/or polarization of the PNA-bound water molecules in the Cys-T7 and Cys-Bckb7 systems. The C 1s spectra for Cys-T7 consist of three main emission lines at 285.1 eV, 285.4 eV, and 288.4 eV. The Cys-Bckb7 emission also contains three lines located at 284.9 eV, 286.4 eV, and 288.3 eV. The lines at 285.1 eV and 284.9 eV respectively correspond to the carbon atoms in C—C and CdC. The latter bonds are present only in Cys-T7; hence, the resulting peak is higher in the Cys-T7 spectrum than in the Cys-Bckb7 spectrum. The total number of carboncarbon bonds, regardless of the bond order, is 1.95 larger in Cys-T7 than in Cys-Bckb7, which agrees well with the 2.3:1 ratio between the peak heights. The other two lines of the C 1s spectra are characteristic of carbon in C—H and C—N bonds (the 286.4 eV lines)41 and in CdO bonds (the 288.4 eV/288.3 eV lines).41,53 The N 1s spectra for Cys-T7 and Cys-Bckb7 consist of emission lines at 400.9 eV and 400.6 eV, respectively, which are attributed to the CN bonded nitrogen. Both spectra also contain a line at 400 eV, attributed to the nitrogen in the amino and amide groups of PNA.41,53 The 400.9 eV line in the Cys-T7 spectrum has a higher intensity than the 400.6 eV line in the CysBckb7 spectrum, reflecting the presence of the nucleobases. These N 1s assignments are in good agreement with earlier results by Mateo-Marti et al.54 The absence of S 2p emissions at about 162 eV39 (as seen in Figure 1) is consistent with the “standing up” orientation of most molecules in the PNA SAM. Previous studies of Cys-T7 SAMs formed on Au electrodes9 showed that the SAMs contain about 90% “standing up” molecules and 10% “lying down” molecules. Furthermore, the S 2p signal is weak even in the “lying down” molecules due to the low ionization cross section of S.38 In addition, S atoms represent less than 1% of the atoms in Cys-T7 and Cys-Bckb7 molecules, which adds to the difficulty to detect S. An estimate of the film thickness can be made from the attenuation of the Au 4f spectra (Figure 1). The peak intensities of the core level emission at 84 eV were fitted by the exponential function

I ¼ I0 expðdRÞ

ð1Þ

where I is the measured intensity of the covered substrate, I0 is the intensity of the uncovered substrate, d is the thickness of the 17130

dx.doi.org/10.1021/jp201602j |J. Phys. Chem. C 2011, 115, 17123–17135

The Journal of Physical Chemistry C

Figure 7. Cys-T7 and Cys-Bckb7 spectra after inelastic background subtraction (cf. Figure 2). DOS curves from MD simulation B are superimposed as gray filled spectra. The HOMO spectral features are in close agreement between the calculations and the measured UP spectra.

thin film, and R is the mean free path of the emitted electrons, which was estimated to be 36 Å.55 The I/I0 intensity ratios determined from the peak intensity of the core level emission at 84.1 eV were 0.45 and 0.34 for Cys-T7 and Cys-Bckb7, respectively. On the basis of these values for I/I0 and R, the average film layer thickness was determined to be 29 Å and 38 Å for Cys-T7 and Cys-Bckb7, respectively. The larger thickness result for the Cys-Bckb7 film suggests that the Cys-Bckb7 film is also denser than the Cys-T7 film. Indeed, the absence of nucleobases in CysBckb7 may allow the PNA molecules in that film to adopt a straighter conformation than the helical conformations expected for Cys-T7, thereby promoting denser packing. The larger density of the Cys-Bckb7 film also agrees with the smaller amount of water detected in that film (see above). The film thickness values above represent rough estimates only because eq 1 assumes that the film is perfectly flat and homogeneous. Pinholes and other defects typically increase the substrate signal compared to the signal of the overlayer, resulting in an underestimated thickness of the deposited film. However, ellipsometry measurements on the Cys-T7 film showed a thickness of 33 ( 6 Å which is in good agreement with the above values. Furthermore, the thickness values are consistent with the UPS data. UPS has a higher surface sensitivity (∼1020 Å) because of the lower energy of the emitted photoelectrons, which have a shorter mean free path than typical XPS electrons. The Au emission is significantly suppressed in the UP spectra measured after either incubation, indicating a SAM thickness of more than 20 Å. B. Electronic Structure. Information about the electronic structure of the Au/Cys-T7 and Au/Cys-Bckb7 interfaces can be obtained from the analysis of the UP spectra measured before

ARTICLE

and after PNA deposition (Figure 2). In principle, the hole injection barrier between the molecular overlayer and the metal substrate can be determined directly by measuring the difference between the energy of the highest energy molecular electronic states and that of the metal Fermi edge. Using HOMO cutoff energies extracted from the background-removed spectra for both interfaces (Figure 7), the hole injection barriers Φh are determined to be 2.81 eV for the Au/Cys-T7 interface and 3.23 eV for the Au/Cys-Bckb7 interface. Notably, the hole injection into the PNA HOMO may proceed not only directly but also through a “stepping stone” state at 1.5 eV, which corresponds to the antibonding orbital of the thiolAu bond.12,39 In order to compare the experimental and theoretical DOS spectra, the latter were shifted along the energy axis to achieve the best overlap (Figure 7). It is standard practice to adjust the energy axis of calculated DOS to match experimentally determined DOS, since the two use different reference points: the vacuum energy level and the Fermi edge, respectively. Furthermore, due to the inevitably occurring interface dipole between the substrate and the molecule of interest, the molecular energy reference point in the experiment depends on the particular substrate, which is not included in the calculations. A good agreement between the calculated and measured spectra is apparent for the highest-energy occupied electronic states of PNA, i.e., for the binding energy between 2 eV and 6 eV (Figure 7). The calculations allow the assignment of the spectral features to the individual MOs and parts of the PNA molecule as described above. Further down the energy axis, the number and height of the theoretical DOS peaks differ from the experimental data. The experimental spectrum for the Cys-T7 shows one high, broad peak at 8 eV and a small shoulder at around 9.5 eV, while the computed spectrum shows two low intensity peaks at approximately the same energies. The Cys-Bckb7 spectrum also shows one high, broad peak located at around 9 eV with a shoulder at 7.5 eV, while the computed spectrum shows two lower intensity peaks, one at 7.5 eV and the other at 9 eV with a shoulder at 9.5 eV. The most likely reason for these discrepancies is that water molecules were excluded from the QM computations, whereas the experimental DOS includes the electronic states of the residual water (cf. Figure 2). The presence of residual water in the PNA film should lead to the broadening of the PNA DOS peaks. Including water in the QM calculations as point charges indeed resulted in the desired amount of broadening, as calculations on the double-stranded PNA systems showed (Figure S5, Supporting Information). Furthermore, the PNA conformations in the film may show more variety than the straight-axis P-helical conformation sampled in the simulations. Therefore, more coupling interactions between MOs, including those on different PNA molecules, would cause increased orbital delocalization and a broadening of orbital energies. The contributions of each molecular orbital to the HOMO emissions are shown in Figure 8 in relation to the Cys-T7 UP spectrum and the calculated DOS. From the calculations, energies of 3.13 ( 0.16 eV (HOMO), 3.43 ( 0.13 eV (HOMO-1), 3.59 ( 0.12 eV (HOMO-2), 3.72 ( 0.11 eV (HOMO-3), 3.84 ( 0.1 eV (HOMO-4), 3.95 ( 0.11 eV (HOMO-5), and 4.08 ( 0.11 eV (HOMO-6) were determined for the different HOMOs. It can be seen that the theoretical calculation and the experimental results are in good agreement, while it is also evident that the lowest HOMO emission (3.13 ( 0.16 eV) originating from T1, 17131

dx.doi.org/10.1021/jp201602j |J. Phys. Chem. C 2011, 115, 17123–17135

The Journal of Physical Chemistry C

Figure 8. Energy distributions for individual MOs (HOMO through HOMO-6) of the complete-PNA system in simulation B. The dotted black line shows the sum of all the selected MOs. The over 1 eV spread between the centers of the MO distributions illustrates the static broadening of the DOS peak due to the basebackbone interactions.

the nucleobase located closest to the cysteine group (compare to Figure 4), defines the HOMO emission onset. The measured hole injection barriers between the Au Fermi level and molecular HOMOs are 2.81 eV for Cys-T7 and 3.23 eV for Cys-Bckb7 (see above). The corresponding electron injection barriers between the Au Fermi level and the molecular LUMO of Cys-T7 and Cys-Bckb7 can be estimated from the hole injection barriers and the theoretical values for the HOMOLUMO gap of the molecular overlayer, which constitute 6.69 eV for Cys-T7 and 8.15 eV for Cys-Bckb7. However, the latter values represent the HOMOLUMO gaps of the neutral molecules, whereas charge transfer across the interface depends on the “transfer gap” between the corresponding HOMO and LUMO polaron states, i.e., the HOMO of a positively (+1) and the LUMO of a negatively (1) charged molecule. The transfer gap can be estimated by subtracting the electron and the hole polaron energy from the theoretical HOMOLUMO gap. The hole injection barriers derived from photoemission spectroscopy measurements already include the polaron energy because the final state of photoemission spectroscopy measurements is the positive ion in its (electronically) polarized surrounding. Hence, the corresponding LUMO can be calculated directly, if the transfer gap is known. According to Conwell et al., the charge of the ribose diphosphate backbone of DNA does not affect the hole polaron binding energy because the effect of static polarization is already accounted for in the binding energy of the ground state HOMO.56 Therefore, the polarization energy of DNA can be used as an estimate for the corresponding energy for PNA, despite the electrically neutral backbone of the latter. Conwell et al. calculated the hole polaron binding energy for a “dry” double-stranded DNA to be about 0.2 eV5759 and for a dry single-stranded DNA of alternating G and T bases to be about 0.13 eV.58 The water polarization increases the hole polaron binding energy to about 0.5570.6 eV.59 Since the bases on a

ARTICLE

single strand are more exposed to water than those on a double strand, it can be assumed that the polaron binding energy of a solvated single-stranded DNA is at least similar to that for double-stranded DNA, i.e., about 0.6 eV. Our measurements indicate that Cys-T7 and Cys-Bckb7 in the films are partially solvated, and consequently, the average hole polaron binding energy should be in between the values for the dry and solvated molecules. The average of the above values, 0.37 ( 0.24 eV, may therefore be a reasonable estimate. Assuming a (0.1 eV error for the energy values extracted from the photoemission spectra (cf. Figure 2) and assuming that the electron and hole polarons have a similar binding energy as the peptide backbone and that nucleobases have a similar screening ability, the HOMOLUMO transfer gap can be estimated as 5.95 ( ((0.24)2 + (0.1)2)1/2 eV = 5.95 ( 0.26 eV (assuming independent uncertainties) for CysT7 and 7.41 ( 0.26 eV for isolated Cys-Bckb7 molecules surrounded by a thin shell of solvent. The polarons on PNA suffer an additional energy loss in close contact with the Au surface as compared to PNA in bulk solvent, because of the much larger dielectric screening of the polarons by the metal surface, compared to water. This effect is often called “polarization barrier lowering”22,60 or “final state screening”, in photoemission spectroscopy nomenclature.6163 The additional energy loss causes both the hole and the electron injection barriers to be lowered by an amount that depends on the difference between the screening capabilities of the molecule and of the substrate surface. Recent measurements by Magulick et al. on ribonucleic acid (RNA)/Au interfaces22 showed that the decrease of the polarization barrier is a substantial 1.0 ( 0.1 eV per either electron or hole injection barriers. If Cys-T7 and Cys-Bckb7 have a similar screening ability to RNA, then the estimated HOMOLUMO transfer gaps Eg in the vicinity of the Au surface would be reduced to 3.95 ( ((0.24)2 + (0.1)2 + (0.1)2)1/2 eV = 3.95 ( 0.28 eV for Cys-T7 and 5.41 ( 0.28 eV for Cys-Bckb7. Using these transfer gap values, the orbital line-up at Au/CysT7 and Au/Cys-Bckb7 interfaces was determined, including both the electron injection barriers and the interface dipole potentials (Figure 9). The electron injection barriers constitute 1.14 ( ((0.24)2 + (0.1)2 + (0.1)2)1/2 eV = 1.14 ( 0.28 eV for Au/CysT7 and 2.18 ( 0.28 eV for Au/Cys-Bckb7 (Figure 9). Thus, the injection barriers between PNA molecules and the Fermi level of the Au surface are significant for both electrons and holes, even for the hole injection proceeding through the “stepping-stone” state. It should be pointed out that the Fermi level in the molecular layers as shown in Figure 9 does not represent the bulk value. It is rather determined by the orbital line-up in combination with the Fermi level of the Au substrate due to the absence of free charge carriers in the molecular layers. In semiconductor terminology, this corresponds to a depletion layer width far exceeding the thickness of the SAM. The ionization energies Eion of 7.13 eV for Cys-T7 and 7.07 eV for Cys-Bckb7 are determined for each system by adding the HOMO binding energy and the work function resulting from the UP spectra (Figures 2,7, and 9, and Table 2). It should be noted that these Eion values are valid only in the direct vicinity of the Au surface because they are affected by the additional polarizability of the Au surface. If the above estimate for the lowering of the binding energy by the polarization of the Au surface is correct, the ionization energy of isolated molecules should be about 1 eV larger than the above values. 17132

dx.doi.org/10.1021/jp201602j |J. Phys. Chem. C 2011, 115, 17123–17135

The Journal of Physical Chemistry C

ARTICLE

axis. In the approximation of a continuous double layer of charges, D can be related to ΔU as ΔU ¼

Figure 9. Orbital line-up at the (a) Au/Cys-T7 and (b) Au/Cys-Bckb7 interfaces as determined from UPS measurements and theoretical calculations. The dotted lines indicate the uncertainty of the estimated LUMO energies.

Finally, the interface dipole potentials for both Cys-T7 and Cys-Bckb7 are determined. Using the ionization energies above and the work function of 5.4 eV measured for the sputtered Au surface prior to deposition, interface dipoles of 1.08 eV for CysT7 and 1.56 eV for Cys-Bckb7 were determined (Table 2 and Figure 9). These values are consistent with the value of 1.03 eV measured for SAMs of L-cysteine on Au surfaces.39 The dipoles mainly originate from (1) the interaction of the Au electron wave functions with the organic overlayer, (2) the formation of the chemical bond between the thiol group and the Au surface, and (3) molecule intrinsic dipole potentials.14 Phenomena 1 and 2 (the “bond dipole”14) lead to a localized net charge transfer between substrate and overlayer, which creates an electrical dipole potential at the interface and shifts the electronic states of the molecular overlayer down relative to the substrate states. In the first approximation, the bond dipole and the intrinsic molecular dipoles are additive, i.e., eD ¼ BD þ ΔU

ð2Þ

where eD is the total interface dipole potential, BD is the bond dipole potential, and ΔU is the intrinsic molecular dipole potential.14 The presented electronic structure calculations yielded the ensemble-average molecular dipole D of 19.4 ( 5.9 D for Cys-T7 and 19.1 ( 4.9 D for Cys-Bckb7, as projected on the PNA helical

D3a εr 3 ε0

ð3Þ

where a and εr are the surface coverage and the dielectric permittivity of the PNA film and ε0 is the vacuum permittivity (cf. eq 3 in ref 14). Using a surface coverage of ∼80 pmol/cm2, as measured for Cys-T7 by cyclic voltammetry,9 and εr = 24 results in ΔU = 0.91.8 eV for either Cys-T7 or Cys-Bckb7. This value is in good agreement with the measured value of eD = 1.08 eV/ 1.56 eV for Cys-T7/Cys-Bckb7. It can be concluded that the interface dipole potential of the PNA film is mostly due to the backbone dipole of PNA. It should be noted that the calculated value of ΔU can easily vary by more than 50% due to the uncertainty of the parameters. The measured surface coverage is affected by an error of (10 pmol/cm2, while the dielectric permittivity may increase several times from the assumed value of 24, depending on the amount of the residual solvent in the PNA layer. The dipole potential of a Cys molecular layer on Au was already determined to be 1.03 eV.39 Taken at face value, that should be equal to the BD term for the PNA films here. However, the BD term should be roughly proportional to the surface coverage.14 It is evident that the surface coverage of a pure Cys monolayer is likely to be several times higher than that of either Cys-Bckb7 or Cys-T7 due to the small size of Cys molecules; a 10 difference may be a realistic assumption. Therefore, the BD term should amount to only a few tenths of eV, which is significantly smaller than either the intrinsic molecular dipole or the net interface dipole of the PNA film. The difference in surface coverage is also likely to be the reason for the observed difference between the interface dipole potentials of Cys-T7 (1.08 eV) and Cys-Bckb7 (1.56 eV). Assuming that both terms contributing to eD are roughly proportional to the surface coverage (cf. ref 14 and eq 3 above), it can be concluded that the surface coverage of the Cys-Bckb7 film is 1.56/1.08 ≈ 1.44 times larger than that of the Cys-T7 film. Indeed, the missing nucleobases reduce the effective diameter of the PNA molecule by about a factor of 2 (cf. Scheme 1).

’ CONCLUSIONS Self-assembled monolayers of Cys-appended PNA 7-mers of thymine (Cys-T7) and abasic PNA backbone (Cys-Bckb7) were investigated with ultraviolet and X-ray photoemission spectroscopy. In combination with molecular-dynamics-based electronic structure calculations, these studies revealed the orbital line-up of the PNA molecules bound to the Au substrate (Figure 9). The measured density of the highest occupied electronic states closely matched the calculated density of states of both PNAs, allowing the unambiguous assignment of the emission spectral features. The calculations showed that the PNA backbone must be accounted for in order to compute the PNA density of states correctly; the customary bases-only approximation is insufficient in this case. The orbital line-up showed significant barriers for both hole and electron injection from Au to PNA, as well as large interface dipoles (1.081.56 eV). These results suggest that charge transfer between PNA SAMs and the Au substrate may be difficult due to the required amounts of energy to overcome the injection barriers. 17133

dx.doi.org/10.1021/jp201602j |J. Phys. Chem. C 2011, 115, 17123–17135

The Journal of Physical Chemistry C

’ ASSOCIATED CONTENT

bS

Supporting Information. A detailed description of methods and protocols for PNA molecular dynamics simulation and electronic structure computations used in this study, a more detailed analysis of the PNA electronic structure fluctuations than that presented in the main manuscript, more comparisons between simulations A and B, and a discussion of the solvent effect on the PNA DOS. This information is not essential for understanding the main manuscript and is intended for a reader interested in the details of the presented study. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Phone: 1-813-974-8463. Fax: 1-813974-5250.

’ ACKNOWLEDGMENT The authors gratefully acknowledge funding by the National Science Foundation (NSF DMR 0906922 to R.S. and NSF CHE 0628169 to C.A., D.H.W., and D.N.B.). H.J.H. and R.V. acknowledge partial support through the Research Experience for Undergraduates program at the USF College of Engineering. S.B. acknowledges partial support by a fellowship provided by The Colin U. Miller and Mary Hay Miller Charitable Trust for the Advancement of Education. A.B. and D.N.B. acknowledge the computational time from the Duke Shared Cluster Resource and fruitful discussions with Ravi Venkatramani and Shahar Keinan. We thank Jeffrey R. Reimers for the CNDO computer code. ’ REFERENCES (1) Lukeman, P. S.; Mittal, A. C.; Seeman, N. C. Chem. Commun. 2004, 1694. (2) Odenthal, K. J.; Gooding, J. J. Analyst 2007, 132, 603. (3) Seeman, N. C. Annu. Rev. Biochem. 2010, 79. (4) Singh, R. P.; Oh, B.-K.; Choi, J.-W. Bioelectrochemistry 2010, 79, 153. (5) Zhang, G.-J.; Chua, J. H.; Chee, R.-E.; Agarwal, A.; Wong, S. M.; Buddharaju, K. D.; Balasubramanian, N. Biosens. Bioelectron. 2008, 23, 1701. (6) Peptide Nucleic Acids; Nielsen, P. E., Ed., 2nd ed., Horizon Bioscience: Wymondham, UK, 2004. (7) Egholm, M.; Buchardt, O.; Nielsen, P. E.; Berg, R. H. J. Am. Chem. Soc. 1992, 114, 1895. (8) Paul, A.; Bezer, S.; Venkatramani, R.; Kocsis, L.; Wierzbinski, E.; Balaeff, A.; Keinan, S.; Beratan, D. N.; Achim, C.; Waldeck, D. H. J. Am. Chem. Soc. 2009, 131, 6498. (9) Paul, A.; Watson, R. M.; Lund, P.; Xing, Y. J.; Burke, K.; He, Y. F.; Borguet, E.; Achim, C.; Waldeck, D. H. J. Phys. Chem. C 2008, 112, 7233. (10) Venkatramani, R.; Davis, K. L.; Wierzbinski, E.; Bezer, S.; Balaeff, A.; Keinan, S.; Paul, A.; Kocsis, L.; Beratan, D. N.; Achim, C.; Waldeck, D. H. J. Am. Chem. Soc. 2011, 133, 62. (11) Venkatramani, R.; Keinan, S.; Balaeff, A.; Beratan, D. N. Coord. Chem. Rev. 2011, 255, 635. (12) Salomon, A.; Cahen, D.; Lindsay, S.; Tomfohr, J.; Engelkes, V. B.; Frisbie, C. D. Adv. Mater. 2003, 15, 1881. (13) Scott, J. C. J. Vacuum Sci. Technol., A 2003, 21, 521. (14) Heimel, G.; Romaner, L.; Zojer, E.; Bredas, J.-L. Acc. Chem. Res. 2008, 41, 721. (15) Waldrop, J. R.; Grant, R. W. Phys. Rev. Lett. 1979, 43, 1686. (16) Ishii, H.; Sugiyama, K.; Ito, E.; Seki, K. Adv. Mater. 1999, 11, 605.

ARTICLE

(17) Seki, K.; Ito, E.; Ishii, H. Synth. Met. 1997, 91, 137. (18) Schlaf, R.; Merritt, C. D.; Crisafulli, L. A.; Kafafi, Z. H. J. Appl. Phys. 1999, 86, 5678. (19) Schlaf, R.; Schroeder, P. G.; Nelson, M. W.; Parkinson, B. A.; Merritt, C. D.; Crisafulli, L. A.; Murata, H.; Kafafi, Z. H. Surf. Sci. 2000, 450, 142. (20) Cascio, A. J.; Lyon, J. E.; Beerbom, M. M.; Schlaf, R.; Zhu, Y.; Jenekhe, S. A. Appl. Phys. Lett. 2006, 88, No. 062104. (21) Lyon, J. E.; Rayan, M. K.; Beerbom, M. M.; Schlaf, R. J. Appl. Phys. 2008, 104, No. 073714. (22) Magulick, J.; Beerbom, M. M.; Schlaf, R. J. Appl. Phys. 2008, 104, 123701. (23) Hufner, S. Photoelectron Spectroscopy: Principles and Applications; Springer-Verlag: Berlin, Heidelberg, New York, 2003. (24) Peptide Nucleic Acids; Nielsen, P. E., Ed., 2nd ed., Horizon Bioscience: Wymondham, UK, 2004. (25) Schlaf, R.; Murata, H.; Kafafi, Z. H. J. Electron Spectrosc. Relat. Phenom. 2001, 120, 149. (26) Beerbom, M. M.; Lagel, B.; Cascio, A. J.; Doran, B. V.; Schlaf, R. J. Electron Spectrosc. Relat. Phenom. 2006, 152, 12. (27) Dam, N.; Doran, B. V.; Braunagel, J. C.; Schlaf, R. J. Phys. Chem. B 2005, 109, 748. (28) Kohlscheen, J.; Emirov, Y. N.; Beerbom, M. M.; Wolan, J. T.; Saddow, S. E.; Chung, G.; MacMillan, M. F.; Schlaf, R. J. Appl. Phys. 2003, 94, 3931. (29) Kojima, I.; Kurahashi, M. J. Electron Spectrosc. Relat. Phenom. 1987, 42, 177. (30) Kandt, C.; Ash, W. L.; Peter Tieleman, D. Methods 2007, 41, 475. (31) Scott, H. L. Curr. Opin. Struct. Biol. 2002, 12, 495. (32) Rasmussen, H.; Sandholm Kastrup, J.; Nederby Nielsen, J.; Nielsen, J. M.; Nielsen, P. E. Nat. Struct. Biol. 1997, 4, 98. (33) He, W.; Hatcher, E.; Balaeff, A.; Beratan, D. N.; Gil, R. R.; Madrid, M.; Achim, C. J. Am. Chem. Soc. 2008, 130, 13264. (34) Zeng, J.; Hush, N. S.; Reimers, J. R. J. Am. Chem. Soc. 1996, 118, 2059. (35) Keinan, S.; Venkatramani, R.; Balaeff, A.; Beratan, D. N. J. Phys. Chem. C 2010, 114, 20496. (36) Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K. M.; Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J. W.; Kollman, P. A. J. Am. Chem. Soc. 1995, 117, 5179. (37) Voityuk, A. A. Chem. Phys. Lett. 2006, 427, 177. (38) Scofield, J. H. J. Electron Spectrosc. Relat. Phenom. 1976, 8, 129. (39) Beerbom, M. M.; Gargagliano, R.; Schlaf, R. Langmuir 2005, 21, 3551. (40) Magulick, J.; Beerbom, M. M.; Schlaf, R. Thin Solid Films 2008, 516, 2396. (41) Magulick, J.; Beerbom, M. M.; Schlaf, R. J. Phys. Chem. B 2006, 110, 15973. (42) Fisher, G. B.; Gland, J. L. Surf. Sci. 1980, 94, 446. (43) Mateo-Marti, E.; Briones, C.; Pradier, C. M.; Martin-Gago, J. A. Biosens. Bioelectron. 2007, 22, 1926. (44) Grozema, F. C.; Tonzani, S.; Berlin, Y. A.; Schatz, G. C.; Siebbeles, L. D. A.; Ratner, M. A. J. Am. Chem. Soc. 2008, 130, 5157. (45) Rosch, N.; Voityuk, A. A. Quantum chemical calculation of donor-acceptor coupling for chare transfer in DNA. In Long-Range Charge Transfer in DNA II; Schuster, G. B., Ed.; Springer-Verlag: Berlin, Heidelberg, New York, 2004; p 37. (46) Sadowska-Aleksiejew, A.; Rak, J.; Voityuk, A. A. Chem. Phys. Lett. 2006, 429, 546. (47) Hatcher, E.; Balaeff, A.; Keinan, S.; Venkatramani, R.; Beratan, D. N. J. Am. Chem. Soc. 2008, 130, 11752. (48) Voityuk, A. A. Chem. Phys. Lett. 2007, 439, 162. (49) Tong, G. S. M.; Kurnikov, I. V.; Beratan, D. N. J. Phys. Chem. B 2002, 106, 2381. (50) Senthilkumar, K.; Grozema, F. C.; Guerra, C. F.; Bickelhaupt, F. M.; Lewis, F. D.; Berlin, Y. A.; Ratner, M. A.; Siebbeles, L. D. A. J. Am. Chem. Soc. 2005, 127, 14894. 17134

dx.doi.org/10.1021/jp201602j |J. Phys. Chem. C 2011, 115, 17123–17135

The Journal of Physical Chemistry C

ARTICLE

(51) Chen, X.; Zhang, L.; Zhang, L.; Sun, W.; Zhang, Z.; Liu, H.; Bu, Y.; Cukier, R. I. J. Phys. Chem. Lett. 2010, 1637. (52) Chandra, A. K.; Nguyen, M. T.; Zeegers-Huyskens, T. J. Phys. Chem. A 1998, 102, 6010. (53) Magulick, J.; Beerbom, M. M.; Lagel, B.; Schlaf, R. J. Phys. Chem. B 2006, 110, 2692. (54) Mateo-Marti, E.; Briones, C.; Roman, E.; Briand, E.; Pradier, C. M.; Martin-Gago, J. A. Langmuir 2005, 21, 9510. (55) Laibinis, P. E.; Bain, C. D.; Whitesides, G. M. J. Phys. Chem. 1991, 95, 7017. (56) Conwell, E. M.; Bloch, S. M.; McLaughlin, P. M.; Basko, D. M. J. Am. Chem. Soc. 2007, 129, 9175. (57) Conwell, E. M.; Bloch, S. M. J. Phys. Chem. B 2006, 110, 5801. (58) Conwell, E. M.; Rakhmanova, S. V. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 4556. (59) Kurnikov, I. V.; Tong, G. S. M.; Madrid, M.; Beratan, D. N. J. Phys. Chem. B 2002, 106, 7. (60) Amy, F.; Chan, C.; Kahn, A. Org. Electron. 2005, 6, 85. (61) Horn, K.; Frank, K. H.; Wilder, J. A.; Reihl, B. Phys. Rev. Lett. 1986, 57, 1064. (62) Jacobi, K. Phys. Rev. 1988, B38, 5869. (63) Schlaf, R.; Parkinson, B. A.; Lee, P. A.; Nebesny, K. W.; Armstrong, N. R. Surf. Sci. 1999, 420, L122. (64) Shields, G. C.; Laughton, C. A.; Orozco, M. J. Am. Chem. Soc. 1998, 120, 5895. (65) Sen, S.; Nilsson, L. J. Am. Chem. Soc. 2001, 123, 7414. (66) Foloppe, N.; MacKerell, A. D. J. Comput. Chem. 2000, 21, 86.

17135

dx.doi.org/10.1021/jp201602j |J. Phys. Chem. C 2011, 115, 17123–17135