W. Ewen Smith

Control of enhanced Raman scattering using a DNA-based assembly process of dye-coded nanoparticles

Enhanced Raman scattering from metal surfaces has been investigated for over 30 years. Silver surfaces are known to produce a large effect, and this can be maximized by producing a roughened surface, which can be achieved by the aggregation of silver nanoparticles. However, an approach to control this aggregation, in particular through the interaction of biological molecules such as DNA, has not been reported. Here we show the selective turning on of the surface enhanced resonance Raman scattering effect on dye-coded, DNA-functionalized, silver nanoparticles through a target-dependent, sequence-specific DNA hybridization assembly that exploits the electromagnetic enhancement mechanism for the scattering. Dye-coded nanoparticles that do not undergo hybridization experience no enhancement and hence do not give surface enhanced resonance Raman scattering. This is due to the massive difference in enhancement from nanoparticle assemblies compared with individual nanoparticles. The electromagnetic enhancement is the dominant effect and, coupled with an understanding of the surface chemistry, allows surface enhanced resonance Raman scattering nanosensors to be designed based on a natural biological recognition process.

Surface-enhanced raman scattering (SERS) and surface-enhanced resonance raman scattering (SERRS): a review of applications

Surface-enhanced Raman scattering (SERS) and surface-enhanced resonance Raman scattering (SERRS) can provide positive identification of an analyte or an analyte mixture with high sensitivity and selectivity. Better understanding of the theory and advances in the understanding of the practice have led to the development of practical applications in which the unique advantages of SERS/SERRS have been used to provide effective solutions to difficult analytical problems. This review presents a basic theory and illustrates the way in which SERS/SERRS has been developed for practical use.

Phthalocyanines : structure and vibrations

Raman scattering for a range of metal phthalocyanines using excitation frequencies from 457.9 to 1064 nm have been interpreted in the light of the recent DFT calculation on zinc phthalocyanine. The intention was to determine the features of the spectra that might be used for in situ analysis of specific phthalocyanines. B1 bands were found to be the most intense. The frequency of one band with a large C–N–C ring displacement is particularly sensitive to the metal ion size. The effect is determined not only by the size of the ion but also by the effect it has on ring shape. First and second order overtone bands of zinc and copper phthalocyanines are broadly similar, with some coupling differences at about 2000 cm−1. The region between 1350 and 1550 cm−1 has been little studied previously. It shows a remarkable sensitivity to the metal ion present and provides a specific signature for each phthalocyanine studied. In contrast, a study of α-, β-, γ- and e-copper phthalocyanines using 514.5 nm excitation showed very few differences, suggesting that intra- rather than intermolecular markers are most efficiently determined by Raman scattering. The study enables the interpretation of the Raman spectra of the phthalocyanines in terms of molecular structure and due to the resonant enhancement involved will enable the in situ identification of specific phthalocyanines in matrices.

Enhanced oligonucleotide–nanoparticle conjugate stability using thioctic acid modified oligonucleotides

Metallic nanoparticles of gold functionalized with oligonucleotides conventionally use a terminal thiol modification and have been used in a wide range of applications. Although readily available, the oligonucleotide–nanoparticle conjugates prepared in this way suffer from a lack of stability when exposed to a variety of small molecules or elevated temperatures. If silver is used in place of gold then this lack of stability is even more pronounced. In this study we report the synthesis of highly stabilized oligonucleotide–nanoparticle conjugates using a simple oligonucleotide modification. A modified solid support was used to generate 3′-thioctic acid modified oligonucleotides by treatment with an N-hydroxysuccimidyl ester of thioctic acid. Unusually, both gold and silver nanoparticles have been investigated in this study and show that these disulphide-modified oligonucleotide probes offer significant improvements in nanoparticle stability when treated with dithiothreitol (DTT) compared with monothiol analogues. This is a significant advance in oligonucleotide–nanoparticle conjugate stability and for the first time allows silver nanoparticles to be prepared that are more stable than standard gold-thiol functionalized nanoparticles. This opens up the possibility of using silver nanoparticles functionalized with oligonucleotides as an alternative to gold.

Multiplexed detection of six labelled oligonucleotides using surface enhanced resonance Raman scattering (SERRS).

The labelling of target biomolecules followed by detection using some form of optical spectroscopy has become common practice to aid in their detection. This approach has allowed the field of bioanalysis to dramatically expand; however, most methods suffer from the lack of the ability to discriminate between the components of a complex mixture. Currently, fluorescence spectroscopy is the method of choice but its ability to multiplex is greatly hampered by the broad overlapping spectra which are obtained. Surface enhanced resonance Raman scattering (SERRS) holds many advantages over fluorescence both in sensitivity and, more importantly here, in its ability to identify components in a mixture without separation due to the sharp fingerprint spectra obtained. Here the first multiplexed simultaneous detection of six different DNA sequences, corresponding to different strains of the Escherichia coli bacterium, each labelled with a different commercially available dye label (ROX, HEX, FAM, TET, Cy3, or TAMRA) is reported. This was achieved with the aid of multivariate analysis, also known as chemometrics, which can involve the application of a wide range of statistical and data analysis methods. In this study, both exploratory discriminant analysis and supervised learning, by partial least squares (PLS) regression, were used and the ability to discriminate whether a particular labelled oligonucleotide was present or absent in a mixture was achieved using PLS with very high sensitivity (0.98-1), specificity (0.98-1), accuracy (range 0.99-1), and precision (0.98-1).

Free radical pathology in chronic arterial disease

The generation of toxic oxygen metabolites is more usually associated with inflammation. However, pathological free radical reactions can cause tissue damage by adversely affecting prostacyclin (PGI2) synthesis allowing initiation of coagulation. We have assessed changes in the red cell defence to toxic oxygen metabolite generation, viz measurement of glutathione concentration (GSH) and superoxide dismutase activity (SOD). GSH and SOD were measured in 20 patients with peripheral arterial disease, 22 patients with vasculitis, and 11 patients with angina, and compared to 17 matched controls. The 53 subjects with arterial disease had significantly lower SOD levels: in contrast GSH levels were significantly higher. Extracellularly plasma thiol levels (PSH) were low and caeruloplasmin (Cp) levels were high. We suggest that free radical pathology exists not only in inflammatory vascular disease but also in atherosclerosis.

A theoretical study of the structure and vibrations of 2,4,6-trinitrotolune

Abstract Theoretical calculations of the structure, internal rotations and vibrations of 2,4,6-trinitrotolune, TNT, in the gas phase were performed at the B3LYP/6-31G* and B3LYP/6-311+G** levels of theory. Two genuine energy minimum structures were found. In both structures the 4-nitro group is planar to the phenyl ring, while the 2,6-nitro groups are slightly out of plane with the phenyl ring due to steric interaction with the methyl group. The two structures are related by internal rotations of the methyl and 2, or 6-nitro group. The lowest energy route for interconversion between them is a concerted motion of the methyl group and 2 or 6 nitro group in a ‘cog wheel’ type of mechanism. The geometry of the low energy structure A is closest to that observed in the crystal structures of TNT, where all three nitro groups are out of plane with the phenyl ring. FTIR and Raman spectra of solid TNT and 13C, 15N enriched TNT are presented and assigned with the help of the B3LYP/6-311+G** calculations on A. The lower level B3LYP/6-31G* calculation fails to predict the correct vibrational coupling between the nitro and phenyl groups. The B3LYP/6-311+G** calculation gives a good prediction of the nitro vibrations and the isotopic shifts observed for TNT isotopomers.

Chromophore containing bipyridyl ligands. Part 1: supramolecular solid-state structure of Ag(I) complexes

The solid-state structures of a series of azine or azo chromophore containing bipyridyl ligand complexes of Ag(I) salts have been determined by single-crystal X-ray diffraction. The supramolecular structures are dominated by one-dimensional chains formed through pyridyl–Ag–pyridyl bonding, but the packing of these chains through non-covalent intermolecular interactions is unpredictable. Ag⋯anion interactions are shown to be important, especially for nitrate and perchlorate species, but these may be supported or replaced by Ag⋯Ag, Ag⋯solvent, Ag⋯azine or Ag⋯ π contacts. The molecular structures of the ligands show little alteration on complex formation, except for the AgNO3 complex of N,N′-bis-pyridin-4-ylmethylene-hydrazine where the normally planar azine ligand adopts a twisted geometry.

A DFT analysis of the vibrational spectra of nitrobenzene

Abstract Raman and FTIR, spectra of nitrobenzene, nb, and its isotopomers, nb- 15 N, nb- 13 C 6 and nb- d 5 , were obtained and the fundamental vibrational modes assigned with the aid of a B3LYP/6-311+G** calculation, without the need for scaling of the force constants. The changes in vibrational coupling between the nitro and benzene groups upon certain isotopic substitutions are well modelled by the calculation, which is able to reproduce the isotopic shifts in frequencies for the nitro vibrations, as well as changes in IR intensities.

Characterization of active site structure in CYP121. A cytochrome P450 essential for viability of Mycobacterium tuberculosis H37Rv.

Mycobacterium tuberculosis (Mtb) cytochrome P450 gene CYP121 is shown to be essential for viability of the bacterium in vitro by gene knock-out with complementation. Production of CYP121 protein in Mtb cells is demonstrated. Minimum inhibitory concentration values for azole drugs against Mtb H37Rv were determined, the rank order of which correlated well with Kd values for their binding to CYP121. Solution-state spectroscopic, kinetic, and thermodynamic studies and crystal structure determination for a series of CYP121 active site mutants provide further insights into structure and biophysical features of the enzyme. Pro346 was shown to control heme cofactor conformation, whereas Arg386 is a critical determinant of heme potential, with an unprecedented 280-mV increase in heme iron redox potential in a R386L mutant. A homologous Mtb redox partner system was reconstituted and transported electrons faster to CYP121 R386L than to wild type CYP121. Heme potential was not perturbed in a F338H mutant, suggesting that a proposed P450 superfamily-wide role for the phylogenetically conserved phenylalanine in heme thermodynamic regulation is unlikely. Collectively, data point to an important cellular role for CYP121 and highlight its potential as a novel Mtb drug target.