Diode laser based time-resolved detection and identification of individual mononucleotide molecules in aqueous solution

We applied a short-pulse diode laser emitting at 637 nm with a repetition rate of 30 MHz in combination with a confocal microscope to study bursts of fluorescence photons from individual labeled mononucleotide molecules in water. A newly synthesized oxazine dye (MR121) and the commercially available carbocyanine dye Cy5 were used as fluorescent labels. Multichannel scaler traces, the fluorescence autocorrelation function and fluorescence decay times determined by time-correlated single-photon counting have been measured simultaneously. The time-resolved fluorescence signals of the two mononucleotides were analyzed and identified by a maximum likelihood estimator. The results showed out that 60 detected photons per transit of a single molecule are sufficient to distinguish two labeled mononucleotides in water with a misclassification of less than 10% via their characteristic fluorescence lifetimes of 1.07+/-0.27 ns (Cy5-dUTP) and 1.89+/-0.34 ns (MR121-dUTP).