ADVANCED VIBRATIONAL SPECTROSCOPIC STUDIES OF BIOLOGICAL MOLECULES

Transcription

1 ADVANCED VIBRATIONAL SPECTROSCOPIC STUDIES OF BIOLOGICAL MOLECULES A thesis submitted to the University of Manchester for the degree of Doctor in Philosophy in the Faculty of Life Sciences 2012 Saeideh Ostovar pour

2 Table of Contents List of Contents... 2 List of tables... 6 List of figures... 7 Abstract Declaration Copyright statement Abbreviations List Glossary Acknowledgements Chapter Introduction Conformational analysis of biological molecules Infrared and Raman spectroscopies Principles of Raman spectroscopy Resonance Raman spectroscopy Raman spectroscopy of biomolecules Raman Optical Activity (ROA) ROA spectroscopy of biomolecules Surface-Enhanced Raman Scattering The Electromagnetic Mechanism The Chemical Mechanism (CE) Biological applications of SERS Surface enhanced Raman optical activity (SEROA) References Chapter 2 Use of a Hydrogel Polymer for Reproducible Surface Enhanced Raman Optical Activity (SEROA) 2.0 Declaration

3 Table of Contents 2.1 Abstract Introduction Experimental Results and Discussion Conclusion References Supplementary Information Colloid Preparation Sample Preparation for Raman and ROA Measurements Atomic Force Microscopy SERS Time Dependence Chapter 3 Induced Chirality to Non-chiral Surfaces of Silver Silica Nanotags 3.0 Declaration Abstract Introduction Experimental Results and Discussion Conclusion References Supplementary Information Chapter 4 Phosphorylation Detection and Characterization in Ribonucleotides Using Raman and Raman Optical Activity (ROA) Spectroscopies 4.0 Declaration Abstract Introduction Experimental Results and Discussion Conclusion

4 Table of Contents 4.6 Acknowledgement References Supplementary Information Colloid Preparation Surface enhanced Raman spectroscopy (SERS) References Chapter 5 Study of Experimental and Computational Raman and Raman Optical Activity (ROA) Spectra of Cyclic and Linear L-Ala-L-Ala in Solution 5.0 Declaration Abstract Introduction Experimental Computational methods Results and Discussion Conclusion References Supplementary Information Chapter 6 Combined Experimental and Computational Study of Raman and Raman Optical Activity (ROA) Spectra of Linear and Cyclic L-Ser-L-Ser in Solution 6.0 Declaration Abstract Introduction Experimental Computational methods Results and Discussion Conclusion References Supplementary Information

5 Table of Contents Chapter Conclusion Future work References Chapter 8 Appendix 8.0 Declaration ,600 words 5

6 List of Figures List of Tables Table 1.1 Advantages and disadvantages of Raman spectroscopy use Table 1.2 Important spectral regions of protein vibrations Table S2.1 Raman and ROA band assignments of L- and D-ribose in aqueous solution Table 4.1 Raman band assignments of adenosine, AMP, ADP, ATP, A(2)MP, A(2,3)MP, A(3)MP and A(3,5)MP Table 4.2 ROA band assignments of adenosine, AMP, ADP, ATP, A(3,5)MP, A(2,3)MP, A(2)MP and A(3)MP Table S4.1 SERS band assignments of adenosine, AMP, ADP, ATP, A(2)MP, A(2,3)MP, A(3)MP and A(3,5)MP Table 5.1 Calculated and experimental wavenumber band assignments for Raman and ROA of cyclic and linear L-Ala-L-Ala in H 2 O Table 5.2 Calculated and experimental wavenumber band assignments for Raman and ROA of cyclic and linear L-Ala-L-Ala in D 2 O Table S5.1 Calculated and experimental bond lengths (Å) for cyclic and linear L-Ala-L-Ala Table S5.2 Calculated and experimental bond angles ( o ) for cyclic and linear L-Ala-L-Ala Table S5.3 Calculated and experimental torsion angles ( o ) for cyclic and linear L-Ala-L-Ala Table 6.1 Calculated and experimental Raman and ROA bands for cyclic and linear L-Ser-L-Ser in H 2 O Table 6.2 Calculated and experimental Raman and ROA bands for cyclic and linear L-Ser-L-Ser in D 2 O Table S6.1 Calculated and experimental bond lengths (Å) for cyclic and linear L-Ser-L-Ser Table S6.2 Calculated bond angles ( o ) for cyclic and linear L-Ser-L-Ser Table S6.3 Calculated torsion angles ( o ) for cyclic and linear L-Ser-L-Ser

7 List of Figures List of Figures Figure 1.1 An energy level diagram showing the transitions involved in Raman scattering Figure 1.2 Schematic diagram of the basic ROA experiment which measures a small difference in the intensity of Raman scattering in right (R) and left (L) circularly polarized light from chiral molecules Figure 2.1 Raman (I R + I L, top) and SCP ROA (I R I L, bottom) spectra of D- and L-ribose in aqueous. Sample concentrations were 2.66 M at ph 5.46 (D-) and 5.60 (L-ribose), data collection time was min, and laser power at the sample W for each Figure 2.2 Raman spectrum of polycarbopol in solution (A), SERS spectra before (B) and after addition of L- and D-ribose (0.25 mg ml -1 ) in the presence of silver citrate reduced colloid and K 2 SO 4 at M concentration, data collection time: 20 min (C), SERS spectra of L- and D-ribose (0.25 mg ml -1 ) in the presence of polycarbopol polymer, data collection time: 20 min (D), ROA spectrum of polycarbopol polymer in solution, sample concentration 40 mg ml - 1, data collection time: 218 min (E), SEROA spectra of silver Figure S2.1 Figure S2.2 Figure S2.3 Figure S2.4 citrate reduced colloids in presence of aggregating salt before (F) and after addition of L- and D-ribose, data collection time: 35 min (G), SEROA spectra of L- and D-ribose with addition of polycarbopol, datacollection time of 35 min (H) A schematic diagram of SEROA sample preparation with use of the polycarbopol polymer Dual views of AFM images of silver citrate reduced colloids only (A), polycarbopol polymer only (B) and a mixture of silver citrate reduced colloids and polycarbopol polymer (C) Time dependence SERS study for D-ribose molecule with and without addition of polymer to SERS solution Example repeats of SEROA spectra for D- (top) and L-ribose (bottom) with the same conditions for each spectrum, where concentration of each sample was 0.25 mg/ml and K 2 SO 4 7

8 List of Figures concentration was M, with addition of 20 mg/ml of polycarbopol, data collection time of 35 mins Figure S2.5 Raman (I R + I L, A and B) and SCP ROA (I R I L, E and F) spectra of D- and L-tryptophan in aqueous. Sample concentrations were 50 mg/ml at ph 1.90 (D-) and 1.58 (L-tryptophan), data collection time was 4-8hrs, and laser power at the sample W for each. SERS spectra after addition of D- and L-tryptophan (C and D), ( M) in the presence of silver hydroxyalamine reduced colloid and MgSO 4 at M concentration, data collection time: 5 min, SEROA spectra of D- and L-tryptophan (G and H) with addition of polycarbopol, data collection time of 35 min Figure 3.1 SERRS spectra of nanotag (tri-functional benzotriazole dye) without silica coated silver colloids (A), with silica coated silver colloids (B) and SERROA spectra of A (C) and B (D), data collection time of 35 min and laser power at source 0.20 W Figure 3.2 SERRS spectra of D- and L-ribose that attached to silver silica nanotag (A), SERROA of D- and L-ribose replicates 1 (B) and batch 2 (C), data collection time of 35 min and laser power at source 0.20 W Figure 3.3 SERRS spectra of D- and L-tryptophan that attached to silver silica nanotag (A), SERROA spectra of D- and L-tryptophan (B), data collection time of 35 min and laser power at source 0.20 W Figure S3.1 Structure of tri-functional benzotriazole dye Figure 4.1 Raman spectra of adenosine (ph= 12.95), AMP (ph= 6.02), ADP (ph= 5.18), ATP (ph= 4.20), A(2)MP (ph= 3.13), A(2,3)MP (ph=5.54), A(3)MP (ph= 8.10) and A(3,5)MP (ph=6.67) in solution. The concentration for each sample was 100 mg/ml and laser power was 0.6 W at the sample Figure 4.2 ROA spectra of adenosine (ph= 12.95), AMP (ph= 6.02), ADP (ph= 5.18), ATP (ph= 4.20), A(2)MP (ph= 3.13), A(2,3)MP (ph=5.54), A(3)MP (ph= 8.10) and A(3,5)MP (ph=6.67) in solution. The concentration for each sample was 100 mg/ml and laser power at the sample was 0.6 W

9 List of Figures Figure S4.1 Figure 5.1 Figure 5.2 Figure 5.3 Figure 5.4 Figure 6.1 Figure 6.2 Figure 6.3 SERS spectra of adenosine ribonucleotides and adenosine in the presence of silver citrate reduced colloid. Analyte concentrations were 1x10-5 mg/ml, K 2 SO 4 concentration was M, data collection time was 50 seconds, and laser power was 0.20 W at the laser The chemical structure with atom numbering scheme (left) and calculate minimum energy conformation (right) of linear L-Ala-L- Ala The chemical structure with atom numbering scheme (left) and calculate minimum energy conformation (right) of cylic L-Ala-L- Ala Experimental and computed Raman (top) and ROA (bottom) spectra of linear (ph= 7.0) and cyclic L-Ala-L-Ala (ph= 7.0) in aqueous solution. The concentration for each sample was 50 mg/ml and laser power was 0.6 W at the sample. The marker bands that are induced upon cyclization are highlighted by shading Experimental and computed Raman (top) and ROA (bottom) spectra of linear (ph= 7.0) and cyclic L-Ala-L-Ala (ph= 7.0) in D 2 O. The concentration for each sample was 50 mg/ml and laser power was 0.6 W at the sample. The marker bands that are induced upon cyclization are highlighted by shading The chemical structure with atom numbering scheme (left) and calculate minimum energy conformation (right) of linear L-Ser-L- Ser The chemical structure with atom numbering scheme (left) and calculate minimum energy conformation (right) of cyclic L-Ser-L- Ser Experimental and computed Raman (top) and ROA (bottom) spectra of linear (ph= 7.0) and cyclic L-Ser-L-Ser (ph= 7.0) in aqueous solution. The concentration for each sample was 50 mg/ml and laser power was 1.2 W at the laser. The marker bands that are induced upon cyclization are highlighted by shading

10 List of Figures Figure 6.4 Experimental and computed Raman (top) and ROA (bottom) spectra of linear (ph= 7.0) and cyclic L-Ser-L-Ser (ph= 7.0) in D 2 O. The concentration for each sample was 50 mg/ml and the laser power was 0.6 W at the sample. The marker bands that are induced upon cyclization are highlighted by shading

11 Abstract Abstract Raman optical activity (ROA) is a powerful probe of the structure and behaviour of biomolecules in aqueous solution for a number of important problems in molecular biology. Although ROA is a very sensitive technique for studying biological samples, it is a very weak effect and the conditions of high concentration and long data collection time required limit its application for a wide range of biological samples. These limitations could possibly be overcome using the principle of surface enhanced Raman scattering (SERS). The combination of ROA with SERS in the form of surface enhanced ROA (SEROA) could be a solution for widening the application of ROA. In the last few years, the generation of reliable SEROA spectra of biomolecules has been problematic due to non-homogenous colloidal systems forming and low signal-to-noise ratios which complicated detection of the true SEROA signal from the analyte. L- and D-enantiomers give full or partially mirror image chiroptical spectra, this property of enantiomers can be employed to prove the chiroptical activity of the SEROA technique. In this thesis we employed a hydrophilic polycarbopol polymer as stabilising media which has led to the first report of mirror image SEROA bands for enantiomeric structures. This new technique of incorporating the hydrogel polymer as a means to stabilise the colloidal system has proven to be reliable in obtaining high quality SEROA spectra of D- and L-enantiomers of ribose and tryptophan. In an extension of the hydrogel-stabilised SEROA work, we also demonstrate that single nanoparticle plasmonic substrate such as silver silica nanotags can enhance the weak ROA effect. These dye tagged silica coated silver nanoparticles have enabled a chiral response to be transmitted from a chiral analyte to the plasmon resonance of an achiral metallic nanostructure. The measurement of mirror image SERROA bands for the two enantiomers of each of ribose and tryptophan was confirmed for this system. The generation of SEROA for both systems was achieved and confirmed SEROA as a new sensitive tool for analysis of biomolecular structure. In a related project, Raman and ROA spectra were measured for adenosine and seven of its derivative ribonucleotides. Both of these spectroscopic techniques are shown to be sensitive to the site and degree of phosphorylation, with a considerable number of marker bands being identified for these ribonucleotides. Moreover, the SERS studies of these ribonucleotides were also performed. The obtained SERS spectra were shown similar features that confirm these analytes interact with the surface in a similar manner, hence limiting the structural sensitivity of this method towards phosphate position. Short dipeptides such as diketopiperazine (DKP) have been investigated during the last decades as both natural and synthetic DKPs have a wide variety of biological activities. Raman and ROA spectra of linear and cyclic dialanine and diserine were measured to charecterize their solution structures. Density functional theory (DFT) calculations were carried out by a collaborator to assist in making vibrational band assignments. Considerable differences were observed between the ROA bands for the cyclic and linear forms of both dialanine and diserine that reflect large differences in the vibrational modes of the polypeptide backbone upon cyclicization. In this study, the ROA spectra of cyclic dialanine and diserine have been reported for the first time which demonstrated that ROA spectroscopy when utilised in combination with computational modelling clearly provides a potential tool for characterization of cyclic peptides. 11

12 Declaration Declaration No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning. 12

13 Copy right Statement Copyright Statement 1. The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the Copyright ) and he has given The University of Manchester certain rights to use such Copyright, including for administrative purposes. 2. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made. 3. The ownership of certain Copyright, patents, designs, trade marks and other intellectual property (the Intellectual Property ) and any reproductions of copyright works in the thesis, for example graphs and tables ( Reproductions ), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions. 4. Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property and/or Reproductions described in it may take place is available in the University IP Policy (see policies/intellectualproperty.pdf), in any relevant Thesis restriction declarations deposited in the University Library, The University Library s regulations (see and in The University s policy on presentation of These. 13

14 Abbreviations List Abbreviations List AMP ADP A(2)MP A(2,3)MP A(3)MP A(3,5)MP ATP CID CP Cyclic L-Ala-L-Ala Cyclic L-Ser-L-Ser DCP DNA EM IR Linear L-Ala-L-Ala Linear L-Ser-L-Ser NMR SCP SEROA SERROA SERRS SERS Adenosine 5 -monophosphate Adenosine 5 -diphosphate Adenosine 2 -monophosphate Adenosine cyclic 2,3 - monophosphate Adenosine 3 -monophosphate Adenosine 3,5 -cyclic monophosphate Adenosine 5 -triphosphate Circularly intensity difference Circular polarization Cyclic L-Alanine-L-Alanine Cyclic L-Serine-L-Serine Dual circularly polarised Deoxyribonucleic acid Electromagnetic Infrared Linear form of L-Alanine-L-Alanine Linear form of L-Serine-L-Serine Nuclear magnetic resonance Scattered circular polarisation Surface-enhanced Raman optical activity Surface enhanced resonance Raman optical activity Surface enhanced resonance Raman scattering Surface-enhanced Raman scattering 14

15 Glossary Glossary Anti-stokes scattering Chirality Colloids D-configuration Elastic scattering Enantiomers Inelastic scattering L-configuration Nanoparticles Nucleoside Nucleotides Purine Pyrimidine Raman scattering Rayleigh scattering Raman scattering event where the scattered photon has more energy than incident radiation Property of molecules with a handedness to their chemical Structure (i.e. molecules which cannot super impose on its mirror image) and possessing optical activity Type of chemical mixture when one substance is dispersed evenly throughout another Dextrorotatory- configuration rotating the plane of vibration of polarized light to the right Specific form of scattering where the energy of incident photon is equal to the energy of scattered photon. Molecules that are optical isomers, or mirror images, of one another. Enantiomers can be distinguished by the direction in which they rotate the plane of polarization of polarized light Raman scattering where the energy of scattered photon is not equal to Incident photon Laevorotatory-Rotating the plane of vibration of polarized light to the left Particles whose diameter is between 1 and 100 nm are glycosylamines consisting of a nucleobase (often referred to simply base) bound to a ribose or deoxyribose sugar Nucleic acids base, sugar and phosphate Heterocyclic aromatic organic compound which is consisting of a pyrimidine ring fused to an imidazole ring Nitrogenous organic base, containing two nitrogen atoms at position 1 and 3 of the six-member ring The phenomenon change in wavelength of a photon as result of inelastic scattering of light by molecule Occurs as result of elastic collision between the photons and molecules in the samples (no energy change in energy level of the analyte) 15

16 Glossary ROA Single plasmonic nanoparticles substrate Stokes scattering Surface plasmons Raman optical activity one form of vibrational spectroscopies which is reliant on the difference in intensity of Raman scattered right and left circularly polarized light due to molecular Chirality Metallic nanoparticles which have localized surface plasmon resonances that match the excitation wavelengths of lasers used in Raman scattering event where those photon scattered with less energy than the incident radiation The oscillations that occur as result of the interaction of light beam with conduction electrons held in a lattice by the presence of positive charge from the metal centre 16

17 Acknowledgements Acknowledgements I would like to thank to my supervisor Dr Ewan Blanch for his endless enthusiasm for making this project feasible. It was a great experience working with him. To all group mates, Clare, Myra, Ben, Christian, Grant, Nicola, Lorna, Heather, Kieaibi and my friends Zahra and Soumya for making my last three years here a wonderful experience, to Steve Prince, David Ellis and Elon Correa for giving me advice on different subjects. I am truly fortunate to know you all. To my parents, Jafar, Suosan and my grandmother for their endless support, encouragement and love that made me believe in myself and pursue my dreams. Without you all this would not be achievable. My final and greatest appreciation is for my sister Soheyla, without her nothing was possible. 17

18 Chapter 1 Chapter 1 Introduction 18

19 Chapter Introduction 1.1 Conformational analysis of biological molecules The characterisation of the three-dimensional (3D) structures of biological molecules and their relationship to their functions has made a tremendous impact on all subsequent biochemical investigations [1,2]. X-ray protein crystallography is currently the primary methodology used for determining the 3D structure of biological molecules at near-atomic or atomic resolution, the other notable atomic resolution technique being nuclear magnetic resonance (NMR) [1-4]. However, typically around 20 40% of all protein molecules, including many important proteins, are difficult or impossible to crystallize, and hence their structures have not been accessible by crystallography [1,2]. NMR has limitations on the size and type of molecules that can be structurally characterized [3,4]. Overcoming these limitations has therefore necessitated the development of novel approaches for structural biology. One of these novel approaches is the employment of vibrational spectroscopy which is concerned with the interaction of electromagnetic radiation with matter. Vibrational spectroscopic techniques, such as infrared (IR) [5-7] and Raman [8-10] spectroscopies have been widely applied in different fields of science such as biochemistry and biomolecular structure analysis. Infrared spectroscopy measures the light intensity of light absorbed in the infrared region of the electromagnetic spectrum. The electric dipole of the molecule must change during a molecular vibration in order for the molecule to absorb infrared radiation [5]. In the mid- and far-infrared spectral regions, the absorption occurs where the frequencies of light and molecular vibrations are equal which causes promotion of the molecule to a vibrationally excited state [5]. Raman spectroscopy is also based on vibrational 19

20 Chapter 1 transitions that give rise to narrow spectral features characteristic of the investigated sample [11] and measures the intensity of light that is inelastically scattered from the molecule. Raman and infrared spectroscopies possess several advantages in contrast to other analytical techniques since they are non-invasive, the requirement for sample mass/volume is minimal and more importantly, there is no requirement for chemical labelling/probes. Protein aggregation, stability, conformational changes induced by different factors, the accurate prediction of structure and folding can all be assessed by vibrational spectroscopy [5-11]. 1.2 Infrared and Raman spectroscopies Light is a form of electromagnetic (EM) radiation composed of electric and magnetic waves that are oriented perpendicular to each other when they oscillate in single planes. The interaction of electric waves with matter can either lead to the absorbance or scattering of the incident light. If the energy of the photon matches that of the energy difference between the ground and excited states of a molecule, the photon may be absorbed and the molecule is promoted to a higher energy excited state. The energy change resulting from this phenomenon is measured by absorbance spectroscopy through the detection of the energy lost from the initial radiation. Infrared spectroscopy is a technique derived from the vibrations of atoms of a molecule. An infrared spectrum is commonly obtained by passing infrared radiation through a sample and determining what fraction of the incident radiation is absorbed at a particular energy. The energy at which any peak in an absorbance spectrum appears corresponds to the frequency of a vibration of the sample molecule. This 20

21 Chapter 1 interaction of infrared radiation with matter can be explained by changes in the molecular dipole associated with vibrations and rotations [5-7,11]. Light scattering occurs when there is no necessity for a photon to possess an energy matching the energy difference between the ground and excited vibrational levels. This phenomenon of change in wavelength as a result of inelastic scattering of light by matter was first observed by the Indian scientist C.V. Raman in 1928 [12]. When the incident photon collides with a molecule, the electron distribution is perturbed which results in the Raman effect. The majority of photons are scattered elastically which is known as Rayleigh scattering, with no change in wavelength of the scattered photon [11]. However, a transfer of energy can occur either from the incident photon to the molecule or from the molecule to the incident photon, if nuclear motion is induced. This results in inelastic scattering of the photon for which the energy of the incident photon is different to that of the scattered photon, termed Raman scattering [13]. In the energy level diagram shown in Figure 1.1, at room temperature most molecules are present in the lowest energy vibrational level [11], and with a monochromatic light beam of photon energy hv and wavelength λ the Raman scattering process from the ground vibrational state m leads to the absorption of energy to a higher excited vibrational state n. This difference in energy between the incident and scattered photons is represented by Stokes and anti-stokes lines. The scattered photon for Stokes lines has a lower energy than the incident photon, and for anti-stokes lines the incident photon has less energy than the scattered photon [11]. Variation in energy from the excitation state is correlated to the vibrational energy spacing in the ground state of the molecule, hence, the vibrational energy of the molecule can be a probe of molecular chemistry of the sample via quantification of 21

22 Chapter 1 the frequencies of Stokes and anti-stokes lines [14]. Vibrational motion is sensitive to chemical modification and therefore the molecular chemistry of samples can be studied. In Raman spectroscopy typically the Stokes scattering is used for analysis of molecular structure since the anti-stokes scattering is weak. Virtual State Stokes Rayleigh Anti Stokes Vibrational Levels n m Ground Electronic State Figure 1.1: An energy level diagram showing the transitions involved in Rayleigh and Raman scattering, adapted from [11]. 22

23 Chapter Principles of Raman spectroscopy: As an oscillating beam of light interacts with a molecule, the electron cloud of the molecule is perturbed periodically with the same frequency as the electric field of the incident wave. The perturbation of the electron cloud results in a periodic separation of charge within the molecules that is termed an induced dipole moment. The oscillating induced dipole moment is a source of EM radiation, thereby resulting in scattered light [15]. As discussed above, the electric field associated with the laser radiation induces a dipole moment in the molecule which is proportional to the electrical field strength E and to the molecular polarizability α (the ability of the electron charge distribution to be distorted by an electric field) that depends on the molecular structure and the nature of the molecular bonds. The strength of the induced dipole moment μ is given by [15], (1) Because of the vector nature of the dipole moment and electric field, α is not a simple constant, but can be written as a tensor by taking account of the contributions with respect to the three Cartesian axes x, y and z. All three components of E contribute to each of the three components of μ shown in tensor form, as they refer to the Cartesian axis directions [11]. (2) 23

24 Chapter 1 In the case of aqueous solution samples, there is no ordering of the axes of the molecule to the polarization direction of the light but it is possible to measure these from polarization measurements. In practical situations, the ratio of depolarization is determined where the intensity of a given band is measured with respect to the plane of polarization of the incident light being parallel or perpendicular to the scattered light. The average polarizability can also be described in terms of isotropic (with the analyzer parallel to the plane of the incident radiation) and anisotropic (with the analyzer perpendicular to the plane) components of the tensors as represented in equations (3) and (4), where and represent isotropic and anisotropic terms, respectively [11,14], (3) (4) Resonance Raman spectroscopy Historically, coloured compounds were avoided by most Raman spectroscopists. This was mainly due to decomposition of the sample by the powerful lasers used that prevents Raman analysis as a result of strong fluorescence [16,17]. However, if the frequency of the laser beam is close to the frequency of an electronic transition, scattering enhancement of up to 10 6 can be observed [11,16-17]. This results in a more sensitive technique in contrast to conventional Raman spectroscopy since a chromophore provides more efficient scattering that is selective for the molecule involving the chromophore [16,17]. The resonance scattering can provide both electronic and vibrational information concerning the molecule of interest. 24

25 Chapter 1 Resonance Raman scattering can occur when an incident laser beam has an excitation frequency close to that of an electric transition. A tuneable laser beam can be used for excitation, and the frequency would correspond to the energy difference between the ground vibrational state and the first or second vibronic state of the excited state. The resonance condition is met when the energy difference between the lowest vibrational state of the ground electronic state and the resonant vibronic state is of the same energy as the excitation resulting from the incident light. The obtained enhancement of Raman scattering is mainly due to an increase in polarizability [16,17]. Elucidation of structural information from deep within complex biological samples was enabled through development of this technique [18-20] Raman spectroscopy of biomolecules The molecular information provided by Raman spectroscopy is the same as that from infrared spectroscopy. However, the Raman effect has advantages over IR absorption for aqueous environments since less interference occurs from the solvent [5-10]. This advantage is beneficial for studying biological samples in solution since water in most cases is a pre-requisite for functioning in the surrounding physiological environment. Raman spectroscopy has a number of advantages and disadvantages compared to other analytical techniques for studying biological samples, which are summarised in Table 1.1. Raman spectroscopy still remains a practical method for probing the interplay between structure, dynamics and function of biomolecules [21-25]. Understanding the precise structure of biomolecules in terms of their vibrational spectra can have a large impact on discovery of exact physiological function in living systems. The 25

26 Chapter 1 vibrational modes of biomolecules that can be studied by vibrational spectroscopies such as Raman are characteristic of their molecular structure. However, due to the large number of vibrational modes in biomolecules, it is a complex task to elucidate detailed information based on the measurements of vibrational spectra. Even so, important information on secondary structure elements may frequently be derived [21-25]. Table 1.1: Advantages and disadvantages of Raman spectroscopy [5-11].u Column2 Advantages Distinct characteristic vibrations that can be used as finger prints for qualitative/ quantitative identification Lack of interference with other vibrational bands which results in narrower absorption bands from the laser beam No or minimal sample preparation required Disadvantages Weak effect Interference from fluorescence Decomposition of coloured samples as a result of heating Minimal volume Minimum absorption by water molecules Non-invasive Can be used for a wide range of conditions e.g. aqueous, gas, solid, tissue Table 1.2, displays the most notable spectral regions from protein vibrations, which are called amide I, II and III [26-28]. The spectral information obtained in these regions is a sensitive indicator of the presence of secondary structure within biomolecules such as proteins and peptides where they have been used to estimate the amount of α-helix and β-sheet content [26-28]. 26

27 Chapter 1 Table 1.2: Important spectral regions for protein vibrations [26-28] Band Wavenumber (cm -1 ) Vibrational Assignment Amide I C=O Stretching Amide II N-H bend/ C-N stretching Amide III N-H bend/ C-N stretching/ C α -H deformation Bands in the vicinity of , 1300 and 1340 cm -1 in the amide I and III regions indicate α-helical conformations whereas bands in the vicinity of 1670, 1700 and cm -1 usually indicate β-sheet conformations [26-28]. 27

28 Chapter Raman Optical Activity (ROA) The vibrational optical activity of chiral molecules, exemplified by Raman optical activity (ROA), was predicted by Atkins and Barron in 1969 [29]. They noticed a new optical process involving interference between light waves scattered through the polarizability and optical activity tensors of a chiral molecule that was first experimentally measured by Barron, Bogaard and Buckingham, in 1973, who observed a small difference in the intensity of Raman scattering in right- and leftcircularly polarized light from α-phenylethylamine and α-phenylethanol [30]. This observation was independently verified by Hug in 1975 (Figure 1.2). x Raman I R + I L ω y z R L ROA I R - I L Figure 1.2: Schematic diagram of the basic SCP ROA experiment which measures as a small circular component in the scattered light in right (R) and left (L) using unpolarized incident light (adapted from [11]). The ROA measurement can be represented in terms of the circular intensity difference (CID) that is defined by; (5) 28

29 Chapter 1 where R I and L I are scattered Raman intensities in right and left circularly polarized light, respectively. ROA measures the optical activity related to Raman scattering and the chirality associated with molecular vibrational transitions [32], where a chiral molecule is one that is not super-imposable on its mirror image. The two mirror image forms of a chiral molecule are referred to as enantiomers [33]. Chiral molecules scatter left- and right-circularly polarized light to different degrees which leads to the resultant ROA spectrum. Unlike conventional Raman spectroscopy, in which only the electric dipole interacts with the incident light, in ROA spectroscopy, contributions from magnetic dipole and electric quadrupole optical activity tensors must also be considered. The oscillating electric dipole, magnetic dipole and electric quadrupole moments are characteristic of the scattered radiation field induced in a molecule by the incident light. The electric dipole, magnetic dipole are described by, m and electric quadrupole moments (6) (7) (8) where particle i at distance r i has charge e i, mass m i, linear momentum p i and the Kronecker delta,, is a function of two variables which is equal to 1 if they are equal and 0 otherwise [31,32]. 29

30 Chapter 1 The molecular multipole moments and quantum mechanical expressions for the dynamic molecular property tensors can be defined by the fields and field gradients that are assessed at the origin of the molecule. The field and field gradients are derived from the time-dependent perturbation theory and are defined as, Electric dipole-electric dipole tensor: (9) Electric dipole-magnetic dipole optical activity tensor: (10) Electric dipole-electric quadrupole tensor: (11) where n and j represent, respectively, the initial and virtual intermediate states of the molecule, is their angular frequency separation and is Plank s jn j n constant [31]. Circularly polarized light scattering and diffraction are caused by the electric dipole-electric dipole tensor, optical rotation in aqueous solution is generated by the electric dipole-magnetic dipole tensor G ; and A is the electric dipole-electric quadrupole tensor which leads to additional contributions to optical rotation in oriented samples [34,35]. By averaging the different polarizability-polarizability and polarizability-optical activity tensors components for all possible orientations of a molecule, we can 30

31 Chapter 1 consider their tensorial components that are invariant to axis rotations as shown in equations The isotopic invariants are defined as, and (12) and the anisotropic invariants as [34, 35], (13) (14) (15) The scattering angle can be varied, for example we can have forward ( 0 ) or backward ( 180 ) scattering. Right angle scattering can also be measured using a linear polarization analyzer in the scattered beam to select either the perpendicular (x) or parallel (z) transmission axis to the scattering plane (yz). CID expressions for different scattering geometries can be written in terms of, G and A, (16) (17) 31

32 Chapter 1 (18) (19) When a molecule consists of idealized axially symmetric bonds, for which 2 2 ) ( ) and G 0 ( G A, a simple bond polarizability theory explains that ROA is generated entirely by anisotropic scattering in which case the CID expressions reduce to : (20) and (21) These equations illustrate that ROA scattering intensities are maximized in the backward direction and are zero in forward scattering. This is unlike the case of conventional Raman spectroscopy, where the forward and backward scattering intensities are the same [34,35] ROA spectroscopy of biomolecules By understanding the connection that exists between protein structure and function, the behaviour of proteins can be studied. A range of techniques have been applied to the clarification of 3D structures of proteins, ranging from prediction based on the sequence and physico-chemical properties of the constituent amino acids to high resolution methods for the detection of atoms and determination of their molecular 32

33 Chapter 1 coordinates. As a result, investigations of protein structure (at primary, secondary, tertiary and quaternary levels) are important as probes of protein function in living organisms [36]. For a number of important problems in molecular biology such as protein folding, protein-protein interactions, and protein-nucleic acid interactions, quantitative measurement of the secondary structure provides significant insight into structural features critical to biological function [36,37]. Almost all biological compounds are chiral, so it is logical to investigate them not only by Raman spectroscopy but also by ROA, where additional features can be determined. Although ROA is a very weak effect with -values typically being ~ , it provides more structural information than Raman spectroscopy as ROA spectral details are more sensitive to stereochemistry [31]. ROA has been measured for a wide range of biological molecules including proteins, carbohydrates [37], nucleic acids [38, 39] and viruses [40]. The ROA spectra of proteins are dominated by bands originating in the peptide backbone which directly reflect their solution conformations [37]. The bands from side chains are usually not as significant in the ROA spectra of polypeptides and proteins as they are in the conventional Raman spectra [41] since the largest ROA signals are often associated with vibrational coordinates from the most rigid and chiral parts of the biomolecules [31]. 33

34 Chapter Surface-Enhanced Raman Scattering Surface-enhanced Raman scattering (SERS) is a powerful tool for determining chemical information about molecule substrates. The enhanced Raman signals in SERS are due to enhanced electromagnetic fields that result from adsorption of molecules on nanotextured metallic surfaces [42]. The SERS observation was first reported by Fleischman and co-workers, Hendra and McQuillan in 1974 [43]. In 1977 two separate papers by Jeanmaire and Van Duyne, and by Albrecht and Creighton confirmed the observation of a surface Raman spectrum of pyridine adsorbed on electrochemically roughened silver electrodes as a result of successive oxidation-reduction cycles [44,45]. The SERS method can increase the intensity of the Raman signal with enhancement 2 6 factors of in scattering efficiency over conventional Raman scattering [45]. It has been reported that the enhancement of Raman signals can be up to or greater for some experiments which proposes the possibility of single molecule detection levels [46,47]. Silver and gold are the typical substrates used in SERS technique and in various metal forms, for instance colloids, roughened electrodes, deposited layers and nanoshells [48]. While the type and preparation methods of metal substrates have an effect on the outcome of SERS signals, other factors such as temperature, pressure, nature of the analyte, aggregating agents and laser power can also have significant influence on SERS signals [49-51]. Several theories have been suggested to explain the mechanisms involved in SERS enhancement. The enhancement occurs due to increases in both the molecular polarizability of adsorbed species and the local electric field in the vicinity of the metallic surface [52]. However, the exact nature of SERS is still unknown, though it 34

35 Chapter 1 is now accepted that the electromagnetic and charge transfer (chemical) enhancements are the two most important mechanisms [50] The Electromagnetic Mechanism Various electromagnetic (EM) theories have been developed over the past decades. Complete electrodynamic calculations have been performed for simpler systems and the effects of dielectric responses have been discussed by Moskovits [52]. EM enhancement only depends on the characteristics and morphology of the metal surface [53]; therefore the same enhancement factor of vibrational modes should be obtained for the same surface morphology [54]. In order to explain the theory, the morphologies of roughened metal surface need to be understood. Electrons circulate on the metal surface which is held in a lattice by the presence of positive charges. This electron density on the surface expands in a significant distance from the surface which has the freedom of movement in the lateral direction [55,56]. As the incident electromagnetic radiation interacts with the electron density that surrounds the atomic lattice sites of the metal, vibrations in the molecule are initiated, resulting in a collective oscillation which is known as a plasmon [50,57-58]. Surface plasmons from small uniform particles or from single periodic roughness features of a surface have a resonance frequency which results in scattering of electromagnetic radiation [57]. The dielectric constant of the metal has a direct effect on the frequency of the surface plasmon oscillation. The resonance frequency should match with the visible frequencies of Raman scattering in order to generate the SERS enhancement [57]. To facilitate enhancement it is necessary for the oscillation of the plasmon to be perpendicular to the surface plane which is usually achieved by roughening of the surface [58]. This results in an increase of the coupling concentration of the electromagnetic field in certain regions on metallic surfaces [58]. An analyte 35

36 Chapter 1 molecule on or near the metal surface interacting with a surface plasmon experiences a large electromagnetic field, resulting in enhancement of the vibrational modes in the Raman spectrum [58]. EM theory cannot entirely explain the mechanisms involved in SERS; in particular it predicts the uniform enhancement of all Raman active bands. However, this is not the case in practice since some intense bands that can be observed in Raman spectra weaken or disappear in their corresponding SERS spectra. Therefore, mechanisms other than EM must be implicated in the SERS phenomenon in order to fully explain SERS enhancement The Chemical Mechanism (CE) Other studies suggest that there is a second enhancement mechanism for SERS which operates independently from the EM mechanism [48,59]. Different molecules with identical polarizability, adsorbed onto the same metallic substrate under the same experimental conditions, demonstrate different enhancement factors [48]. This would suggest that an EM mechanism is not the only mechanism involve in SERS enhancement. Further evidence in support of the chemical mechanism comes from potential-dependent electrochemical experiments. When an electrode potential is scanned at a fixed laser frequency, or the laser frequency is scanned at fixed potential, broad resonance is observed [48]. Enhancement resulting from the chemical mechanism occurs due to the formation of a chemical bond between the atomic scale of metal roughness and the adsorbed analyte [10]. This chemical contribution generates surface species which consist of the analyte and surface metal atoms [10]. This in turn enables feasibility of charge transfer from the metal surface into the analyte which causes an increase in the 36

37 Chapter 1 molecular polarizability of the analyte due to interaction with the metal s electrons [59]. Basically, these observations can be explained by resonant intermediates in Raman scattering in which either the electronic states of the adsorbate are shifted and broadened by their interaction with the surface, or the formation of a new electronic state arising from chemisorption [50]. The highest occupied molecular orbital and lowest unoccupied molecular orbital of the adsorbate are symmetrically disposed in energy with respect to the Fermi level of the metal [50]. In this case, charge transfer excitations can occur either from the metal substrate to the molecule or from the molecule to the metal substrate at the about half the energy of the intrinsic intramolecular excitation of the adsorbate [10,50]. Most charge transfer excitations in SERS take place at visible wavelengths since the molecule s lowest-lying excitation energy is in the near ultraviolet region [59]. As discussed above, it is very difficult to separate the contribution effects resulting from EM and chemical mechanisms on systems which support SERS enhancement. Although the majority of evidence suggests that both mechanisms play a key role in SERS enhancement, EM enhancement has the greater effect on enhancement as the charge transfer enhancement of Raman signals drops off by from the surface [10]. 3 1 r with distance r Biological applications of SERS Metal substrates can be applied to obtain more precise information for structural determination of nucleic acids and peptides as they can give more enhanced signals in short illumination periods (less than 1s), lower laser power and sample concentrations. SERS spectra of different biomolecules such as amino acids, nucleotides, peptides, enzymes, DNA and RNA have been reported using different 37

38 Chapter 1 metal substrates e.g. Ag and Au [60-77]. One of the main advantages of SERS for analysis of biomolecules is the reduction of the luminescent background that often obscures Raman scattering from biological molecules [78]. The compatibility of the metal substrate with biomolecules and the morphology of the surface play an important role in SERS activation. This compatibility facilitates a better coupling between adsorbed sample molecules and the metallic surface for enhancement of Raman scattering. The potential for application of SERS for analysis of biological components is illustrated by its use in the diagnosis of tissue lesions [79,80], analysis of blood components and study of tissues [79,80]. A highly sensitive and selective SERS detection has been reported for DNA using plasmonic nanoparticle substrates, highlighting the potential of this approach as a rapid genetic analysis tool for understanding biological process, for unlocking the underlying molecular cause of diseases and for development of biosensors [81]. 38

39 Chapter Surface enhanced Raman optical activity (SEROA) Existing problems with ROA spectroscopy for studying biological molecules are principally that the conditions required include high sample concentrations ( mg/ml) and long acquisition periods in comparison to those required for Raman and SERS [82]. Although ROA spectroscopy gives more incisive information about stereochemistry of biomolecules, it suffers from being a weak effect hence preventing the application of ROA to a wider range of target molecules at present. 6 Alternatively, SERS generates signals that can be ~ 10 larger than the conventional Raman signals [45]. The combination of ROA with SERS in the form of surface enhanced Raman optical activity (SEROA) is a promising solution for widening the application of ROA to other biomolecules that are not easily accessible to other structural methods, such as unfolded proteins and viruses. In recent decades, various theoretical aspects of surface enhancement of ROA signals have been investigated. Efrima proposed that measurements of ROA are possible for a molecule adsorbed on the metal surface which contains information on the local electric field, their gradients and, in general, local dielectric properties of the metalsolution interface [83,84]. The model relies upon large electric field gradients close to the metal surface where a molecule is subjected to a larger electric field than in the bulk solution. Enhancement of the ROA signal can be achieved by this model as the electric dipole-electric quadrupole contribution is predicted to be large. According to Efrima s calculations, SEROA spectra can be influenced by several properties of electromagnetic radiation once it interacts with metal surface. These include the magnitude, direction, spatial dependence and polarization of the electromagnetic radiation. In summary, Efrima proposed that SEROA can be obtained if certain conditions are met [83,84]. These are the existence of an electromagnetic field 39

40 Chapter 1 gradient near the surface of the metal surface, a phase difference between the electric field and its gradient and finally induction of resonance where the interaction between the molecule and the metal surface occurs [83,84]. Hecht and Barron also considered enhancement of the ROA signal using a metal substrate, through the approximation of a pure electric dipole surface ROA model [85,86]. They also investigated the possibility of a SEROA spectrum being generated by an achiral molecule. They postulated that when an achiral molecule adsorbs onto the metal surface and is randomly oriented, no ROA signal can be achieved. This is mainly due to cancellation of the signal as a result of different enantiomeric projections. However, if they align in a manner to form an ordered surface, obtaining a SEROA signal from an achiral molecule is feasible. A decade later Janesko and Scuseria considered the effect of averaging over all orientations of the metal surface, using three different models; a dipolar sphere, quadrupole sphere and a dipolar nanorod [87]. In contrast with Efrima s models, they predicted significantly smaller CID values. Etchegoin et al. [88] have proposed the generation of SEROA signals by modelling of single SERS experiments. In their theoretical work, they have predicted that the high enhancements associated with hot spots for SERS single molecule detection affect the behaviour of circularly polarized light in the vicinity of the surface plasmons [88]. Given that ROA is a weaker effect than conventional Raman, they predicted that SEROA signals may be small and difficult to distinguish from background noise. Also, the detected SEROA signal comes from the electric field of a number of different molecules that may have different polarization directions which may result in cancelling of the SEROA signals and prevent reliable measurement of a SEROA spectrum. 40

41 Chapter 1 Various theoretical studies modelling ROA responses in the vicinity of metal substrates have facilitated a better understanding of the fundamental phenomenon of SEROA [89]. The first simulation of SEROA using the time-dependent density function theory has recently been proposed by Janesko and Scuseria where interaction of adenine with a Ag cluster was investigated [90]. They predicted an enhancement factor of 10 4 for both SERS and SEROA and concluded that the observed enhancement due to charge transfer is larger than that of SERS. The chemical effect of analyte-colloid binding on the metal surface was also calculated [90]. The results suggest that observed SEROA bands, in terms of signs and intensities, are very sensitive to analyte-metal orientation. As a result, they proposed that future SEROA experiments may require utilising ordered monolayers of chiral analytes to minimise this orientation effect [90]. Yang et al. [91] have studied the interaction of electromagnetic fields with light and the phenomena of enhancement of magnetic and electric field gradients. They have highlighted the potential applicability of SEROA as a chiroptical analytical tool. Halas et al. [92] also developed a SEROA model for molecules moving near spherical metal nanoshells where the excitation profiles for a simple chiroptical model was analyzed in detail and suggested a preferred excitation wavenumber. Very recently, the matrix polarization theory was employed to model the SEROA spectra of ribose and cysteine molecules and enabled comparison with experimental results [93]. Findings showed a strong distance dependence of enhancement between molecules and the metal surface along with dependence of the ROA ratio and Raman intensities (CID) on distance and rotational averaging. Findings from this study confirmed that maximum enhancement can be obtained by colloidal aggregates rather than asymmetry of individual particles and validated the experimental 41

42 Chapter 1 observation of L- and D-ribose which was reported as part of this thesis [94]. The importance of controlling the colloid-sugar distance was emphasised which was done in the experimental set up for SEROA measurement of the ribose molecule. Currently, few experimental studies have been reported to validate the technique with a high degree of certainty. Kneipp et al. [95] have claimed to observe a SEROA spectrum of adenine molecule adsorbed onto silver colloidal nanoparticles. They correlated two SEROA bands to the most enhanced peak in the SERS spectrum of adenine. Since adenine is an achiral molecule, they suggested that symmetry of the molecule can be lost once it is adsorbed onto the metal surface which then becomes chiral. They have also postulated that the adenine molecule aligns in the same orientation on the metal surface that then result in prevention of signal cancelling from various pro-chiral attachments. Abdali and Blanch [82] in their review article dismissed these results and suggested that they are artefacts as Kneipp et al. neglected the problems associated with changes in the polarization state modelled by Etchegoin et al. [84]. These problems arise from reflection of electromagnetic light from the metal surface that modify circularly polarized light and create elliptically polarized light, which may result in the generation of birefringent artefacts [84]. Also, the generation of an ROA signal under these conditions would require a significant proportion of circularly polarized light, since a significant proportion of linearly polarized light would result in SEROA artefacts. Abdali et al. reported SEROA spectra of two resonant molecules; cyctochrome c and myoglobin as well as a nonresonant molecule; Met-enkephalin [96-98]. However, it is difficult to verify these results since there may be artefacts arising from the parent SERS bands and also there were no enantiomers of these compounds available to 42

43 Chapter 1 verify SEROA mirror image band responses which are very important for validating of chiroptical techniques such as SEROA. More recently, Osinska et el. have reported the measurement of mirror image SEROA spectra of L- and D-cysteine using an electrochemically roughened solid silver based system [99]. Although this was an interesting observation, this study did not consider a number of details concerning the experimental procedure utilised. These results cannot be deemed reliable since the authors reported that SERS spectra of L- and D-cysteine for the same experiment could not be observed. Instead they reported SERS spectra for a different colloid-based experiment with no corresponding SEROA. Since any SEROA measurement must logically be weaker than the corresponding SERS measurement, and therefore more difficult, this calls into doubt the reliability of these spectra. It appears that they did not measure SEROA but rather the solution-phase ROA, as sample concentrations were very high in their study, complicated by the presence of large reflection-based artefacts from the metal surface. In 2008 [100] and 2009 [101], two PhD theses presented attempts to prove SEROA as a technique, but which were unsuccessful in both cases due to the complexity of the process. However, both illustrated the importance of controlling the experimental SERS conditions such as colloidal type, analytes, aggregating agents, ph and concentration since they have a direct effect on obtaining not only reliable SERS but also any associated SEROA spectra. The time-dependent nature of the enhancement process was shown to have a significant effect on the obtained results laying the platform for this thesis. SEROA can still be considered an unproven technique. The main objective of this PhD thesis is to prove and develop reliable SEROA. Obtaining a more consistent 43

44 Chapter 1 colloidal system may assist to validate SEROA as a feasible technique and this could be achieved by controlling the colloidal aggregation over long time periods using a hydrogel polymer as a stabilising agent. The measurement of mirror image bands for two enantiomers of each of ribose and tryptophan were undertaken in order to prove the validity of SEROA measurements, and this work is presented in Chapter 2. Silver silica nanotags, which have been proven to provide strong SERS enhancement, as well as stable metal nanoparticles were both used to assess the potential application of SERROA as a nanoprobe for biomolecule analysis. The chiroptical properties of these nanotags were confirmed by the measurement of mirror image surface enhanced resonance Raman optical activity (SERROA) spectra of the two enantiomers of each of ribose and tryptophan which is demonstrated in Chapter 3. As part of the PhD research undertaken a number of other studies were performed to investigate outstanding problems in biomolecular structure using analytical spectroscopies. Chapter 4 presents work in which adenosine and seven of its derivative ribonucleotides were studied by Raman, ROA and SERS in order to identify spectral markers of site-specific phosphorylation in nucleic acids. Raman and ROA spectroscopies in combination with computational modelling were used to study the structural changes in short linear dipeptides, specifically diserine and dialanine, due to cyclization; are presented in Chapters 5 and 6. The conclusion chapter highlights the importance of optimization of the correct experimental protocols for obtaining reliable SERS and SEROA spectra. The conclusion also discusses how that combination of four different spectroscopic techniques, Raman, ROA, SERS and SEROA is more advantageous for studying of biological samples since they can provide more structural information on the nature of biomolecules along with their chirality. 44

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48 Chapter L. Hecht, L. D. Barron, Journal of Molecular Structure. 1995, 348, L. Hecht, L. D. Barron, Chemical Physics Letters. 1994, 225, B. G. Janesko, G. E. Scuseria, Journal of Chemical Physics. 2006, 125, P. G. Etchegoin, C. Galloway, E. C. Le Ru, Physical Chemistry Chemical Physics. 2006, 8, P. Bour, The Journal of Chemical Physics. 2007, 126, B. G. Janesko, G. E. Scuseria, The Journal of Physical Chemistry C. 2009, 113, N. Yang, Y. Tang, A. E. Cohen, Nano Today. 2009, 4, R. Lombardini, R. Acevedo, N. J. Halas, B. R. Johnson, Journal of Physical Chemistry C. 2010, 114, V. Novak, J. Sebestík, P. Bour, Journal of Chemical Theory and Computation. 2012, 8, S. Ostovar Pour, S. E. J. Bell, E. W. Blanch, Chemical Communications. 2011, 47, H. Kneipp, J. Kneipp, K. Kneipp, Analytical Chemistry. 2006, 78, S. Abdali, C. Johannessen, J. Nygaard, T. Nørbygaard, Journal of Physics: Condensed Matter. 2007, 19, S. Abdali, Journal of Raman Spectroscopy. 2006, 37, C. Johannessen, P. C. White, S. Abdali, Journal of Physical Chemistry A, , K. Osińska, M. Pecul, A. Kudelski, Chemical Physics Letters. 2010, 496, A. J. Hobro, PhD thesis, Structural Investigation of RNA through Application of Raman, Raman Optical Activity and Surface Enhanced Spectroscopies. 2008, University of Manchester, UK N. R. Yaffe, PhD thesis, Raman Spectroscopic Studies of Biological Molecules. 2009, University of Manchester, UK. 48

49 Chapter 2 Chapter 2 Use of a Hydrogel Polymer for Reproducible Surface Enhanced Raman Optical Activity (SEROA) 49

50 Chapter Declaration This chapter consists of one published full paper: S. Ostovar Pour, S. E. J. Bell, E. W. Blanch, Chemical Communication. 2011, 47, The manuscripts have been incorporated in a format identical to that for journal submission, except for minor adjustments to incorporate them into this thesis. As first author on this publication I carried out all of the associated experimental and spectroscopic analysis. The polymer was provided by Dr Steve Bell at the Queen s University of Belfast. 50

51 Chapter 2 Use of a Hydrogel Polymer for Reproducible Surface Enhanced Raman Optical Activity (SEROA) Saeideh Ostovar Pour,* a Steven E. J. Bell b and Ewan W. Blanch a Received 30th November 2010, Accepted 25th February 2011 a Faculty of Life Sciences, Manchester Interdisciplinary Biocentre, The University of Manchester, 131 Princess Street, Manchester, UK M1 7DN. E.Blanch@manchester.ac.uk; Fax: +44 (0) ; Tel: +44 (0) b School of Chemistry and Chemical Engineering, The Queen s University of Belfast, Belfast, UK BT9 5AG. s.bell@qub.ac.uk ; Fax: +44(0) ; Tel: +44(0) Abstract We present surface enhanced Raman optical activity (SEROA), as well as Raman, SERS and ROA, spectra of D- and L-ribose. By employing a gel forming polyacrylic acid to control colloid aggregation and associated birefringent artefacts we observe the first definitive proof of SEROA through measurement of mirror image bands for the two enantiomers. 2.2 Introduction As a result of its sensitivity to chirality, Raman optical activity (ROA), which measures a small difference in the intensity of vibrational Raman scattering from chiral molecules in right- and left-circularly polarized light [1,2] is a powerful probe of the structure and behaviour of biomolecules in aqueous solution [3 7]. However, ROA is a very weak effect being ~3 5 orders of magnitude smaller than the parent Raman scattering. The conditions of high concentration and long data collection time required for ROA currently limit its application for a wide range of 51

52 Chapter 2 biological samples. These limitations could possibly be overcome using the principles of surface enhanced Raman scattering (SERS) [8 10] in which a sample in the presence of surface plasmons localized on a neighbouring nanostructured feature of a metal surface can interact with the incident light leading to large enhancement of the Raman scattering. However, the generation of reliable SEROA spectra of biomolecules has been problematic due to difficulties in controlling spectral artefacts and low signal-to-noise ratios which complicate detection of true SEROA signals. Although several papers have presented possible SEROA spectra [11, 12] currently a proof demonstrating mirror image SEROA spectra from opposite enantiomers has not been reported. Recently, observation of SEROA spectra for the L- and D-enantiomers of cysteine has been claimed [13]; however the authors stated that no corresponding SERS spectra could be measured under the same conditions. Therefore, no surface enhancement had occurred in the earlier study [13] either SERS or SEROA with the observed spectral features probably being reflection associated birefringent artefacts. Thus, SEROA has still not been confirmed as an experimental technique. SEROA spectral features depend on SERS experimental conditions, since they reflect the stability of colloids over time periods longer than those typically used in conventional SERS [14] Contributing factors, including the concentration of analyte and aggregating agents, ph, type of colloid and time dependence, have been studied in order to determine the effects of these parameters on SERS spectra [15 17] as they should also be optimal for measuring SEROA [18]. However, it has proven difficult to stabilise the extent of aggregation in colloidal systems sufficiently to control the fluctuation of bands in SEROA experiments, complicating validation of observed spectral features. Etchegoin et al. modelled the effect of surface plasmons on circularly polarized light [19]. Their calculations suggest that large artefacts in 52

53 Chapter 2 SEROA spectra would be highly sensitive to the nature of colloid colloid interactions, explaining the origin of the intense and fluctuating features often observed in SEROA experiments. Slowing down changes in the aggregation state to minimize changes in colloidal interactions should improve the reliability and reproducibility of SEROA spectra. In this study we employ a hydrophilic polyacrylic acid polycarbopol polymer as a stabilising medium. This polymer has small Raman and surface-enhanced Raman cross sections, minimising interference from background signals, does not significantly change the UV-vis absorption spectra when added to silver colloids and is known to stabilise even aggregated colloids for extended periods of time [20,21]. We report SERS and SEROA spectra, along with the Raman and ROA spectra of D- and L-ribose measured in the presence of citrate-reduced silver colloids and the polycarbopol polymer, providing the first definitive observation of SEROA. 2.3 Experimental Silver nitrate (99%), sodium borohydride (99%), sodium citrate (99%), sodium hydroxide (99%), potassium sulphate (99%), D- and L-ribose (99%) were purchased from Sigma- Aldrich UK and used without further purification. Citrate reduced silver colloids were prepared by reduction of silver nitrate with citrate ions [22] see supplementary information for details. The polycarbopol polymer was purchased from B.F. Goodrich Ltd and used without further purification to form the polymer-sol mixture. The Raman (I R + I L ), scattered circularly polarized (SCP) ROA (I R - I L ), SERS (I R + I L ) and SCP SEROA (I R - I L ) spectra were all measured using a ChiralRAMAN SCP spectrometer (BioTools Inc., Jupiter FL) operating in the backscattering configuration 53

54 Chapter 2 at an excitation wavelength of 532 nm with spectral resolution of 7 cm -1. Raman and ROA spectra were taken with laser power of W at the sample with data collection times of 4 6 h. The laser power for SERS and SEROA was 0.25 W at the sample with data collection times of 35 min. The details of sample, aggregating agent and colloid concentration are given in each figure legend. All SERS samples were prepared to 1 ml, the sample was left to sit for 15 min in order to obtain maximum SERS enhancement, which was determined from time dependence measurements, and then 20 mg of polycarbophil polymer powder was added and stirred vigorously for a few seconds, then left for 60 min in order to allow full hydration and swelling of the polymer prior to data collection. 2.4 Results and Discussion The Raman and ROA spectra in aqueous solution obtained for D- and L-ribose are shown in Figure 2.1. All spectra (Raman, ROA, SERS and SEROA) presented in this study are raw data without any smoothing, base lining, normalization or any other data pretreatment. The Raman and ROA band assignments for both enantiomers of ribose are summarized in Table S2.1 in supplementary information [23 25]. The Raman and ROA spectra of D-ribose are in excellent agreement with those reported by Wen et al. [23] and those measured recently by Dr C. Johannessen in Glasgow (personal communication). We have repeated the Raman and ROA spectra for L- ribose, but these have not been previously reported. Mirror image responses are observed for most ROA bands, though it is not known why no ROA band appears near 877 cm -1 for L-ribose, though this spectrum is reproducible. Figure 2.2 A and E presents the Raman and ROA spectra, respectively, of polycarbopol in solution, measured at the same concentration as used in the SEROA 54

55 Chapter 2 experiments, with SERS and SEROA spectra of D- and L-ribose shown in Figure 2.2 C, D, G and H, respectively, before and after addition of polycarbopol polymer. The Raman spectrum of polycarbopol shows that the polymer does not generate any significant Raman signal as this polymer has a very small Raman cross section [20], and only the spectrum of water is evident. Although the polycarbopol subunit is chiral, it has a low Raman cross-section [21] so helping to minimise its ROA spectrum. Together, this leads to the ROA spectrum of polycarbopol being very weak, barely above the noise level. Figure 2.2 B and F shows spectra for the combination of silver colloids, aggregating salt and polycarbopol (no analyte). The two stronger bands at ~1394 and 1452 cm -1 in the SERS spectrum are a fingerprint of the sol with polycarbopol. The corresponding SEROA spectrum has negative features which are very noisy, that arise from both the polycarbopol and the interaction of plasmon resonances with circularly polarized light. Figure 2.2 C and D presents the experimental SERS spectra of D- and L-ribose before and after, respectively, the addition of the polymer. The SERS spectra for D- and L-ribose shown in Figure 2.2 C and D were obtained using the optimum type of aggregating agent (K 2 SO 4 ), its concentration (20 mm) and ph (8.7). The optimum concentration of the polycarbopol polymer was found to be 20 mg ml -1 which generated a viscous solution that was dilute enough to pipette but thick enough to control the aggregation of colloids, and gave rise to strong SERS signals for an extended period of time. The spectra demonstrate that the SERS signals for the two enantiomers are similar both in the presence (Figure 2.2 D) and the absence (Figure 2.2 C) of the polymer. All bands measured in the conventional SERS experiments appear at the same position in the presence of the polymer with only small differences in relative intensities of bands, confirming that the polymer does 55

56 Chapter 2 not interfere with signals from ribose molecules. Time dependence measurements, see Figure S2.3 in supplementary information, show that SERS intensity is stable for over 35 min with the polymer, but for only 10 min without polymer. We conclude, therefore, that the addition of the polymer increases the stability of the aggregated colloids, allowing measurement of reliable SERS signals from the analyte. The SEROA spectra of D- and L-ribose measured in the absence of polymer are shown in Figure 2.2 G. These spectra present a common problem that can occur in attempts to measure SEROA spectra. The SEROA spectrum of D-ribose gives rise to a mix of +ve and -ve bands, which are what may be expected in a chiroptical measurement, but in the spectrum of L-ribose all of the bands are negative in sign, due to difficulties in controlling the highly birefringent background signal. Therefore, we do not observe a mirror image response in Figure 2.2 G for any of the purported SEROA bands generated by the two enantiomers due to the large birefringence generated by the surface plasmons from the aggregating colloids, making it difficult to have confidence in the reliability of either of these two spectra. Furthermore, though the SERS spectra presented in Figure 2.2 C, which are insensitive to this problem, could be reproduced many times, the corresponding SEROA spectra demonstrated poor reproducibility both from sample-to-sample and as a function of time. Figure 2.2 H shows the SEROA spectra of D- and L-ribose with polycarbopol polymer. Both D- and L-ribose give highly reproducible SEORA spectra (see Figure S2.4 in supplementary information for replicate measurements) with positive and negative bands. Critically, despite baseline variations, mirror image bands are now observed for the two enantiomers. The SEROA spectrum of D-ribose displays a number of bands that clearly show the opposite sign to their L-ribose counterparts. The +ve SEROA bands for D-ribose at 1247, 1273 and 1315 cm -1 correspond to the - 56

57 Chapter 2 ve SEROA bands for L-ribose at 1242, 1270 and 1310 cm -1, respectively. A complex -ve/+ve/-ve triplet exhibited by D-ribose from ~ cm -1 is nicely replicated as a +ve/-ve/+ve triplet by L-ribose with similar band shapes and intensities, as is the +ve/-ve couplet for D-ribose from ~ cm -1. A strong +ve SEROA band at 1571 cm -1 for D-ribose gives rise to an equivalent -ve feature for L-ribose. The regions between cm -1 and below 1000 cm -1 reveal a number of weak features that appear to show opposite sign for the two enantiomers, though variations in local baselines due to residual birefringent background signals complicate their analysis. However, several features in the SEROA spectrum for D-ribose do not lead to a mirror image for the L-enantiomer, most notably the +ve bands at ~1014 and 1539 cm -1, while there are also no counterpart features to the -ve SEROA bands displayed by L-ribose at ~1699 and 1739 cm -1. The reasons for these differences are not known, but they are reproducible (Figure S2.4, supplementary information) so do not originate from variable birefringent artefacts or shot noise. In order to verify the reliability of this method further, L- and D-tryptophan were also measured, Figure S2.5 (spectra provided in supplementary information). In both case L- and D-tryptophan provided mirror image response in SEROA Spectra. 2.5 Conclusion We have demonstrated the first experimental proof of SEROA by recording SEROA spectra for two enantiomers, D- and L-ribose, along with their corresponding SERS and ROA spectra. Addition of the polycarbopol polymer provides a solution to the problem of how to stabilize the aggregated colloids, and so reduce the effect of plasmon resonance induced changes in circularly polarized light that typically plague 57

58 Chapter 2 SEROA experiments. This strategy will allow the potential of SEROA to be more effectively explored. 58

59 Chapter References 1. P. W. Atkins, L. D. Barron, Molecular Physics. 1969, 16, L. D. Barron, M. P. Bogaard, A. D. Buckingham, Journal of the American Chemical Society. 1973, 95, L. D. Barron, L. Hecht, E. W. Blanch Molecular Physics. 2004, 102, L. D. Barron, Current Opinion in Structural Biology. 2006, 16, T. Uchiyama, M. Sonoyama, Y. Hamada, R. K. Dukor, L. A. Nafie, F. Hayashi, K. Oosawa, Vibrational Spectroscopy. 2008, 48, L. D. Barron, E. W. Blanch, I. H. McColl, C. D. Syme, L. Hecht, K. Nielsen, Spectroscopy. 2003, 17, E. W. Blanch, L. Hecht, L. D. Barron, Methods. 2003, 29, D. L. Jeanmaire, R. P. Van Duyne, Journal of Electroanalytical Chemistry. 1977, 84, K. Kneipp, Y. Wang, H. Kneipp, L. T. Perelman, I. Itzkan, R. Dasari, M. S. Feld, Physical Review Letters. 1997, 78, E. Koglin, H. H. Lewinsky, J. M. Sequaris, Surface Science. 1985, 58, C. Johannessen, P. C. White, S. Abdali, Journal of Physical Chemistry A. 2007, 111, N. A. Brazhe, A. R. Brazhe, O. V. Sosnovtseva, S. Abdali, Chirality. 2009, 21, E307 E K. Osinska, M. Pecul, A. Kudelski, Chemical Physics Letters. 2010, 496, S. Abdali, Journal of Raman Spectroscopy. 2006, 37, A. J. Hobro, S. Jabeen, B. Z. Chowdhry, E. W. Blanch, Journal of Physical Chemistry C. 2010, 114, N. R. Yaffe, E. W. Blanch, Vibrational Spectroscopy. 2008, 48, N. R. Yaffe, A. Ingram, D. Graham, E. W. Blanch, Journal of Raman Spectroscopy. 2009, 41, S. Abdali, E. W. Blanch, Chemical Society Reviews. 2008, 37, P. G. Etchegoin, C. Galloway, E. C. Le Ru, Physical Chemistry Chemical Physics. 2006, 8, S. E. J. Bell, S. J. Spence, Analyst. 2001, 126, S. E. J. Bell, N. M. S. Sirimuthu, Analyst. 2004, 129, P. C. Lee, D. Meisel, Journal of Physical Chemistry. 1982, 86, Z. Q. Wen, L. D. Barron, L. Hecht, Journal of the American Chemical Society. 1993, 115, P. Carmona, M. Molina, Journal of Raman Spectroscopy. 1990, 21, M. Mathlouthi, A. M. Seuvre, J. L. Koenig, Carbohydrate Research. 1983, 122,

60 Chapter 2 Raman 8.8x10 10 D - Ribose L - Ribose I R - I L I R + I L 6.6x x x x ROA D- Ribose L- Ribose wavenumber (cm -1 ) Figure 2.1: Raman (I R + I L, top) and SCP ROA (I R I L, bottom) spectra of D- and L- ribose in aqueous. Sample concentrations were 2.66 M at ph 5.46 (D-) and 5.60 (Lribose), data collection time was min, and laser power at the sample W for each. 60

61 Chapter 2 6.9x10 8 A 4.6x x B I R + I L 7.5x x10 9 D-ribose L- ribose C 1.2x x10 8 D 3.5x x10 4 E F -5.0x10 4 I R - I L -1.0x G -6.5x x H Wavenumber (cm -1 ) Figure 2.2: Raman spectrum of polycarbopol in solution (A), SERS spectra before (B) and after addition of L- and D-ribose (0.25 mg ml -1 ) in the presence of silver 61

62 Chapter 2 citrate reduced colloid and K 2 SO 4 at M concentration, data collection time: 20 min (C), SERS spectra of L- and D-ribose (0.25 mg ml -1 ) in the presence of polycarbopol polymer, data collection time: 20 min (D), ROA spectrum of polycarbopol polymer in solution, sample concentration 40 mg ml -1, data collection time: 218 min (E), SEROA spectra of silver citrate reduced colloids in presence of aggregating salt before (F) and after addition of L- and D-ribose, data collection time: 35 min (G), SEROA spectra of L- and D-ribose with addition of polycarbopol, data collection time of 35 min (H). 62

63 Chapter Supplementary Information n * Polycarbopol Figure S2.1: A schematic diagram of SEROA sample preparation with use of the polycarbopol polymer Colloid Preparation Citrate-reduced silver colloids were prepared by reduction of silver nitrate with citrate ions (Lee and Meisel method), 21 where g of AgNO 3 was dissolved in 500 ml of distilled H 2 O and heated to boiling point, then 10 ml of 1% trisodium citrate solution was added drop wise to the mixture. Heating was continued for another hour with constant stirring and then the solution was allowed to cool to room temperature. Approximately 300 ml of a green-grey solution was obtained at ~0.5 M concentration. All glassware used to prepare the colloids was washed prior to use with aqua regia followed by gentle scrubbing with a 2% Helmanex solution and thorough rising with distilled water Sample Preparation for Raman and ROA Measurements Samples of D- and L-ribose for Raman and ROA spectra were prepared by dissolving into distilled water at 100 mg/ml, then were microcentrifuged for 5 minutes at 3000 rpm (1000 g) to minimize dust particles prior to loading into quartz microflourescence cells. 63

64 Chapter Atomic Force Microscopy Micrographs were obtained using a Veeco Picoforce Multimode AFM with standard extender module, Nanoscope IIIA controller and a Picoforce scanner. Each AFM plate was prepared by adding 50 µl of sample to freshly cleaved mica and left at room temperature for 30 minutes. The mica was then rinsed carefully under distilled water for approximately 10 seconds and dried under a gentle stream of nitrogen. AFM was carried out in air in tapping mode with a scan size of 5 microns and a scan rate of 0.5 Hz using a Silicon TAP300 AFM cantilever and tip (oscillated at approximately 260 khz). Figure S2.2 presents AFM images of silver citrate reduced colloids before and after addition of polycarbopol polymer. The micrograph of polycarbopol without metal colloids (Figure S2.2 B) shows a very smooth surface whereas the metal colloids give rise to a very rough surface (Figure S2.2 A). Addition of polymer to metal colloids does not induce significant change to morphologies of the metal particles that are observed in Figure S2.2 C and confirms that aggregation is significantly reduced. 64

65 Chapter 2 Figure S2.2: Dual views of AFM images of silver citrate reduced colloids only (A), polycarbopol polymer only (B) and a mixture of silver citrate reduced colloids and polycarbopol polymer (C). Random regions of different micrographs were selected and the diameters of nanoparticles contained within were measured by using the measuring tool in the Nanoscope 7.2 software. The average particle sizes of silver colloids with and without polycarbopol polymer were ~67 and 70 nm, respectively, so the particle size in the polymer gel was very similar to that observed for normal silver colloids. However, the individual colloidal particles are much more distinct in the AFM images upon addition of the polymer. This is not an issue as the maximum SERS enhancement was obtained before controlling the aggregation process. The micrographs in combination with the time dependent SERS data (Figure S2.3), which are discussed below, confirm that the addition of polycarbopol to silver colloid controls the aggregation process of the nanoparticles. 65