On-line fluorescence lifetime determinations in capillary electrophoresis.

Abstract

A near-infrared time-correlated single-photon counting instrument was developed for on-line fluorescence lifetime determinations of components separated by capillary electrophoresis (CE). The lifetimes of the migrating components were determined using maximum likelihood estimators, which are computationally easy to perform and yield values with high precision and favorable accuracies in the limit of low photocounts within the decay profile. The laser source used in the present system was a passively mode-locked Ti-sapphire laser with a single-photon avalanche diode serving as the fast detector. The instrument response function of this system was determined to be 165 ps (fwhm). Electrophorectic separation of two near-IR dyes, DTTCI (cationic) and IR-125 (anionic), in a 95:5 methanol/water running buffer (pH = 9.5) produced electrophoretic peak widths at the base of approximately 1 -9 s, which set the integration time for collection of the decay profiles. At a loading level of 1.42 zmol for IR-125 and 49 zmol for DTTCI, lifetime values were determined to be 482 +/- 14 ps for IR-125 and 943 +/- 23 ps for DTTCI, which agreed favorably with the lifetimes determined for these dyes using static measurements at high concentrations. To minimize background resulting from scattered photons in ultradilute conditions, which introduces bias into the lifetime determination, the calculation was initiated at a fixed time delay with respect to the excitation pulse. To demonstrate the feasibility of making lifetime determinations in capillary gel electrophoresis, where the gel can produce high scattering backgrounds, the lifetimes of C-terminated fragments produced from the M13mp 18 template and labeled at the 5' end of a universal M13 sequencing primer with a near-IR fluorescent tag were determined. The collection of lifetimes for 30 different peaks in the electropherogram yielded a mean value of 581 ps and a standard deviation of +/- 9 ps.