Optical Characterisation of DNA Bases. on Silicon Surfaces

Transcription

1 Optical Characterisation of DNA Bases on Silicon Surfaces von der Fakultät für Naturwissenschaften der Technischen Universität Chemnitz genehmigte Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) vorgelegt von M. Sc. Phys. Simona Dorina Silaghi geboren am 14 Februar 1977 in Gherla eingereicht am 02 März 2005 Gutachter: Prof. Dr. Dietrich R.T. Zahn Prof. Dr. Michael Hietschold Dr. Uwe Rossow Tag der Verteidigung: 17 Juni 2005

2 Bibliografische Beschreibung 2 Bibliografische Beschreibung M. Sc. Phys. Simona Dorina Silaghi Optical Characterisation of DNA Bases on Silicon Surfaces Technische Universität Chemnitz Dissertation (in englischer Sprache), 2005 Im Rahmen dieser Arbeit werden DNA-Basen-Moleküle (Thymin, Cytosin, Adenin und Guanin) auf H-passivierten Si(111)-Substraten mittels theoretischer Berechnungen und optischen Spektroskopien charakterisiert. Für ein einzelnes DNA-Basen-Molekül wurden quantenchemische Berechnungen von Elektronenübergängen und vibronischen Moden durchgeführt. Zusätzlich wurden die vibronischen Eigenschaften von Metall(Ag,In)/Cytosin- Komplexen sowie die Adsorption von einzelnen Cytosin-Molekülen auf H:Si(111)- Oberflächen studiert. Die biomolekularen Schichten von DNA-Basen wurden durch organische Molekularstrahldeposition (OMBD) im Ultrahochvakuum auf flachen und vicinalen H:Si(111)- Oberflächen hergestellt. Die Morphologie, Struktur und Kristallinität von DNA-Basen- Schichten wurden mittels Rasterkraftmikroskopie (AFM), Röntgenbeugung (XRD) und Röntgenreflektometrie (XRR) charakterisiert. Die Vibrationseigenschaften von biomolekularen Schichten wurden experimentell durch Infrarotspektrokopie untersucht. Metall(Ag,In)/Cytosin/H:Si(111)-Heterostrukturen wurden mittels oberflächenverstärkter Ramanstreuung (SERS) charakterisiert. In dieser Arbeit wurden erstmals die optischen Konstanten und die dielektrischen Funktionen von dicken DNA-Basen-Schichten auf ebenen H:Si(111)-Oberflächen mittels spektroskopischer Ellipsometrie (SE) bestimmt. Ebenfalls zum ersten Mal wurden dünne biomolekulare Schichten auf vicinalen H:Si(111)-Oberflächen durch Reflektionsanisotropiespektroskopie (RAS) charakterisiert. Schlagwörter Biomoleküle, flaches und vicinales Si(111), Organische Molekularstrahldeposition, Ag, In, Grenzfläche, Rasterkraftmikroskopie (AFM), Röntgenbeugung (XRD), Röntgenreflektometrie (XRR), Infrarotspektrokopie, oberflächenverstärkte Ramanstreuung (SERS), spektroskopische Ellipsometrie (SE), Reflektionsanisotropiespektroskopie (RAS).

3 Table of Contents 3 Table of Contents Bibliografische Beschreibung...2 Table of Contents...3 List of Abbreviations Introduction ab initio Calculations of DNA Base Molecules Density Functional Theory Computation objectives Geometry optimisation Electronic transitions Vibrational properties Calculated optical properties of single DNA bases Thymine Geometry optimisation Molecular orbitals. Electronic transitions Vibrational properties Cytosine Geometry optimisation Molecular orbitals. Electronic transitions Vibrational properties Cytosine/metal complexes Cytosine/Silicon Adenine Geometry optimisation Molecular orbitals. Electronic transitions Vibrational properties Guanine Geometry optimisation Molecular orbitals. Electronic transitions Vibrational properties Summary Structural Properties of DNA Base Films Sample preparation Silicon substrates. Low Energy Electron Diffraction Biomolecular and metal films Morphology and Structure Atomic force microscopy (AFM) characterisation of DNA base films X-ray diffraction (XRD) characterisation of DNA base films X-ray reflectivity (XRR) of guanine films Vibrational Spectroscopy: Infrared and Raman Spectroscopies Theoretical background Infrared absorption Raman scattering Surface-enhanced Raman scattering Electromagnetic enhancement Charge transfer enhancement Molecular vibrations... 50

4 Table of Contents Experimental details FTIR experimental set-up Raman experimental set-up Infrared results Thymine Cytosine Adenine Guanine Surface-enhanced Raman spectroscopy results Interaction of cytosine with metals SER spectra of cytosine films on flat H:Si(111) surfaces SER spectra of cytosine films on vicinal H:Si(111)-6 surfaces Summary Spectroscopic Ellipsometry Theoretical background Light polarisation using different formalisms The Jones formalism. Jones vectors and matrices The formalism of Stokes vectors and Mueller matrices Rotating analyser ellipsometer in Mueller matrix formalism The effective dielectric function. Kramers-Kronig consistency Dielectric function of silicon Evaluation of the experimental SE data Cauchy model Surface roughness model Oscillator model Experimental details ex situ Variable Angle Spectroscopic Ellipsometry (VASE) in situ Vacuum-Ultraviolet Spectroscopic Ellipsometry (VUV-SE) Optical constants of DNA base films Thymine ψ and spectra Optical constants of thymine Cytosine ψ and spectra Optical constants of cytosine Adenine ψ and spectra Optical constants of adenine Guanine ψ and spectra Optical constants of guanine Summary Reflectance Difference / Anisotropy Spectroscopy Theoretical Background Description of the RAS spectrometer using the Jones formalism r The RAS signal r Surface optical anisotropy RAS signal of silicon surfaces Experimental details RAS monitoring of DNA Base films on vicinal H:Si(111) Thymine Cytosine Adenine Guanine Summary

5 Table of Contents 5 7 Concluding Remarks References List of Tables List of Figures Erklärung Curriculum Vitae List of Publications Acknowledgements...129

6 List of Abbreviations 6 List of Abbreviations ν δ β ρ ω AFM asym BESSY CASSCF CT DFT DNA DTGS EM GGA FTIR HOMO ip IR KBr LEED LUMO MCT MSA MIR-SE MSE OMBD oop NIM NIR-Vis-UV-SE PEM RAS RCA RDS SDA SE SERS SIOA sqz TD-DFT TMP UHV VASE VUV-SE XRD XRR Stretching vibration Deformation vibration Scissoring vibration Rocking vibration Wagging vibration Atomic Force Microscopy Asymmetric stretching Berliner Elektronenspeicherring Gesellschft für Synchrotronstrahlung m.b.h. Complete Active Space Self-Consistent Field Charge Transfer Density Functional Theory Deoxyribonucleic Acid Deuterated Triglycine Sulphate Electromagnetic Enhancement Generalized Gradient Approximation Fourier Transform Infrared Highest Occupied Molecular Orbital in-(molecular) plane Infrared Potassium Bromide Low Energy Electron Diffraction Lowest Unoccupied Molecular Orbital Mercury Cadmium Telluride Multi-Sample Analysis Middle Infrared Spectroscopic Ellipsometry Mean Square Error Organic Molecular Beam Deposition out-of-(molecular) plane Normal Incidence Monochromator Near Infrared-Visible-Ultraviolet Spectroscopic Ellipsometry Photoelastic Modulator Reflectance Anisotropy Spectroscopy Radio Corporation America Reflectance Difference Surface Dielectric Anisotropy Spectroscopic Ellipsometry Surface-Enhanced Raman Spectroscopy Surface Induced Optical Anisotropy Squeezing vibration Time-Dependent Density Functional Theory Trimethyl Poshpate Ultra-High Vacuum Variable Angle Spectroscopic Ellipsometry Vacuum Ultraviolet Spectroscopic Ellipsometry X-Ray Diffraction X-Ray Reflectivity

7 Chapter 1. Introduction 7 Chapter 1 1 Introduction In the last years, the long-range charge transport in DNA was investigated for applications in nano-electronic technologies [Fin01]. Electrical transport measurements on micrometer-long DNA "ropes" [Fin99] and on DNA molecular films [Oka98] indicated that DNA has a metallic conductivity. On the other hand, the electrical transport measurements performed on poly (guanine)-poly (cytosine), a double-stranded DNA polymer, showed semiconducting behaviour with a large band gap [Por00]. Charge migration through DNA takes place via the overlap of the π orbitals in adjacent base pairs in a single strand. It was shown that irregular base-pair sequences lead to localisation of charge carriers and reduce the transfer rate of electrons. Therefore, DNA base molecules, adenine, thymine, guanine, and cytosine may be involved as charge transport molecules in biomolecular electronic devices. Their electronic properties are similar to those of inorganic wide band gap materials, e.g. GaN, with the absorption onset in the near ultra-violet (UV) region as it will be revealed in this work. The temperature dependence of the electrical admittance and permittivity of thin cytosine layers showed that these films exhibit electrical ordering in the temperature range between 220 and 260 K [Sot80]. A recent field effect transistor study based on a modified DNA base revealed that the prototype bio-transistor gives rise to a better voltage gain compared to carbon nanotubes (CNTs) [Mau03]. However, the ability to fabricate structures with characteristic dimensions of a few nanometers is a key prerequisite for future applications in nanoelectronics or as functional materials on the nanometer scale. With the reduced size of future molecular devices the effects of dimensionality are becoming more and more important. Because silicon can be patterned in many ways, it is possible to use it as a versatile template for combining organic molecules and biomolecules with silicon electronics. It is known that the flat Si(111) has a 3- fold symmetry inducing the growth of three equivalent superstructure domains by symmetry. The vicinal Si(111) surfaces can limit overlayer growth to a single domain. Moreover, vicinal surfaces should favour the nucleation along the step edges, thus being potential substrates in controlling the ordering of molecules in so-called molecular nano-wires. In view of the above mentioned applications, the aim of this work is to optically characterize DNA base molecules on both flat and vicinal hydrogen passivated Si(111) surfaces.

8 Chapter 1. Introduction 8 This work is structured as follows. Chapter 2 contains the ab initio calculations performed within density functional methods (DFT) on single DNA base molecules. The calculated optimized geometries, electronic transitions as well as the vibrational properties are discussed. Chapter 3 presents the substrate preparation via wet-chemically processes. The surface reconstruction of both flat and vicinal H:Si(111) is characterized by low energy electron diffraction (LEED). The growth parameters of the DNA bases and metal (Ag, In) films are introduced. The structure and morphology of the biomolecular films is further investigated by means of atomic force microscopy (AFM), X-ray diffraction (XRD), and X-ray reflectivity (XRR) methods. Chapter 4 introduces the experimental vibrational properties of DNA base films on flat H:Si(111) and of metal (Ag, In)/cytosine/H:Si(111) heterostructures characterized by means of infrared (IR) and surface-enhanced Raman (SER) spectroscopies. For the first time, the optical constants and dielectric functions of the DNA base films are determined by employing spectroscopic ellipsometry from infrared to ultraviolet as it is described in chapter 5. The molecular ordering of thin DNA base films on various vicinal H:Si(111) surfaces is uniquely probed by means of reflectance anisotropy spectroscopy in chapter 6. In the end, chapter 7 provides the concluding remarks of this work.

9 Chapter 2. ab initio Calculations of DNA Base Molecules 9 Chapter 2 2 ab initio Calculations of DNA Base Molecules 2.1 Density Functional Theory Beyond Hartree-Fock approximation, the great advantage of density functional theory stems from the inference of correlation effects. More exactly, the density functional approach is based on a strategy of modeling the electron correlation via general functionals of the electron density. Following the work by Kohn and Sham, the approximate functionals employed by current DFT methods separate the electronic energy into several terms: E = ET + EV + EJ + EX C (2.1) where E T is the kinetic energy term, E V includes terms describing the potential energy of the nuclear-electron attraction and of the repulsion between pairs of nuclei, E J is the electronelectron repulsion term, and E XC is the exchange-correlation term and includes the remaining part of the electron-electron interactions. The energy sum ET + EV + E J corresponds to the classical energy of the charge distribution ρ. The exchange-correlation term EXC accounts for the exchange energy arising from the antisymmetry of the quantum wavefunctions and for the dynamic correlation in the motions of individual electrons. Hohenberg and Kohn [Hoh64] demonstrated that E XC is entirely determined by the electron density: r r r r r E ( ρ) = f ρ ( ), ρ ( ), ρ ( ), ρ ( ) d XC ( ) 3 α β α β where ρ, ρ are referring to the corresponding α, β spin densities. α β E XC is usually divided into components, referred to as the exchange and correlation parts, but actually corresponding to the same-spin and mixed-spin interactions, respectively: ( ρ ) ( ρ) ( ρ) XC X C (2.2) E = E + E (2.3) Pure DFT methods are defined by pairing an exchange functional with a correlation functional. For example, the well-known BLYP functional pairs Becke s gradient-corrected exchange functional with the gradient-corrected correlation functional of Lee, Yang and Parr [Lee88]. Recently, Becke has formulated functionals which include a mixture of Hartree-Fock and DFT exchange along with DFT correlation, conceptually defining E XC as: HF HF DFT DFT E = const. E + const. E (2.4) XC X XC

10 Chapter 2. ab initio Calculations of DNA Base Molecules Computation objectives ab initio calculations performed in this work were carried out based on density functional theory methods using the B3LYP functional implemented in the software package Gaussian Geometry optimisation Optimized geometry computations of single DNA bases were performed by employing DFT/B3LYP method in combination with the basis set G(d, p). A different basis set, namely LANL2DZ, was employed for the optimization of the metal-molecule complexes. This takes into account possible relativistic effects of the metal atoms [Hay85a, b; Wad85]. On the other hand, the adsorption of a DNA base at the step edge of a slab H:Si(111) was approached with the basis set 3-21G basis set Electronic transitions Time-dependent density-functional theory TD-DFT was used to study the electronic spectra i.e. excitation energies, oscillator strengths, and transition moment directions of single DNA base molecules. This method provides a formally rigorous extension of Hohenberg Kohn Sham density-functional theory, which is time-independent, to the situation where a system, initially in its ground stationary state, is subject to a timedependent perturbation modifying its external potential. An evaluation of the performance of TD-DFT for the calculation of high-lying bound electronic excitation energies of molecules is in detail described by Casida et al. [Cas98]. TD-DFT results are directly compared with DFT/GGA calculations performed by M. Preuss [Pre04] Vibrational properties Based on the fully relaxed optimized geometries of the single DNA bases as well as of metal/dna base complexes, the calculations of vibrational frequencies were performed in order to understand the IR and surface enhanced Raman (SER) spectroscopy results.

11 Chapter 2. ab initio Calculations of DNA Base Molecules Calculated optical properties of single DNA bases Thymine Geometry optimisation Thymine is one of the four essential components of DNA molecule. The pyrimidine molecule containing 15 atoms (C 5 H 6 N 2 O 2 ) binds to adenine molecule via hydrogen bridges forming one of the strands in the double helix DNA. The optimized geometry using the DFT method B3LYP/ G(d,p) is shown in figure 2.1 with an arbitrarily chosen rectangular coordinate system. The corresponding atomic coordinates of the optimized geometry are included in table 2.1. The molecule has an average deviation from the plane of about Å due to the hydrogen pyramidalisation of the C 9 atom. The nine atoms except the hydrogen atoms in the thymine molecule are coplanar. The presented atomic coordinates are in a very good agreement with those reported in [Tsu97] using ab initio SCF MO calculations where the ring atoms are also coplanar. Furthermore, the average deviation from the plane of Å is due to the CH 3 group. The X-ray diffraction (XRD) study [Oze69] on thymine anhydrate crystals revealed that the ring atoms of a thymine molecule are nearly coplanar with an average deviation from the plane of about Å. Figure 2.1 Molecular structure of thymine projected in the (x, y) plane with the z axis perpendicular to the molecular plane. Table 2.1 Atomic positions of thymine. The calculated intramolecular bond distances and selected bond angles of thymine molecule are indicated in figure 2.2. The theoretical values are compared to the experimental values determined from XRD measurements [Oze69]. The length of the hydrogen bonds lies between and Å. The average deviation in bond lengths between the theoretical and experimental values is of about Å while for bond angles it is approximately 1.3. The crystal structure of thymine is shown in figure 2.2. The unit-cell containing four molecules is monoclinic with the space group P2 1 /c. The molecules are nearly planar within

12 Chapter 2. ab initio Calculations of DNA Base Molecules 12 the (001) plane with the long crystal axis-a parallel to the long molecular axis which connects the C 2 O 8 C 5 C 9 bonds. The tilt angle of molecular plane was estimated to be about 5 with respect to the (001) plane. The planes of neighboring molecules in a layer are inclined at an angle of 6.60 with each other. The average interplanar spacing between adjacent layers in the (010) plane is about 3.36 Å. Pairs of molecules i.e. dimers are connected by the N-H O- C hydrogen bonds and they form infinite chains along the b axis. Figure 2.2 Molecular arrangement of thymine molecules in the unit cell taken from [Oze69]. The crystal structure is monoclinic with the space group P2 1 /c. The unit-cell dimensions were reported as follows: a=12.87 Å, b=6.83 Å, c=6.70 Å, β=105 and Z=4 where Z is the number of molecules. The calculated internal coordinates using B3LYP/ G(d,p) together with those determined from XRD are indicated on the molecular structure shown in the plane (001). The projection upon the plane (100) of the crystal structure shows four molecules in the unit cell. The plane of the molecule is nearly parallel to (001) plane. Additionally, Ozeki et al. concluded that the thymine anhydrate crystals grow as plates with (100) and (001) planes as prominent faces [Oze69] Molecular orbitals. Electronic transitions Figure 2.3 shows the HOMO (highest occupied molecular orbital) and LUMO (lowest occupied molecular orbital) of the thymine molecule. The side views of both molecular orbitals reveal their π and π* characters. Selected electronic transitions between occupied and unoccupied molecular orbitals are shown in figure 2.4. These are calculated with respect to the vacuum level (E vacuum =0).

13 Chapter 2. ab initio Calculations of DNA Base Molecules 13 LUMO HOMO Figure 2.3 HOMO and LUMO of thymine reproduced in both top and side views. Figure 2.4 Selected energy levels (with respect to the vacuum level, E=0) of thymine indicating with arrows the main electronic transitions obtained with TD-DFT/B3LYP. The first one hundred electronic transitions were computed using the TD-DFT/B3LYP method in combination with the basis set G(d,p). From these only the strongest five are taken into account for further discussions. They are indicated by arrows in figure 2.4 while an overview on the attempted assignment of electronic transitions of thymine is given in table 2.2. The calculated electronic transitions obtained from TD-DFT/B3LYP calculations are compared with respect to those obtained using the CASSCF method [Lor95] and the DFT/GGA method [Pre04]. The energy positions are given together with the corresponding oscillator strengths and the direction of the transition dipole moments. The polarization direction of the transition dipole moment in respect to the reference molecular axis of thymine using the DeVoe-Tinoco convention [DeV62] is schematically shown in figure 2.5 (b). In addition, the theoretical electronic spectra are compared to the experimental absorption spectra reported so far in literature together with the extinction coefficient determined for a thymine film from the ellipsometry results introduced in chapter 5. For a better assessment, figure 2.5 depicts the experimental absorption spectra of thymine together with the electronic spectra as achieved from both TD-DFT/B3LYP and DFT/GGA methods. The electronic transitions obtained using TD-DFT/B3LYP are in relatively close agreement with those obtained by employing CASSCF method while DFT/GGA yields much smaller energy transition values. Since the calculations are performed for a single molecule, i.e. practically gas-phase, without taking into account any surrounding medium or intermolecular

14 Chapter 2. ab initio Calculations of DNA Base Molecules 14 interactions, the calculated electronic transitions are typically higher compared to the experimental results. Electronic Transitions of Thymine E (f/k; θ) I II III IV V Reference Theory DFT/B3LYP 4.99 (0.13; +5 ) 5.99 (0.07; +13) 6.25(0.13; -38) 7.46 (0.35;+19) 7.72 (0.1; +3) this work CASSCF 4.9 (0.17; +15) 5.9 (0.17; -19) 6.1 (0.15; +67) 7.1 (0.85; -35) [Lor95] DFT/GGA 3.76 (0.21;) 4.73 (0.04;) 5.0 (0.15;) 5.98 (0.28;) 6.58 (0.07;) [Pre04] Experiment Absorption gas phase >6.7 [Cla65a] in TMP [Cla65a,b] in H 2 O [Cla65a]; [Voe63] film/lif [Yam68] Polarised Reflection single crystal 4.5 (; -12 / +70) 5.8 ( ; -31 / +91) [Nov86]; [Ane75] Linear Dichroism in PVA 4.6( ; -31 / +51) [Mat82] Circular Dichroism in H 2 O [Voe68] Magnetic Circular Dichroism in H 2 O [Voe68] Spectroscopic Ellipsometry film/h:si(111) 4.44, this work Table 2.2 Summary of the theoretical and experimental electronic transitions ( E/eV) of thymine. The corresponding oscillator strengths (f) or the extinction coefficients (k) and the angular directions (θ/ ) of the electronic transitions are given in the brackets. Thus, the first electronic transition at 4.99 ev (TD-DFT/B3LYP), 4.9 ev (CASSCF), and 3.76eV (DFT/GGA) is assigned to the HOMO LUMO transition as π π* type. The corresponding transition moment angle defined with respect to the line connecting the CH3 group and C 2 O 8 bond (see figure2.5 (b)) takes the following values of +5 and +15 as predicted from TD-DFT/B3LYP and CASSCF, respectively. The experimental absorption bands were found at 4.8 ev in gas phase [Cla65a, b], 4.7 ev in various solutions, ev in films, and 4.5 ev for a single crystal. In the case of the extinction coefficient spectrum determined for a thymine film from ellipsometry, the energy positions corresponding to Gaussian oscillators are considered. The red-shift of the absorption from gas phase to single crystal is proportional with the increase of the intermolecular interaction by decreasing the intermolecular distances. The asymmetry of the first band in the extinction coefficient spectrum shown in figure 2.5 (a) appears as a result of the contribution of two oscillator components at 4.44, and 4.64 ev. Single crystal absorption measurements generate different

15 Chapter 2. ab initio Calculations of DNA Base Molecules 15 energy positions for the first absorption band when the incident electric field is polarized along different crystallographic axis. For instance, the absorption along a axis leads to a band centered at 4.5 ev while the absorption measured along b axis gives rise to another band at 4.38 ev. With the help of these polarization-dependent absorption investigations performed on a single crystal one can conclude that the doublet components observed in the case of thymine film are an evidence of the crystalline nature of the film. Moreover, the doublet absorption band observed in the thymine film seems to be mainly polarized in the ab crystallographic plane which corresponds to (001) plane as denoted in figure 2.2. Figure 2.5 (a) Experimental extinction coefficient of the thymine film as determined from the ellipsometry results reported in chapter 5 in comparison to the circular dichroism spectrum of thymine in aqueous solution [Voe68]. (b) The theoretical absorption spectrum of thymine obtained from TD-DFT/B3LYP G(d,p) calculations. The red arrow overlapped on the thymine structure stands for the transition dipole moment polarised under θ angles as given in table 2.2. (c) The theoretical absorption spectrum of thymine obtained from DFT/GGA calculations. The oscillator strengths are described by Gaussian functions with a broadening of 0.2eV. The precise positions of the theoretical electronic transitions are indicated by blue columns. The second calculated electronic transition assigned to HOMO-2 LUMO is located at ~5.99 ev (TD-DFT/B3LYP), 5.9 ev (CASSCF), and 4.73 ev (DFT/GGA) corresponding to the

16 Chapter 2. ab initio Calculations of DNA Base Molecules 16 experimental absorption bands lying in the energy range from 5.7 to 6.1 ev depending on the surrounding environment of thymine molecules. This transition is of π π* type. The next electronic transition at 6.25 ev (TD-DFT/B3LYP), 6.10 ev (CASSCF), and 5.00 ev (DFT/GGA) corresponds to an energy transfer from HOMO to LUMO+2, where LUMO+2 has a π* character. Only the experimental results for thymine in the gas phase showed a distinct absorption band at 6.2 ev [Cla65a]. The fourth calculated electronic transition is found to have the largest oscillator strengths as determined from all three calculations. Its energy position lies at 7.46 ev / 0.35 (TD-DFT/B3LYP), 7.10 ev / 0.85 (CASSCF), and 5.98 ev / 0.28 (DFT/GGA). From TD-DFT/B3LYP computations this excited state is assigned to a π π* type transition between HOMO-2 and LUMO+2 energy levels. The transition dipole moment of this excited state is polarized under +19 from TD-DFT/B3LYP and -35 from CASSCF. The experimental results reveal an absorption band in the energy range between 6.6 and 7 ev (see table 2.2). Finally, the last assigned electronic transition at 7.72 ev with oscillator strength of about 0.1 from TD-DFT/B3LYP and 6.58 ev with an oscillator strength of about 0.07 obtained from DFT/GGA arises mainly due to an energy transfer from HOMO- (σ) to LUMO+5 (σ*). The experimental results showed no further absorption bands above 7 ev. Neglecting the energy shifts, one can notice that the experimental absorption spectrum of thymine in aqueous solution seems to be very similar in lineshape to the simulated electronic spectrum predicted from TD-DFT/B3LYP computations. As can be seen in figure 2.5 (b) the electronic spectrum involves strong in-plane (xy) absorption and weak absorption in the out-of-plane (z) direction. On the other hand, poor agreement in the relative intensities of the bands is found when one compares these calculations with the extinction coefficient of the thymine film. Nevertheless, from DFT/GGA computations neither the overall simulated spectrum nor the different contributions of the extinction coefficient along x, y, or z directions do not describe better the experimental observations Vibrational properties Thymine gives rise to 39 vibrational modes. The molecule belongs to the C s symmetry group with the lowest symmetry. Therefore, both IR and Raman vibrational modes are expected to be active. Starting from the ground state an optimized geometry of the thymine molecule and the vibrational frequencies are calculated using B3LYP functional with the basis set G(d,p). The calculated values are shown below in table 2.3 in comparison to the calculations reported by Santamaria [San99] and the experimental frequencies reported in [Szc00]. Only the assignment of the experimental IR vibrational bands observed below 2000 cm -1 is presented. The experimental spectra of thymine films on silicon surfaces are discussed in the next chapter.

17 Chapter 2. ab initio Calculations of DNA Base Molecules 17 Table 2.3 Vibrational assignment of thymine. [Szc00] [San99] this work a exp. IR BP/ 6-311G b exp. IR pellet c exp. IR film B3LYP/ G(d,p) inplane (ip)/ out-ofplane (oop) Assignment ip ν C2-O8, δ N3-H11,N1-H ~ ip ν C6-O7,C4-C5, δ N1-H10,C9-H / ~ / ip ν C4-C5,δ C4-H12,N3-H11, Fermi resonance doublet ip δ N3-H11, β C9H14H15, ν C4-N3,C6-N ip δ N3-H11, asymν C4-N3, β C9H14H ip ρ CH ip um CH ip asymν N3-C2-C1, ν C5-C6, δ N3-H11, N1-H10, um CH ip δ N1-H10,N3-H ip δ C4-H12, β C9H14H15, ν C4-C ip ν C5-C9, N3-C4, δ C4-H12, δ C9-H ip δ C4-H12, N3-H11,N1-H10,ν N1-C6, N3-C ip ν N1-C6, N3-C4, ρ CH oop γ C4-H12, ρ CH oop ρ CH3, ω C4-H12,N3-H ip def ring, δ N1-H10,C4-H12, ρ CH oop δ C4-H12,, ω CH3 815/ / / ip def ring oop γ C6-O7, ρ CH3, γ ring oop γ C2-O8,N1-H ip ring breathing oop γ N1-H10,N3-H11, ω CH ip ω ring oop γ N1-H10,N3-H ip def ring ip sqz ring Cytosine Geometry optimisation Cytosine is the smallest molecule among the DNA bases. The pyrimidine molecule contains 13 atoms (C 4 H 5 N 3 O) and binds to guanine molecule via hydrogen bridges forming the second strand poly (C-G) in the double helix of the DNA molecule. The optimized geometry predicted by the B3LYP functional in conjunction with the basis set G(d,p) is shown in figure 2.6 with an arbitrarily chosen rectangular coordinate system. The corresponding atomic coordinates of the optimized geometry are included in table 2.4. The molecule is almost planar with a deviation from the planarity of about Å. Surprisingly, the predicted deviation agrees very well with the experimental XRD results obtained by Barker and Marsh, namely the maximum deviation of the ring atoms from the plane is about Å [Bar64].

18 Chapter 2. ab initio Calculations of DNA Base Molecules 18 The calculated intramolecular bond distances and selected bond angles of cytosine molecules are indicated in figure 2.7. The theoretical values are compared to the experimental ones determined from XRD measurements [Bar64]. The average deviation in bond lengths between the theoretical and experimental values is about 0.01 Å while for bond angles it is approximately 0.1. The crystal structure of the cytosine is shown in figure 2.7. The anhydrate crystal is orthorhombic with the space group P The unit cell with the following dimensions a=13.04, b=9.49, and c=3.81 Å contains 4 molecules. The molecules are tilted about 27.5 with respect to the ab plane. The interplanar distance between successive cytosine molecules along c axis is about 3.36 Å. The adjacent molecules form a dihedral angle of about 15. Two features of the hydrogen-bond network are noteworthy: the arrangement of N-H O bonds form spirals up the twofold screw axes parallel to c axis, and the arrangement of N-H O and N-H N bonds form ribbons of molecules along the b axis. Figure 2.6 Molecular structure of cytosine projected in the (x, y) plane with the z axis perpendicular to the molecular plane. (a) (b) Table 2.4 Atomic positions of cytosine. Figure 2.7 (a) Comparison of the calculated internal coordinates of a cytosine molecule using B3LYP/ G(d,p) with the experimental values determined from XRD investigations [Bar64]. (b) Molecular arrangement of cytosine molecules in the unit cell taken from [Bar64]. The crystal structure is orthorhombic with the space group P The unit-cell parameters were reported as follows: a=13.04, b=9.49, c=3.81 Å and Z=4. The projection upon the plane (001) of the crystal structure shows four molecules in the unit cell. The plane of the molecule is tilted by about 27.5 with respect to (001) plane.

19 Chapter 2. ab initio Calculations of DNA Base Molecules Molecular orbitals. Electronic transitions The main molecular orbitals of the cytosine molecule i.e. HOMO and LUMO are depicted in figure 2.8. Also in this case the HOMO has a π character while the LUMO shows a π* character. LUMO HOMO Figure 2.8 HOMO and LUMO of cytosine reproduced in both top and side views. Figure 2.9 Selected energy levels (with respect to the vacuum level, E=0) of cytosine indicating with arrows the main electronic transitions using TD-DFT/B3LYP. Selected energy levels of occupied and unoccupied states calculated with respect to the vacuum level are presented in figure 2.9. The strongest five electronic transitions chosen from the one hundred excited states computed by TD-DFT/B3LYP G(d,p) are indicated with arrows in figure 2.9. Both theoretical and experimental results on the electronic transitions are summarized in table 2.5. As for thymine, the calculated electronic transitions of cytosine obtained using TD-DFT/B3LYP are compared with respect to those obtained using the CASSCF and DFT/GGA methods. The energy positions are given together with the corresponding oscillator strengths and the polarization direction of the transition dipole moments. Figure 2.10 displays the simulation of theoretical electronic transitions of thymine obtained from both type of calculations namely TD-DFT/B3LYP and DFT/GGA together with the experimental absorption spectra. The calculated electronic transitions of a single cytosine molecule using TD-DFT/B3LYP are closer with respect to the experimental results than those obtained by the CASSCF method.

20 Chapter 2. ab initio Calculations of DNA Base Molecules 20 Electronic Transitions of Cytosine E (f/k; θ) I II III IV V Reference Theory DFT/B3LYP 4.64 (0.04; +91 ) 5.43 (0.09; -46 ) 6.20 (0.05;+19 ) 6.60 (0.42;-24 ) 7.87(0.1;-85 ) this work CASSCF 4.81(0.06; +86) 6.68 (0.11; +6) 6.98 (0.03; -27) 7.92 (0.85; -35) [Roo87] DFT/GGA 3.64(0.08;) 4.22 (0.33;) 4.96 (0.36;) 5.47 (0.17;) 6.49(0.27;) [Pre04] Experiment Absorption in TMP [Cla65b] in H 2 O [Voe63] film/lif [Yam68] film [Rak78] Polarised Absorption of Cytosine Monohydrate 4.4 (; +14/48) 5.5 (; -5) [Lew71] single crystal 4.7 (; -11) [Cal70] 4.6 (0.14; +6/54) 5.3 (0.03; ) 5.8 (0.13;+76/-17) 6.3(0.36;+86/-27) [Zal85] Linear Dichroism in PVA 4.6 (; +9) 5.2 (; +7.5/10.5) [Fuc71] in PVA 4.6( ; -46 / +25) 5.2 (; -27/ +6) [Mat82] Circular Dichroism in H 2 O [Spr77] Magnetic Circular Dichroism in H 2 O [Voe68] Spectroscopic Ellipsometry film/h:si(111) this work Table 2.5 Summary of the theoretical and experimental electronic transitions ( E/eV) of cytosine. The corresponding oscillator strengths (f) or the extinction coefficients (k) and the angular directions (θ/ ) of the electronic transitions are given in the brackets. The first calculated electronic transition at 4.64 ev is assigned to the HOMO LUMO transition as π π* type from TD-DFT/B3LYP calculations. The other two methods suggest a HOMO LUMO transition positioned at 3.64 ev (DFT/GGA) and 4.81 ev (CASSCF), respectively. The transition dipole moment of the HOMO LUMO transition is predicted to be oriented under an angle of +91 (TD-DFT/B3LYP) and +86 (CASSCF) with respect to the reference molecular axis of cytosine (see figure 2.10(b)). The experimental investigations performed on cytosine in various environments revealed a first absorption band in the energy range between 4.4 and 4.7 ev. For the extinction coefficient spectrum determined for cytosine from ellipsometry, the energy positions corresponding to Gaussian oscillators are considered. Again, the red-shift of the absorption from gas phase to single crystal is related with the change in the intermolecular interaction. The ellipsometry results give rise to the first Gaussian oscillator positioned at 4.46 ev relatively close to the theoretical position of 4.64 ev