R.E. Littleford

Practical control of SERRS enhancement

The demonstration that quantitative and sensitive analysis can be carried out using surface enhanced resonance Raman scattering (SERRS) prompted a discussion and investigation of the main variables which are within the control of the analyst using colloidal silver as the substrate. Previous papers have dealt with the crucial need to obtain good chemisorption of the analyte to the surface and have reported the use of specially designed dyes for SERRS. One of the most variable processes is the aggregation of the colloid. Here, we investigate the addition of controlled amounts of an organic aggregating agent, poly-L-lysine, at concentrations which reduce the zeta potential in a controlled manner, thus aiding aggregation control. The relationship between the excitation frequency, the surface plasmon resonance frequency of the silver colloid and the frequency of the maximum absorbance of the molecular chromophore is studied using low concentrations of dye and no aggregating agent. Under these conditions, little to no aggregation is expected. The magnitude of the enhancement is strongly dependent on the frequency of the molecular chromophore as well as the plasmon resonance frequency. However, when sodium chloride is used to aggregate the colloid, a larger enhancement is obtained and the strong dependence on the molecular chromophore largely disappears. A much broader enhancement profile is obtained which appears to be related more to the specific enhancement processes caused by aggregation than the frequency of the chromophore. However, the total enhancement for SERRS is higher than for SERS thus indicating that the chromophore is still important to the process.

Novel haem co-ordination variants of flavocytochrome P450 BM3

Bacillus megaterium flavocytochrome P450 BM3 is a catalytically self-sufficient fatty acid hydroxylase formed by fusion of soluble NADPH-cytochrome P450 reductase and P450 domains. Selected mutations at residue 264 in the haem (P450) domain of the enzyme lead to novel amino acid sixth (distal) co-ordination ligands to the haem iron. The catalytic, spectroscopic and thermodynamic properties of the A264M, A264Q and A264C variants were determined in both the intact flavocytochromes and haem domains of P450 BM3. Crystal structures of the mutant haem domains demonstrate axial ligation of P450 haem iron by methionine and glutamine ligands trans to the cysteine thiolate, creating novel haem iron ligand sets in the A264M/Q variants. In contrast, the crystal structure of the A264C variant reveals no direct interaction between the introduced cysteine side chain and the haem, although EPR data indicate Cys(264) interactions with haem iron in solution. The A264M haem potential is elevated by comparison with wild-type haem domain, and substrate binding to the A264Q haem domain results in a approximately 360 mV increase in potential. All mutant haem domains occupy the conformation adopted by the substrate-bound form of wild-type BM3, despite the absence of added substrate. The A264M mutant (which has higher dodecanoate affinity than wild-type BM3) co-purifies with a structurally resolved lipid. These data demonstrate that a single mutation at Ala(264) is enough to perturb the conformational equilibrium between substrate-free and substrate-bound P450 BM3, and provide firm structural and spectroscopic data for novel haem iron ligand sets unprecedented in nature.

In-situ generation and analysis of charge transfer materials using an OTTLE cell and resonance Raman scatteringElectronic supplementary information (ESI) available: BPW91/6-31G(d) predicted vibrational frequencies and intensities for the neutral, radical cation and dication species of 1 and 2. See http://www.rsc.org/suppdata/cp/b4/b402015d/

Poly(aryl)amine based charge transfer materials (CTMs) are essential components in a range of present and future technologies, from the Xerox process to display devices based upon light emitting polymers (LEPs). However, there is a lack of detailed understanding regarding the electronic properties of CTMs in their various neutral and oxidized forms. This paper reports the use of an optically transparent thin layer electrochemical (OTTLE) cell in combination with a Raman microprobe system and DFT calculations to provide information on the molecular and electronic structure of the mono- and di-oxidized derivatives of the classic CTM N,N′-diphenyl-N,N′-bis(3-methylphenyl)(1,1′-biphenyl)-4,4′-diamine (TPD) and the closely related species N,N′-diphenyl-N,N′-bis(2,4-dimethylphenyl)(1,1′-biphenyl)-4,4′-diamine (DMTPD). The resonance Raman scattering profile easily discriminates between the monovalent and divalent cations while DFT calculations permit correlation of the observed vibrational frequencies with localized atomic displacements. The cations are best described in terms of a symmetrical (i.e. fully delocalized) structure. The high sensitivity of the method suggests that it should be appropriate for the observation of low concentrations of the various cations generated from TPD type CTMs during device operation.

Novel heme coordination variants of flavocytochrome P450 BM3

Bacillus megaterium flavocytochrome P450 BM3 is a catalytically self-sufficient fatty acid hydroxylase formed by fusion of soluble NADPH-cytochrome P450 reductase and P450 domains. Selected mutations at residue 264 in the haem (P450) domain of the enzyme lead to novel amino acid sixth (distal) co-ordination ligands to the haem iron. The catalytic, spectroscopic and thermodynamic properties of the A264M, A264Q and A264C variants were determined in both the intact flavocytochromes and haem domains of P450 BM3. Crystal structures of the mutant haem domains demonstrate axial ligation of P450 haem iron by methionine and glutamine ligands trans to the cysteine thiolate, creating novel haem iron ligand sets in the A264M/Q variants. In contrast, the crystal structure of the A264C variant reveals no direct interaction between the introduced cysteine side chain and the haem, although EPR data indicate Cys(264) interactions with haem iron in solution. The A264M haem potential is elevated by comparison with wild-type haem domain, and substrate binding to the A264Q haem domain results in a approximately 360 mV increase in potential. All mutant haem domains occupy the conformation adopted by the substrate-bound form of wild-type BM3, despite the absence of added substrate. The A264M mutant (which has higher dodecanoate affinity than wild-type BM3) co-purifies with a structurally resolved lipid. These data demonstrate that a single mutation at Ala(264) is enough to perturb the conformational equilibrium between substrate-free and substrate-bound P450 BM3, and provide firm structural and spectroscopic data for novel haem iron ligand sets unprecedented in nature.

RAMAN SPECTROSCOPY | Surface-Enhanced

Surface enhanced Raman scattering (SERS) can provide high sensitivity detection of an analyte, specific identification of the analyte in situ in a matrix and identification of several analytes in a solution without separation. The technique uses standard Raman spectrometers. It requires that the analyte is adsorbed on or close to a suitable roughened surface usually of silver or gold. Labeled nanoparticles and designed substrates have both been used successfully. Analysis using SERS detection requires that the experiment is correctly designed and this article outlines the basic theory and the practical requirements for successful analytical procedures to be developed.

SERRS multiplexing from labelled oligonucleotides in a microfluidics lab-on-a-chip

The growing need for accurate and fast methods of DNA detection in the post genomic era has generated the development of a number of new platforms for sample analysis. Two of the most popular approaches have been the use of microarrays of immobilized probes and microfluidics chips. Both approaches require a detection technique to be used with the platform which has a high enough sensitivity for such small analyte volumes. The techniques of choice tend to be mass spectrometry or fluorescence spectroscopy. Here we propose SERRS as an alternative spectroscopy for detection. SERRS and fluorescence have comparable sensitivities but one main advantage of SERRS is that the peaks in the spectra are much narrower and are more easily resolved. This, in principle, opens up the potential for the simultaneous detection of multiple labels to a degree not possible with fluorescence, thus providing an enabling technology for the more complex assays required since the completion of the human genome map. Also, when using SERRS both fluorophores and nonfluorophores are suitable so that a more extensive and simpler labelling chemistry can be employed.