High-Efficiency Molecular Counting in Solution:  Single-Molecule Detection in Electrodynamically Focused Microdroplet Streams

Abstract

We report fluorescence detection of individual rhodamine 6G molecules using a linear quadrupole to focus streams of microdroplets through the waist of a counterpropagating cw Ar(+) laser. Since the terminal velocity scales as the square of the droplet diameter, the droplet-laser interaction time was "tunable" between 5 and 200 ms by using water samples spiked with a small, variable (2-5% v/v) amount of glycerol. Fluorescence bursts from droplets containing single molecules were clearly distinguished from the blanks in real time with an average signal-to-noise ratio of about 10, limited primarily by photobleaching and droplet size fluctuations (<1%). The volume throughput rates associated with this approach (∼10 pL/s) are roughly 10(3) higher than those associated with particle levitation techniques, with minimal sacrifice in sensitivity. Total molecular detection efficiencies of about 80% (at >99% confidence) were obtained for 100 and 15 fM rhodamine 6G solutions, in good agreement with detailed theoretical calculations and statistical limitations.

Optofluidic dye lasers

“…The characteristics of such droplet dye lasers were comprehensively studied and reviewed (Lin et al 1986(Lin et al , 1992aChang and Campillo 1996). Besides the laser applications, microdroplets have also been used to study cavity-induced spontaneous emission modification (Campillo et al 1991;Lin et al 1992b;Barnes et al 1992), single molecule fluorescence (Barnes et al 1993(Barnes et al , 1996Lermer et al 1997), and many nonlinear optical effects such as stimulated Raman scattering, multiorder Stokes emission and stimulated Brillouin scattering (Chang and Campillo 1996, Chaps. 5-7, and references therein).…”

Chemical Characterization of Flowing Polydisperse Aerosols by Raman Spectroscopy

“…Previous studies of aerosols using Raman spectroscopy have focused on either single, levitated particles (Vehring et al 1998;Aardahl et al 1996;Buehler et al 1991;Tang and Fung 1989;Fung and Tang 1989) or streams of uniform, monodisperse particles (Schweiger 1987;Deverakonda et al 1998;Barnes et al 1997;Lermer et al 1997). These studies have elucidated many interesting and novel optical properties of these systems.…”

Ultrasensitive fluorescence-based methods for nucleic acid detection: towards amplification-free genetic analysis

“…4B), 54 oil-in-water microdroplet streams ( Fig. 4C), 55,56 and nanofabricated channels ( Fig. 4D) [57][58][59] have all been used to confine the sample and thus enhance the detection efficiency.…”