Biological substance characterization in water matrices with Raman microspectroscopy

摘要

Raman spectroscopy has been evaluated as a candidate technology for waterborne pathogen detection. Parameters have been investigated that influence the fidelity of Raman spectra of microorganisms and protein biological substances including bacterial species and strains, susceptibility to laser induced photodamage, composition of water matrix, and organism aging in water. An important operating parameter is the laser induced photodamage threshold of a variety of biological materials. The laser induced photodamage may be minimized by operating a 532 nm continuous wave laser excitation at laser power densities below 2300 W/cm~2 for Gram-positive Bacillus atrophaeus (BG) vegetative cells, 2800 W/cm~2 for BG spores, and 3500 W/cm~2 for Gram-negative E. coli organisms. Multivariate principal components analysis was able to discriminate six Gram-positive and Gram-negative organisms as well as five proteins between 5K and 65K mass units. B. thuringiensis, B. cereus, BG spore and vegetative preparations, and E. coli showed minimal aging effects when suspended in distilled and tap water. In general, Raman microspectroscopy of biological substances exhibited minimal spectral variability due to the age of a resting suspension, water matrix, and bacterial strain. The observed signature variability did not prevent the differentiation and characterization of bacterial genus and species and protein substances using Raman spectroscopy.