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

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 × 10−18 cm2 at 266 nm was calculated for tryptophan.