Browsing by Author "Pfukwa, Ngaatendwe Buhle Cathrine"

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    Surface enhanced Raman spectroscopy (SERS) of amino acids
    (Stellenbosch : Stellenbosch University, 2016-03) Pfukwa, Ngaatendwe Buhle Cathrine; Neethling, Pieter H.; Rohwer, Erich G.; Schwoerer, Heinrich; Stellenbosch University. Faculty of Science. Dept. of Physics
    ENGLISH ABSTRACT : Raman spectroscopy (RS) is an invaluable technique for sample identification. This method requires little sample preparation and is not completely non-invasive. The intensity of Raman scattered light can be enormously increased or boosted when a sample molecule is adsorbed on a metallic surface, a technique known as Surface Enhanced Raman spectroscopy (SERS). Since the development of this technique a lot of studies have been done on molecules adsorbed on various types of metallic structures due to the sole purpose of the increase in Raman signal which occurs under such conditions. This has led to the applications of SERS in industry and in basic research. In this study, silver and gold nanospheres of average size 20 nm were successfully synthesised and characterised using UV-Vis (Ultraviolet-visible) spectroscopy and Transmission Electron Microscopy (TEM). Two RS setups were available, a double stage Raman spectrometer using 514.5 nm Ar+ laser as excitation source and a single stage Raman spectrometer using 532 nm frequency doubled Nd:YAG laser as excitation source. The synthesised silver nanospheres were employed in SERS studies on biomolecules (amino acids) using the single stage Raman setup with the aim of advancing SERS as a bio-analytical tool using our in-house developed RS setup. Qualitative analysis was done on amino acid spectra by band profiling and quantitative analysis was performed by carrying out concentration studies so as to determine the detection limit of the measuring instrument. Results are explained based on the setup used and by comparing with what is expected from literature. It was found that amino acids mostly adsorb on a metallic surface via the common carboxylate, amine and R-groups. This is due to the availability of free electron pairs on the oxygen and nitrogen atoms which take part in charge transfer mechanisms and promote chemical enhancement. It was also observed that some amino acids have functional groups which either have strong affinity for metals or have an electronic structure that contribute to chemical enhancement, thus boosting the Raman signal. A low detection limit of 1x10-4 M from amino acid L-Lysine was obtained. Ultimately, these results are new and provide a set of measurements done on four groups of amino acids using gold and two types of silver nanoparticles. These results form a foundation for future studies on larger biological organisations using the setup available in our labs.
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    Vibrational spectroscopy for characterisation of a cyclic antimicrobial peptide
    (Stellenbosch : Stellenbosch University, 2021-12) Pfukwa, Ngaatendwe Buhle Cathrine; Neethling, Pieter H.; Rautenbach, Marina; Parker, Anthony W.; Rohwer, Erich G.; Stellenbosch University. Faculty of Science. Dept. of Physics.
    ENGLISH ABSTRACT: In order to understand antimicrobial peptide action towards membrane surfaces it is imperative to first understand the structure and conformational behaviour of the antimicrobial in solution environments representative of the target membrane. Information on conformational behaviour can be extracted in more detail from analysis of the peptide secondary structure using the structural sensitivity afforded by two dimensional infrared (2D-IR) spectroscopy with which subtle details of peptide structure, which may not be well resolved with one dimensional infrared (1D-IR) spectroscopy, can be obtained. With 2D-IR spectroscopy spectral content is spread over two frequency axes and the high time resolution on femtosecond and picosecond timescales for transient processes provides an advantage over conventional techniques such as X-ray diffraction, circular dichroism (CD) or Ultraviolet spectroscopy. In this study the aims were to evaluate the adaptability of two-dimensional infrared (2D-IR) spectroscopy, together with multivariate data analysis methods such as principal component analysis (PCA), to provide and extract spectral details which can be correlated to changes in structure of a cationic antimicrobial peptide (CAMP) gramicidin S (GS). GS was selected as prototype as it is a well characterised anti-parallel β-sheet peptide, active by disrupting bacterial membranes, consequently causing membrane penetration. Furthermore, a qualitative working relationship between 1D and 2D IR and Raman spectroscopy in corroboration with quantum mechanical (QM) simulations was established towards correlating GS spectral features to conformational secondary structural changes. The structural changes were used to elucidate the conformational behaviour of GS in selected solvent environments which are representative of GS target membrane lipid bilayers. The solvent environments were an aqueous ubiquitous environment (H2O/D2O), a membrane mimetic partially polar environment (1-octanol) and a membrane mimetic strongly H-bonded environment (TFE). Self-association of GS was promoted in H2O/D2O and 1-octanol forming aggregates which persisted in solution at increasing peptide concentration, as revealed from increase in the β-sheet content and loss in β-turns. While in TFE, a strong H-bonding solvent, the GS aggregates were molecularly solvated. These changes were correlated to occur in the residues which contribute greatly to the amphiphilic nature of GS. Further solvent effects on the structural factors contributing to GS bioactivity are discussed. The thermostability of GS was investigated for the temperature range 20-80 ℃. The dissociation of GS aggregates/small oligomers to monomeric structures in 1-octanol was favoured at elevated temperatures. Thermodynamic parameters were extracted and the dissociation process was described by a bimodal profile with two identified melting transitions at Tm1 = 45 oC and Tm2 = 57 oC. Results obtained confirmed that both IR and Raman can provide complementary results as evidenced by their sensitivity towards hydrophilic and hydrophobic structures is GS. Further providing insight towards the type of residue substituents which can be interchanged in future synthesis of GS derivatives, with the aim of increasing the bioactivity of GS whilst lowering its haemolytic effects. The significance of SERS using silver nanoparticles in detection of low peptide concentrations is reported and a GS concentration of 1x10-5 M was detected. Information in thesis demonstrates the novelty and broad use of 1D and 2D IR, RS and SERS techniques in understanding the solvent and temperature induced conformational changes which occur in GS, which provides information on GS structure towards generation of more bioactive GS derivatives for therapeutic purposes.