Thermal lens spectroscopy will be developed as a method for ultrasensitive detection and quantitation of proteins separated by gel electrophresis. Low power (15-20 mW) CW lasers will be used to generate thermal lens signals. Configurations providing spatial resolution to 20-50 microns will be developed. Sources of noise in thermal lens spectroscopy of gels will be investigated, and tactics for noise reduction will be devised. Differential configurations for gel or residual stain background absorption subtraction will be designed. Retroreflection will be used to double delivered laser power, to minimize instrument cost. The system will be applied to Coomassie Brilliant Blue and silver-stained systems. Sensitivity increases of approximately 100X over existing instruments are expected. An experimental thermal lens detector for capillary electrophoresis of proteins will be developed. Low energy pulsed laser radiation will be used as the pump light source to provide 190-200 nm excitation. Approximately 100X increase in sensitivity is expected over conventional spectrophotometry. Raman spectroscopy will be used to study the structure of the silver colloids formed by silver staining. The initial protein-silver ion complexes which initiate the reaction will be elucidated by spontaneous Raman spectroscopy. The complexes present in the completed colloids will be studied by surface-enhanced Raman spectroscopy (SERS). SERS will also be employed to measure approximate amino acid content of denatured proteins separated by SDS page and silver-stained.
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