Surface-enhanced raman spectroscopy and computational chemistry for phytoestrogen research

This thesis examines the vibrational structures of six phytoestrogen isoflavones, namely, daidzein, genistein and their methoxy derivatives: formononetin, biochanin A, prunetin and 4’,7-dimethoxygenistein, with a particular focus on surface-enhanced Raman spectroscopy (SERS). The purpose of this work is to develop a fundamental understanding of some of the structural and vibrational aspects of isoflavones, thereby providing a foundation for applications of vibrational spectroscopy in future phytoestrogen research. Following the first 3 introductory chapters on phytoestrogens, vibrational spectroscopy and experimental techniques, the results are presented over Chapters 4-7, of which, Chapters 4-6 are based on published or accepted articles. Chapter 7, based on unpublished work, applies the knowledge acquired in the earlier chapters to the interpretation of SERS on alternative substrates. Theoretical density functional theory (DFT) calculations are also extensively applied in aiding the interpretation of all spectra explored in this thesis, demonstrating the benefits of computational chemistry in studying vibrational spectroscopy. In Chapter 4, infrared and Raman spectra were applied in the characterization of the structural and vibrational properties of the isoflavones. The analysis was performed through a combined approach, incorporating DFT calculations, vibrational mode correlations between the isoflavone derivatives and also between their sub-structural motifs: phenol and anisole. This provides the necessary background for understanding SERS of the isoflavones in the ensuing chapters. In Chapter 5, investigations of the SERS of daidzein, which has two hydroxyl groups (7-OH and 4’-OH), and its 4’-methoxy derivative, formononetin, on citrate-reduced silver colloids (cit-AgC) are reported. In SERS, the nature of the interaction between the analyte and the metal surface is critical for the effective enhancement of the Raman signals. Through pH dependence and comparative SERS analysis of the two isoflavones, it was revealed that the OH groups needed to be deprotonated in order to interact with the silver surface and become SERS active. Accordingly, the deprotonated 7-OH acts as the site of interaction with cit-AgC on both daidzein and formononetin, and also through 4’-OH deprotonation on daidzein. Bands near 1482 and 1493 cm-1 are identified as characteristic of the anionic interactions with silver via the 7-OH and 4’-OH groups, respectively. The study is extended in Chapter 6 to a series of 5-hydroxyisoflavones: genistein (Figure T1), biochanin A, prunetin and 4’,7-dimethoxygenistein, to reveal a similar pH dependent, anionic interaction via the 7-OH and 4’-OH groups. However, the 5-OH group was found not to be strongly SERS active. Limited solubility in the finely controlled SERS conditions and steric hindrance are suggested as possible reasons for their lack of interaction. Finally, in Chapter 7, a selection of alternative SERS substrates is examined for their effectiveness in aiding isoflavone analysis. Three substrates were selected on the basis of commercial availability and partial reusability: sol-gel SERS substrates, oblique angle deposition (OAD) SERS substrates and Klarite® SERS Detection substrates. Different substrates can have distinct surface environments, which can consequently affect the analyte-metal interactions and the resultant SERS profile. On sol-gel and OAD substrates, which contain silver as the SERS active surface, the isoflavones were found to undergo similar interactions as on cit-AgC, as evidenced by similarities in the measured spectra and, in particular, a number of marker bands. In contrast, the isoflavones were found to adsorb in the neutral form on the gold surface of Klarite, being weakly physisorbed via 7-OH and π-cloud of the chromone group. On all three substrates, the analysis is facilitated by the fundamental understanding acquired in the earlier chapters, thereby demonstrating the applicability of the vibrational characterisations in this work on alternative substrates, and in effect, different environments.