Biomedical subjects
Publications and source records attributed to Karen Faulds.
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.
New methods of measuring biologically relevant concentrations of enzymes are necessary to allow greater understanding of biological systems. We have previously shown that aryl azo benzotriazolyl alkyl esters can act as enzyme substrates, with the progress of the reaction being monitored using SERRS (see Nat. Biotechnol., 2004, 22, 1133, ref. ). This is a wholly novel analytical application of SERRS, and the low detection levels of the technique allow for an ultra-sensitive enzyme assay. Masked enzyme substrates are used that are invisible to SERRS until enzymatic hydrolysis. Turnover of the substrate by the enzyme leads to the release of the surface-seeking dye necessary for SERRS, and intense signals are produced. Here we report an improved synthesis of 2H-benzotriazolyl alkyl esters via nucleophilic substitution of a chloromethyl ester by benzotriazolyl azo dyes, giving up to a ten-fold increase on previously reported yields. Introduction of electron-withdrawing groups to the benzotriazole ring allows control over the SERRS properties of the compounds. This is of great significance in expanding the synthetic flexibility and subsequently the fundamental use of these compounds as ultra-sensitive and selective reporters of enzyme activity.
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.
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.
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 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.
The labeling of biological species using dyes has become common practice to aid in their detection, and immediate positive identification of specific dyes in high dilution is a key requirement. Here the detection by surface-enhanced resonance Raman scattering (SERRS) of eight commercially available dye labels (ROX, rhodamine 6G, HEX, FAM, TET, Cy3, Cy5, TAMRA) attached to oligonucleotide strands is reported. Each of the eight labels was easily detected by using the SERRS from silver nanoparticles to produce a unique, molecularly specific spectrum. The conditions were optimized to obtain the best signal enhancement, and linear concentration graphs at low oligonucleotide concentrations were obtained. At higher concentrations (above approximately 10(-)(8) mol dm(-)(3)), curvature was introduced into the concentration graphs with the exception of rhodamine 6G, TET, and FAM, which gave linearity over the entire concentration range studied. Detection limits as low as 0.5 fmol were obtained, with lower possible if a smaller sample was analyzed. Investigation was also carried out into the effect of a Tris-HCl buffer containing the surfactant Tween 20 to aid in the prevention of surface adhesion of the oligonucleotides to the sample vessels at ultralow concentrations. The Tween 20 allowed lower detection limits to be obtained for each of the labels studied. This study shows that the different dyes commonly used with oligonucleotides can give quantitative SERRS at concentration levels not possible when the same dyes are used with fluorescence detection.
The synthesis of seven monoazo benzotriazole dyes for use in surface enhanced resonance Raman scattering, SERRS, is reported. The dyes are all capable of complexing to the silver surface used to provide the surface enhancement found in SERRS and hence act as 'model' analytes. One dye was examined in detail and showed a quantitative relationship between concentration and signal intensity.