Raman spectroscopy is attracting interest for the rapid identification of bacteria and fungi and is now becoming accepted as a potentially powerful whole-organism fingerprinting technique. However, the Raman effect is so weak that collection times are lengthy, and this insensitivity means that bacteria must be cultured to gain enough biomass, which therefore limits its usefulness in clinical laboratories where high-throughput analyses are needed. The Raman effect can fortunately be greatly enhanced (by some 10(3)-10(6)-fold) if the molecules are attached to, or microscopically close to, a suitably roughened surface; a technique known as surface-enhanced Raman scattering (SERS). In this study we investigated SERS, employing an aggregated silver colloid substrate, for the analysis of a closely related group of bacteria belonging to the genus Bacillus. Each spectrum took only 20 s to collect and highly reproducible data were generated. The multivariate statistical technique of principal components-discriminant function analysis (PC-DFA) was used to group these bacteria based on their SERS fingerprints. The resultant ordination plots showed that the SERS spectra were highly discriminatory and gave accurate identification at the strain level. In addition, Bacillus species also undergo sporulation, and we demonstrate that SERS peaks that could be attributed to the dipicolinic acid biomarker, could be readily generated from Bacillus spores.
We investigate the formation of poly(vinyl alcohol) microparticles by the selective extraction of aqueous polymer solution droplets, templated by microfluidics and subsequently immersed in a non-solvent bath. The role of polymer molecular mass (18-105 kg mol-1), degree of hydrolysis (88-99%) and thus solubility, and initial solution concentration (0.01-10% w/w) are quantified. Monodisperse droplets with radii ranging from 50 to 500 μm were produced at a flow-focusing junction with carrier phase hexadecane and extracted into ethyl acetate. Solvent exchange and extraction result in droplet shrinkage, demixing, coarsening and phase-inversion, yielding polymer microparticles with well-defined dimensions and internal microstructure. Polymer concentration, varied from below the overlap concentration c* to above the concentrated crossover c**, as estimated by viscosity measurements, was found to have the largest impact on the final particle size and extraction timescale, while polymer mass and hydrolysis played a secondary role. These results are consistent with the observation that the average polymer concentration upon solidification greatly exceeds c**, and that the internal microparticle porosity is largely unchanged. However, reducing the initial polymer concentration to well below c* (approximately 100×) and increasing droplet size yields thin-walled (100's of nm) capsules which controllably crumple upon extraction. The symmetry of the process can be readily broken by imposing extraction conditions at an impermeable surface, yielding large, buckled, cavity morphologies. Based on these results, we establish robust design criteria for polymer capsules and particles, demonstrated here for poly(vinyl alcohol), with well-defined shape, dimensions and internal microstructure.
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