On-the-fly fluorescence lifetime detection in HPLC using a multiharmonic Fourier transform phase-modulation spectrofluorometer

Smalley, Maria Brak.; Shaver, Jeremy M.; McGown, Linda B.

doi:10.1021/ac00071a022

Cited by 43 publications

(37 citation statements)

References 6 publications

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“…An even more attractive feature of a fluorescence lifetime measurement is the possibility to distinguish between different analytes [31][32][33][34], which can also serve to identify co-eluting peaks [32][33][34][35][36]. Even for analytes that contain identical UV fluorophores, e.g.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2011

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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.

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Section: Introductionmentioning

confidence: 99%

“…Another approach to determine fluorescence lifetimes on-the-fly was shown by the workgroup of McGown. They used a frequencydomain spectrofluorometer with excitation at 325 nm and determined the lifetimes of chromatographically separated PAHs [35]. The group also presented lifetime detection of fluorescent dyes in CE [43].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2011

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“…In the OFLD experiments, the fluorescence intensity and lifetime data were extracted from the dynamic MHF signal to produce an intensity-lifetime electropherogram. The fluorescence intensity was recovered from the MHF data using an in-house program [13]. The lifetime data were analyzed using NLLS to fit the data to a one-component (monoexponential) model or a two-component (bi-exponential) model in which either both lifetimes were allowed to vary or one lifetime was fixed to the lifetime of the background signal.…”

Section: Analysis Of Lifetime Datamentioning

confidence: 99%

Characterization of acridone dyes for use in four‐decay detection in DNA sequencing

2003

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Four acridone dyes and dye-labeled primers were characterized for use in four-decay DNA sequencing. In the four-decay scheme, fluorescence lifetime replaces spectral ("color") selectivity for distinguishing between four base-specific labels in a single-lane capillary electrophoresis (CE) separation of the DNA fragments. Prior to the introduction of the acridone dyes, a major obstacle to four-decay detection was the lack of four suitable dyes with resolvable lifetimes. The four acridone dyes, whether free in solution or tethered to DNA primer, exhibit significant differences among their lifetimes and are well-suited to use together in four-decay sequencing. The lifetimes of the four dye-labeled DNA primers that were sequentially injected and detected on-the-fly in a 2% POP6 sequencing gel were 4, 6, 11 and 14 ns. A 405 nm violet laser diode provides optimal excitation of the four dyes.

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“…The advantages of frequency domain over time domain for lifetime detection, as well as the advantages of lifetime over fluorescence color detection, have been discussed elsewhere [7,12]. Frequencydomain lifetime detection using multiharmonic, Fourier transform (MHF) technology is particularly well-suited to on-the-fly detection in CE because of its speed and precision [13]. Complete lifetime profiles can be acquired at intervals of several tens of milliseconds during the CE run.…”

Section: Introductionmentioning

confidence: 99%

Mono‐ and bis‐intercalating dyes for multiplex fluorescence lifetime detection of DNA restriction fragments in capillary electrophoresis

et al. 2002

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The use of novel intercalating dyes as labels in DNA restriction fragment analysis by capillary electrophoresis with frequency-domain fluorescence lifetime detection is described. The dyes, including one mono-intercalating dye with three positive charges and three bis-intercalating, homodimeric dyes with four positive charges, were excited by the 488 nm line of an argon ion laser and exhibited lifetimes in the range of 1-3 ns. The separations were performed using a gel containing 1% high-molecular-weight (HMW) hydroxyethylcellulose (HEC) (90,000-105,000) and 0.3% low-molecular-weight (LMW) HEC (24,000-27,000) in Tris-borate-EDTA buffer (TBE). Multiplex lifetime detection of mixtures of dye-labeled DNA restriction fragment digests and size standard fragments was achieved. Compared to previous results obtained with several mono-intercalating dyes of lesser charge (McIntosh, S. L., Nunnally, B. K., Nesbit, A. R., Deligeorgiev, T. G., Gadjev, N. I., McGown, L. B., Anal. Chem. 2000, 72, 5444-5449), the present dyes provided a wider range of lifetimes and better lifetime discrimination in multiplex detection. There was no evidence of dye exchange during the capillary electrophoresis experiment.

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On-the-fly fluorescence lifetime detection in HPLC using a multiharmonic Fourier transform phase-modulation spectrofluorometer

Cited by 43 publications

References 6 publications

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

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

Characterization of acridone dyes for use in four‐decay detection in DNA sequencing

Mono‐ and bis‐intercalating dyes for multiplex fluorescence lifetime detection of DNA restriction fragments in capillary electrophoresis

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