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

Abstract

We present a mathematical description of the S/N ratio in a fluorescence-based protein detector for capillary electrophoresis that uses a pulsed 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 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, 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 cm 2 at 266 nm was calculated for tryptophan.

Capillary electrophoresis hyphenated with UV‐native‐laser induced fluorescence detection (CE/UV‐native‐LIF)

“…Where C 0 is the concentration of the fluorophore, ε 0 is its extinction coefficient, v its velocity in front of the laser beam, R the repetition rate of the laser, A cap is the section of the capillary, L the illuminated length of the capillary, and f is the fraction not photodegradated at each laser pulse. Using this mathematic model, the system was optimized for Trp, Tyr, BSA, and conalbumin, resulting in LOD of 0.7, 5.7, 0.9, and 1.5 nM, respectively . We already indicated that the photobleaching is a factor limiting the sensitivity.…”