Biomedical subjects
Publications and source records attributed to W Ewen Smith.
Silver nanoparticles can be used to provide excellent surface enhanced resonance Raman scattering. Control of the surface chemistry and the use of appropriate protocols enables effective sensing of biomolecules.
Effective correlation of data from a number of analytical techniques over length scales spanning several orders of magnitude is required to more fully investigate the active sites on silver nanoparticles that are responsible for surface-enhanced resonance Raman scattering (SERRS). In this paper, a method is presented that uses fluorescent beads as optical markers to allow direct correlation between a SERRS/fluorescence map and a transmission electron microscope (TEM) collage of the same area. Factors influencing the accuracy of the technique include the flatness of the substrate, the size of the fluorescent beads, and the strength of the signal from the fluorescent beads. When the effect of each of these factors on the technique is addressed, a simple and accurate correlation between the optical spectroscopy and the electron microscopy is achieved. A statistically significant number of particles can then be easily and reliably located and characterized at both optical limits, by SERRS, and with subnanometer resolution in the high-resolution TEM. Examples of HRTEM images and the locations of these particles within the SERRS map/TEM collage are presented. Our findings reveal that the relative SERRS activity of single particles is very low compared to dimers and larger aggregates of particles. The relative activity of dimers is estimated to be 12.4 times greater than single particles, and as the number of particles in the aggregate increase, the relative SERRS activity also increases. The relative SERRS activities of single particles/dimers/trimers/aggregates of 4-9 particles/aggregates of 10-20 are estimated to be 1/12.4/15.6/23.2/43.
A new approach for the detection of DNA using surface enhance resonance Raman scattering (SERRS) is reported. The majority of existing techniques use fluorescence spectroscopy with advanced probe design to provide information on the identity of specific DNA sequences down to single base resolution. A new approach to the labelling of DNA is discussed which uses Michael addition to couple thiolated DNA to dye labels specifically designed to attach to silver surfaces. When combined with existing strategies for sensitive detection of DNA using commercially available labels, a new class of biomolecular probe known as a SERRS Beacon was produced. The detection techniques of fluorescence and surface enhanced resonance Raman scattering (SERRS) are combined to give a sensitive and selective system for use in the development and creation of novel assays for specifically defined targets. It demonstrates improved potential for multiplexing analysis.
A number of silver particles and aggregates of particles were studied using surface enhanced resonance Raman spectroscopy (SERRS), high resolution transmission electron microscopy (HRTEM) and electron energy-loss spectroscopy (EELS). The SERRS mapping/TEM collage method developed previously in our group allows each SERRS active or inactive species to be reliably identified and analysed by each of the techniques in three different instruments. Our aim is to correlate SERRS activity, particle microstructure, chemical composition and electronic properties of each species to gain an insight into the enhancement mechanism. To date, our findings do not reveal any clear link between particle microstructure and SERRS activity. Additionally, the direction of the polarisation of the incident excitation or the presence of interparticle junctions between aggregated particles was not correlated with SERRS activity. However, spectral variations in the EELS data from structurally similar particles and SERRS active and inactive particles suggest that each species is chemically/electronically distinct. Differences in the spectra of single particles, dimers and clusters were also observed. Further analysis of the data, including extraction of the complex dielectric function from the EELS data, will provide an insight into the relationship between these observations and SERRS activity.
This Education article outlines the different ways in which surface enhanced resonance Raman scattering (SERRS) can be used for the detection of DNA. The use of various different SERRS detection strategies that have allowed both sensitive and selective detection to be obtained is covered. Detection of DNA by SERRS involves the use of a dye with the DNA, whether as an intercalator or by direct covalent attachment. This generates strong SERRS signals that indicate the presence of the specific DNA sequence. The SERRS detection of DNA in different molecular biological assays is also discussed.
Comparatively few studies have explored the ability of Raman spectroscopy for the quantitative analysis of microbial secondary metabolites in fermentation broths. In this study we investigated the ability of Raman spectroscopy to differentiate between different penicillins and to quantify the level of penicillin in fermentation broths. However, the Raman signal is rather weak, therefore the Raman signal was enhanced using surface enhanced Raman spectroscopy (SERS) employing silver colloids. It was difficult by eye to differentiate between the five different penicillin molecules studied using Raman and SERS spectra, therefore the spectra were analysed by multivariate cluster analysis. Principal components analysis (PCA) clearly showed that SERS rather than the Raman spectra produced reproducible enough spectra to allow for the recovery of each of the different penicillins into their respective five groups. To highlight this further the first five principal components were used to construct a dendrogram using agglomerative clustering, and this again clearly showed that SERS can be used to identify which penicillin molecule was being analysed, despite their molecular similarities. With respect to the quantification of penicillin G it was shown that Raman spectroscopy could be used to quantify the amount of penicillin present in solution when relatively high levels of penicillin were analysed (>50 mM). By contrast, the SERS spectra showed reduced fluorescence, and improved signal to noise ratios from considerably lower concentrations of the antibiotic. This could prove to be advantageous in industry for monitoring low levels of penicillin in the early stages of antibiotic production. In addition, SERS may have advantages for quantifying low levels of high value, low yield, secondary metabolites in microbial processes.
The surface-enhanced resonance Raman scattering (SERRS) activity of a statistically significant number of silver nanoparticles has been studied using a correlated SERRS mapping and transmission electron microscopy (TEM) method. TEM allowed the nature of each entity to be directly identified, and the SERRS activity was obtained from the corresponding SERRS map. Particles in various states of aggregation were analyzed to establish relative activities. It was established that SERRS activity is dependent on the specific batch of colloid tested. By averaging different colloid batches, it was shown that increasing SERRS activity is observed with increasing numbers of particles in the aggregates. By reducing the surface coverage of the particles to the extent that single moieties could be examined optically, the ratio of the relative activities of single particles, dimers, trimers, and larger aggregates was estimated. High-resolution TEM images of a number of active and inactive particles are reported. However, no clear correlation between microstructure and SERRS activity was observed.
A commercially available fluorimeter with a white light source is used to detect surface enhanced resonance Raman scattering (SERRS). This approach allows facile tunability of the excitation source for SERRS.
The interaction of nitric oxide with cytochrome P450 BM3 from Bacillus megaterium has been analyzed by spectroscopic techniques and enzyme assays. Nitric oxide ligates tightly to the ferric heme iron, inducing large changes in each of the main visible bands of the heme and inhibiting the fatty acid hydroxylase function of the protein. However, the ferrous adduct is unstable under aerobic conditions, and activity recovers rapidly after addition of NADPH to the flavocytochrome due to reduction of the heme via the reductase domain and displacement of the ligand. The visible spectral properties revert to that of the oxidized resting form. Aerobic reduction of the nitrosyl complex of the BM3 holoenzyme or heme domain by sodium dithionite also displaces the ligand. A single electron reduction destabilizes the ferric-nitrosyl complex such that nitric oxide is released directly, as shown by the trapping of released nitric oxide. Aerobically and in the absence of exogenous reductant, nitric oxide dissociates completely from the P450 over periods of several minutes. However, recovery of the nativelike visible spectrum is accompanied by alterations in the catalytic activity of the enzyme and changes in the resonance Raman spectrum. Specifically, resonance Raman spectroscopy identifies the presence of internally located nitrated tyrosine residue(s) following treatment with nitric oxide. Analysis of a Y51F mutant indicates that this is the major nitration target under these conditions. While wild-type P450 BM3 does not form an aerobically stable ferrous-nitrosyl complex, a site-directed mutant of P450 BM3 (F393H) does form an isolatable ferrous-nitrosyl complex, providing strong evidence for the role of this residue in controlling the electronic properties of the heme iron. We report here the spectroscopic characterization of the ferric- and ferrous-nitrosyl complexes of P450 BM3 and describe the use of resonance Raman spectroscopy to identify nitrated tyrosine residue(s) in the enzyme. Nitration of tyrosine in P450 BM3 may exemplify a typical mechanism by which the ubiquitous messenger molecule nitric oxide exerts a regulatory function over the cytochromes P450.
Cytochrome P450 BioI (CYP107H1) from Bacillus subtilis is involved in the early stages of biotin synthesis. Previous studies have indicated that BioI can hydroxylate fatty acids and may also perform an acyl bond cleavage reaction [Green, A. J., Rivers, S. L., Cheesman, M., Reid, G. A., Quaroni, L. G., Macdonald, I. D. G., Chapman, S. K., and Munro, A. W. (2001) J. Biol. Inorg. Chem. 6, 523-533. Stok, J. E., and De Voss, J. J. (2000) Arch. Biochem. Biophys. 384, 351-360]. Here we show novel binding features of P450 BioI--specifically that it binds steroids (including testosterone and progesterone) and polycyclic azole drugs with similar affinity to that for fatty acids (K(d) values in the range 0.1-160 microM). Sigmoidal binding curves for titration of BioI with azole drugs suggests a cooperative process in this case. BioI as isolated from Escherichia coli is in a mixed heme iron spin state. Alteration of the pH of the buffer system affects the heme iron spin-state equilibrium (higher pH increasing the low-spin content). Steroids containing a carbonyl group at the C(3) position induce a shift in heme iron spin-state equilibrium toward the low-spin form, whereas fatty acids produce a shift toward the high-spin form. Electron paramagnetic resonance (EPR) studies confirm the heme iron spin-state perturbation inferred from optical titrations with steroids and fatty acids. Potentiometric studies demonstrate that the heme iron reduction potential becomes progressively more positive as the proportion of high-spin heme iron increases (potential for low-spin BioI = -330 +/- 1 mV; for BioI as purified from E. coli (mixed-spin) = 228 +/- 2 mV; for palmitoleic acid-bound BioI = -199 +/- 2 mV). Extraction of bound substrate-like molecule from purified BioI indicates palmitic acid to be bound. Differential scanning calorimetry studies indicate that the BioI protein structure is stabilized by binding of steroids and bulky azole drugs, a result confirmed by resonance Raman studies and by analysis of disruption of BioI secondary and tertiary structure by the chaotrope guanidinium chloride. Molecular modeling of the BioI structure indicates that a disulfide bond is present between Cys250 and Cys275. Calorimetry shows that structural stability of the protein was altered by addition of the reductant dithiothreitol, suggesting that the disulfide is important to integrity of BioI structure.
The solid-state structures of 43 Li, Na, K, Rb, Mg, Ca and Ba salts of para- and meta-sulfonated azo dyes have been examined and can be categorised into three structural classes. All form alternating organic and inorganic layers, however, the nature of the coordination network that forms these layers differs from class to class. The class of structure formed was found to be primarily governed by metal type, but can also be influenced by the nature and position of the organic substituents. Thus, for the para-sulfonated azo dyes, Mg compounds form solvent-separated ion-pair solids; Ca, Ba and Li compounds form simple coordination networks based on metal-sulfonate bonding; and Na, K and Rb compounds form more complex, higher dimensional coordination networks. Compounds of meta-sulfonated azo dyes follow a similar pattern, but here, Ca species may also form solvent-separated ion-pair solids. Significantly, this first attempt to classify such dyestuffs using the principles of supramolecular chemistry succeeds not only for the simple dyes used here as model compounds, but also for more complex molecules, similar to modern colourants.
A combined surface enhanced resonance Raman scattering (SERRS) and transmission electron microscopy (TEM) method has been developed allowing the same immobilised nanoparticles to be reliably located and studied by both techniques. The method allows large numbers of particles to be analysed by each technique relatively simply and the distribution of Raman enhancement between particles and clusters, as well as the relationship between particle microstructure and Raman enhancement, to be investigated. In addition, the effect of chemical and laser damage on the dye on the surface of the particles and the effect on the particles can be systematically investigated. These effects can cause time dependence fluctuations in Raman signals which could be confused with "blinking" from single molecules. Conditions were identified to enable Raman scattering to be detected without photodegradation to either the analyte molecules or the particles. Measurement outside this range gave rise to alterations in the spectra and to loss of signal. The extent of the damage to the analyte/particle if these conditions are not adhered to gives rise for concern about interpretation of changes in spectra observed unless an attempt is made to assess the limits of the conditions which can be applied before photodegradation or sample drying occurs. The method developed will enable reliable and systematic studies of the enhancement obtained from immobilised single particles by enabling the full power of high resolution TEM to be utilised to aid the development of a reliable SERRS theory.
A series of eleven specially designed benzotriazole monoazo dyes for use in surface enhanced resonance Raman scattering studies are reported. Unlike previous benzotriazole dyes produced for SERRS, these dyes have been synthesised to be trifunctional in nature. The presence of the benzotriazole moiety provides surface complexing properties, the azo linkage provides the colour and hence extra sensitivity and the nucleophilic amine group enables further functionalisation.
This communication contains data from a comparison between the detection limits obtained using surface enhanced resonance Raman scattering (SERRS) and fluorescence detection of dye labelled oligonucleotides. The results show that the detection limits for SERRS are generally at least three orders of magnitude lower than those obtained for fluorescence.
The first use of Kerr gated fluorescence rejection Raman spectroscopy for the analysis of street samples of cocaine in a fluorescing matrix is reported.
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A conserved glutamate covalently attaches the heme to the protein backbone of eukaryotic CYP4 P450 enzymes. In the related Bacillus megaterium P450 BM3, the corresponding residue is Ala264. The A264E mutant was generated and characterized by kinetic and spectroscopic methods. A264E has an altered absorption spectrum compared with the wild-type enzyme (Soret maximum at approximately 420.5 nm). Fatty acid substrates produced an inhibitor-like spectral change, with the Soret band shifting to 426 nm. Optical titrations with long-chain fatty acids indicated higher affinity for A264E over the wild-type enzyme. The heme iron midpoint reduction potential in substrate-free A264E is more positive than that in wild-type P450 BM3 and was not changed upon substrate binding. EPR, resonance Raman, and magnetic CD spectroscopies indicated that A264E remains in the low-spin state upon substrate binding, unlike wild-type P450 BM3. EPR spectroscopy showed two major species in substrate-free A264E. The first has normal Cys-aqua iron ligation. The second resembles formate-ligated P450cam. Saturation with fatty acid increased the population of the latter species, suggesting that substrate forces on the glutamate to promote a Cys-Glu ligand set, present in lower amounts in the substrate-free enzyme. A novel charge-transfer transition in the near-infrared magnetic CD spectrum provides a spectroscopic signature characteristic of the new A264E heme iron ligation state. A264E retains oxygenase activity, despite glutamate coordination of the iron, indicating that structural rearrangements occur following heme iron reduction to allow dioxygen binding. Glutamate coordination of the heme iron is confirmed by structural studies of the A264E mutant (Joyce, M. G., Girvan, H. M., Munro, A. W., and Leys, D. (2004) J. Biol. Chem. 279, 23287-23293).
The effect of excitation frequency and state of aggregation on the sensitivity obtained in ultratrace analysis using colloidal suspensions of silver nanoparticles and surface-enhanced resonance Raman scattering (SERRS) detection is explored to define suitable conditions for quantitative analysis. Two structurally similar dyes, only one of which causes aggregation, were used as analytes without the use of external aggregating agents, thus simplifying the surface chemistry and removing a major source of error. Addition of the nonaggregating dye caused no change in particle charge or size and no time-dependent aggregation as measured by zeta potential and particle size analysis. The most intense single-particle scattering was obtained using excitation at the wavelength of the plasmon resonance. Molecular resonance added approximately 2 orders of magnitude in sensitivity. Addition of the aggregating dye caused a reduction in surface charge of the particles and initiated a time-dependent aggregation process. However, constant SERRS with time is obtained at some excitation wavelengths probably because a constant number of clusters active at these wavelengths is maintained in the dynamic aggregation process. The additional enhancement caused by aggregation and molecular resonance is spread over a range of excitation frequencies. However, electronic spectra suggested that plasmon resonance enhancement would be effective at the longest wavelength of excitation used (785 nm), but there was a significant drop in intensity this far away from the absorbance maximum of the dye (429 nm). Thus, sensitive analysis using suspensions of single nanoparticles is feasible provided the excitation frequency used is close to that of the plasmon resonance frequency. Aggregation adds only an enhancement of approximately 6 in the experiments performed since only some particles in aggregates will have an active plasmon at any one wavelength, but the range of excitation wavelengths at which good enhancement is obtained is wider giving more flexibility if more complexity.