Label‐free analysis in chip electrophoresis applying deep UV fluorescence lifetime detection

Abstract

Herein we introduce deep UV fluorescence lifetime detection in microfluidics applied for label-free detection and identification of various aromatic analytes in chip electrophoresis. For this purpose, a frequency quadrupled Nd:YAG (neodymium-doped yttrium aluminum garnet) picosecond laser at 266 nm was incorporated into an inverse fluorescence microscope setup with time-correlated single photon counting detection. This allowed recording of photon timing with sub-nanosecond precision. Thereby fluorescence decay curves are gathered on-the-fly and average lifetimes can be determined for each substance in the electropherogram. The aromatic compounds serotonin, propranolol, 3-phenoxy-1,2-propanediol and tryptophan were electrophoretically separated using a fused-silica microchip. Average lifetimes were independently determined for each compound via bi-exponential tail fitting. Time-correlated single photon counting also allows the discrimination of background fluorescence in the time domain. This results in improved signal-to-noise-ratios as demonstrated for the above model analytes. Microchip electrophoretic separations with fluorescence lifetime detection were also performed with a protein mixture containing lysozyme, trypsinogen and chymotrypsinogen emphasizing the potential for biopolymer analysis.

Advances in Microfluidic Materials, Functions, Integration, and Applications

“…188, 189 In one format, small aromatic compounds and proteins separated by μCE were effectively detected using 266 nm laser excitation in a fluorescence microscope. 190 In an integrated approach, waveguides formed in a silicon microchip directed excitation light to the detection region and collected transmitted light (Figure 21), allowing analytes separated by μCE and MEKC to be detected by both UV absorbance and native UV-excited fluorescence. 188 …”

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

“…This application could be interesting for complex mixture where the fluorescence lifetime could help to get a better selectivity of detection. For proteins, associations with other molecules or denaturation could be studied in such manner . Finally, a 266 nm continuous wave (cw) laser with 120 mW was used for the native detection of proteins.…”

Recent advances in amino acid analysis by capillary electromigration methods, 2011–2013

“…An interesting review showing most of the developments in the native UV fluorescence detectors, summarizing many articles concerning aromatic AA separations, has been published . Beyreiss et al presented a very interesting UV LIF detector based on lifetime detection . A frequency quadrupled Nd:YAG picosecond laser at 266 nm for excitation was associated to a time‐correlated single photon counting detection.…”