SERS of meso-droplets supported on superhydrophobic wires allows exquisitely sensitive detection of dipicolinic acid, an anthrax biomarker, considerably below the infective dose.

Surface-enhanced Raman measurements of <1 µL analyte/colloid meso-droplets on superhydrophobic wires with hydrophilic tips allowed dipicolinic acid, a spore biomarker for Bacillus anthracis (anthrax), to be detected at 10(-6) mol dm(-3). This is equivalent to 18 spores, significantly below the infective dose of 10(4) spores and 2 orders of magnitude better than previous measurements.

Detection of Protein Glycosylation Using Tip-Enhanced Raman Scattering.

The correct glycosylation of biopharmaceutical glycoproteins and their formulations is essential for them to have the desired therapeutic effect on the patient. It has recently been shown that Raman spectroscopy can be used to quantify the proportion of glycosylated protein from mixtures of native and glycosylated forms of bovine pancreatic ribonuclease (RNase). Here we show the first steps toward not only the detection of glycosylation status but the characterization of glycans themselves from just a few protein molecules at a time using tip-enhanced Raman scattering (TERS). While this technique generates complex data that are very dependent on the protein orientation, with the careful development of combined data preprocessing, univariate and multivariate analysis techniques, we have shown that we can distinguish between the native and glycosylated forms of RNase. Many glycoproteins contain populations of subtly different glycoforms; therefore, with stricter orientation control, we believe this has the potential to lead to further glycan characterization using TERS, which would have use in biopharmaceutical synthesis and formulation research.

Quantitative online liquid chromatography-surface-enhanced Raman scattering of purine bases.

Raman spectroscopy has been of interest as a detection method for liquid chromatographic separations for a significant period of time, due to the structural information it can provide, allowing the identification and distinction of coeluting analytes. Combined with the rapidly advancing field of enhanced Raman techniques, such as surface-enhanced Raman scattering (SERS), the previous low sensitivity of Raman measurements has also been alleviated. At-line LC-SERS analyses, where SERS measurements are taken of fractions collected during or after HPLC separation have been shown to be sensitive and applicable to a wide variety of analytes; however, quantitative, real-time, online LC-SERS analysis at comparable sensitivity to existing methods, applicable to high-throughput experiments, has not been previously demonstrated. Here we show that by introducing silver colloid, followed by an aggregating agent into the postcolumn flow of an HPLC system, we can quantitatively and reproducibly analyze mixtures of purine bases, with limits of detection in the region of 100-500 pmol. The analysis is performed without the use of a flow cell, thereby eliminating previously detrimental memory effects.

Electrochemical modulation of SERS at the liquid/liquid interface.

A surface enhanced Raman scattering system to detect silver nanoparticle adsorption at the water|1,2-dichlorobenzene interface is reported. The Raman response as a function of distance on either side of the interface reveals a reproducible spatial variation, which is potential dependent for a number of adsorption and desorption cycles.

The challenge of applying Raman spectroscopy to monitor recombinant antibody production.

UV resonance Raman (UVRR) spectroscopy combined with chemometric techniques was investigated as a physiochemical tool for monitoring secreted recombinant antibody production in cultures of Chinese hamster ovary (CHO) cells. Due to the enhanced selectivity of the UVRR, spectral variations arising from protein, small molecule substrates, and nucleic acid medium components could be measured simultaneously and we have successfully determined antibody titre. Medium samples were taken during culture of three CHO cell lines: two antibody-producing cell lines and a non-producing cell line, and analysed by UVRR spectroscopy using an excitation laser of 244 nm. Principal component analysis (PCA) was applied to the spectral sets and showed a linear trend over time for the antibody-producing cell lines that was not observed in the non-producing cell line. Partial least squares regression (PLSR) was used to predict antibody titres, glucose utilization and lactate accumulation, and compared very favourably with gold standard data acquired with the much slower techniques of ELISA and liquid chromatography. Further analysis of the UVRR spectral sets using two-dimensional correlation moving windows also revealed that spectral variations due to protein and nucleic acid concentrations in the medium during cell culture varied between each of the three cell lines investigated.

Illuminating disease and enlightening biomedicine: Raman spectroscopy as a diagnostic tool.

The discovery of the Raman effect in 1928 not only aided fundamental understanding about the quantum nature of light and matter but also opened up a completely novel area of optics and spectroscopic research that is accelerating at a greater rate during the last decade than at any time since its inception. This introductory overview focuses on some of the most recent developments within this exciting field and how this has enabled and enhanced disease diagnosis and biomedical applications. We highlight a small number of stimulating high-impact studies in imaging, endoscopy, stem cell research, and other recent developments such as spatially offset Raman scattering amongst others. We hope this stimulates further interest in this already exciting field, by 'illuminating' some of the current research being undertaken by the latest in a very long line of dedicated experimentalists interested in the properties and potential beneficial applications of light.

Portable, quantitative detection of Bacillus bacterial spores using surface-enhanced Raman scattering.

Portable rapid detection of pathogenic bacteria such as Bacillus is highly desirable for safety in food manufacture and under the current heightened risk of biological terrorism. Surface-enhanced Raman scattering (SERS) is becoming the preferred analytical technique for bacterial detection, due to its speed of analysis and high sensitivity. However in seeking methods offering the lowest limits of detection, the current research has tended toward highly confocal, microscopy-based analysis, which requires somewhat bulky instrumentation and precisely synthesized SERS substrates. By contrast, in this study we have improved SERS for bacterial analyses using silver colloidal substrates, which are easily and cheaply synthesized in bulk, and which we shall demonstrate permit analysis using portable instrumentation. All analyses were conducted in triplicate to assess the reproducibility of this approach, which was excellent. We demonstrate that SERS is able to detect and quantify rapidly the dipicolinate (DPA) biomarker for Bacillus spores at 5 ppb (29.9 nM) levels which are significantly lower than those previously reported for SERS and well below the infective dose of 10(4)B. anthracis cells for inhalation anthrax. Finally we show the potential of multivariate data analysis to improve detection levels in complex DPA extracts from viable spores.

Optimization of parameters for the quantitative surface-enhanced Raman scattering detection of mephedrone using a fractional factorial design and a portable Raman spectrometer.

A new optimization strategy for the SERS detection of mephedrone using a portable Raman system has been developed. A fractional factorial design was employed, and the number of statistically significant experiments (288) was greatly reduced from the actual total number of experiments (1722), which minimized the workload while maintaining the statistical integrity of the results. A number of conditions were explored in relation to mephedrone SERS signal optimization including the type of nanoparticle, pH, and aggregating agents (salts). Through exercising this design, it was possible to derive the significance of each of the individual variables, and we discovered four optimized SERS protocols for which the reproducibility of the SERS signal and the limit of detection (LOD) of mephedrone were established. Using traditional nanoparticles with a combination of salts and pHs, it was shown that the relative standard deviations of mephedrone-specific Raman peaks were as low as 0.51%, and the LOD was estimated to be around 1.6 µg/mL (9.06 × 10(-6) M), a detection limit well beyond the scope of conventional Raman and extremely low for an analytical method optimized for quick and uncomplicated in-field use.