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).

Near-infrared laser-induced fluorescence detection in capillary electrophoresis

“…The sequencing ladder was electrokinetically injected onto the column for 1 min at 200 V/cm with the separation carried out using the same field strength. Reprinted from [5], with permission. cated in PMMA.…”

“…Semiconductor diode lasers are attractive excitation sources because they are very compact and inexpensive while supplying ample power (~100 mW) for ultrasensitive detection and also can be operated in a pulsed mode for time-resolved fluorescence [5]. In addition, diode lasers show an operational lifetime that is much longer than the lifetime of most plasma tubes in ion lasers.…”

Near‐infrared time‐resolved fluorescence lifetime determinations in poly(methylmethacrylate) microchip electrophoresis devices

“…One of the advantages of fluorescence lifetime determinations is that the lifetime is independent of concentration and the excitation wavelength [8]. Using fluorescence lifetime discrimination in conjunction with spectral discrimination can offer additional information for analysis and identification of biomolecules, further increasing the information content available from a single assay [9].…”

Fluorescence lifetime imaging system with nm-resolution and single-molecule sensitivity

“…This kind of information is not available from conventional histograms from TCSPC data collected over minutes or hours. To solve the problem one has to continuously collect histograms over very short intervals [4][5][6][7] . Alternatively one can record each fluorescence photon together with its arrival time relative to the start of the experiment or to its predecessor 8 .…”

“…The fluorescence decay times of appropriate fluorescent dyes provide a powerful additional discrimination feature to distinguish the molecules of interest from the background or other species [1][2][3][4][5] .…”