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

• Published in: Analytical chemistry (Washington) Vol. 69; no. 11; pp. 2115 - 2121
• Main Authors: Lermer, N, Barnes, M. D, Kung, C.-Y, Whitten, W. B, Ramsey, J. M
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01-06-1997
• Subjects: Analytical chemistry; Chemistry; Exact sciences and technology; Fluids; Fluorescence; Lasers; Molecules; Spectrometric and optical methods; Trace analysis; Electrodynamics; Molecules; Rhodamine; Flow(fluid); Focusing; Detection limit; CW laser; Measurement method; Droplet; Xanthene dye; Fluorescence spectrometry
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac970093b

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 103 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.