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Optimization of Native Fluorescence Detection of Proteins Using a Pulsed Nanolaser Excitation Source

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Abstract:

We present a mathematical description of the signal-to-noise ratio (S/N) in a fluorescence-based protein detector for capillary electrophoresis that uses a pulsed ultraviolet (UV) laser at 266 nm as an excitation source. The model accounts for photobleaching, detector volume, laser repetition rate, and analyte flow rate. We have experimentally characterized such a system, and we present a comparison of the experimental data with the predictions of the model. Using the model, the system was optimized for test analytes tryptophan, tyrosine, bovine serum albumin (BSA), and conalbumin, producing detection limits (3σ) of 0.67 nM, 5.7 nM, 0.9 nM, and 1.5 nM, respectively. Based on the photobleaching data, a photobleaching cross-section of 1.4 × 10–18cm2 at 266 nm was calculated for tryptophan.

Keywords: CAPILLARY ELECTROPHORESIS; LASER-INDUCED FLUORESCENCE; PHOTOBLEACHING; PROTEIN FLUORESCENCE; PROTEOMICS; PULSED LASER EXCITATION

Document Type: Research Article

DOI: http://dx.doi.org/10.1366/000370210793335016

Affiliations: 1: Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, USA 2: Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, USA. paul_farnsworth@byu.edu

Publication date: November 1, 2010

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