An all solid-state near-infrared time-correlated single photon counting instrument for dynamic lifetime measurements in DNA sequencing applications

Abstract

We have constructed a simple, all solid-state, time-correlated single photon counting device for collecting decay profiles of chromophores attached to DNA fragments moving through a capillary tube filled with a sieving gel under the influence of an applied electric field (capillary electrophoresis). The major components of the instrument consist of an actively pulsed GaAlAs diode laser (λexcitation=780 nm; τp<200 ps; repetition rate=80 MHz; average power=5.0 mW), single photon avalanche diode (dark count rate <50 cps; quantum efficiency=65% at 800 nm) and a PC board containing a constant fraction discriminator, time-to-amplitude converter, and an analog-to-digital converter (maximum processing count rate=3×106 cps). The instrument possessed a response function of approximately 275 ps (full width at half-maximum), adequate for measuring fluorescence lifetimes in the subnanosecond regime. To demonstrate the utility and the sensitivity of the instrument, dynamic measurements of fluorescence lifetimes for near-IR dye-labeled DNA fragments were measured during capillary electrophoresis for the identification of the dye-labeled nucleotide bases via temporal discrimination. The results indicated that in a two-dye experiment, in which only two of the four constituent bases which comprise DNA were labeled with unique fluorescent probes, the characteristic lifetime of the probe could be used to readily identify the terminal nucleotide base. Decay profiles were constructed from roughly 17 500 photoelectrons accumulated over a 2 s counting interval at a loading level of approximately 6.2×10−21 mol (3900 molecules) of DNA per electrophoretic band. The lifetimes of the two labeling dyes were determined to be 669 ps (±42 ps) and 528 ps (±68 ps).