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Accurate Optical Analysis of Single-Molecule Entrapment in Nanoscale Vesicles
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Semiconductor Electronics Division, National Institute of Standards and Technology.
, ‡Wheaton College.
, §Biotechnology Division, National Institute of Standards and Technology.
Section:Abstract
We present a nondestructive method to accurately characterize low analyte concentrations (0−10 molecules) in nanometer-scale lipid vesicles. Our approach is based on the application of fluorescence fluctuation analysis (FFA) and multiangle laser light scattering (MALLS) in conjunction with asymmetric field flow fractionation (AFFF) to measure the entrapment efficiency (the ratio of the concentration of encapsulated dye to the initial bulk concentration) of an ensemble of liposomes with an average diameter less than 100 nm. Water-soluble sulforhodamine B (SRB) was loaded into the aqueous interior of nanoscale liposomes synthesized in a microfluidic device. A confocal microscope was used to detect a laser-induced fluorescence signal resulting from both encapsulated and unencapsulated SRB molecules. The first two cumulants of this signal along with the autocorrelation function (ACF) were used to quantify liposome entrapment efficiency. Our analysis moves beyond typical, nonphysical assumptions of equal liposome size and brightness. These advances are essential for characterizing liposomes in the single-molecule encapsulation regime. Our work has further analytical impact because it could increase the interrogation time of free-solution molecular analysis by an order of magnitude and form the basis for the development of liposome standard reference materials.
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This article has been cited by 4 ACS Journal articles (4 most recent appear below).
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Microfluidic Mixing and the Formation of Nanoscale Lipid Vesicles
Andreas Jahn, Samuel M. Stavis, Jennifer S. Hong, Wyatt N. Vreeland, Don L. DeVoe and Michael GaitanACS Nano2010 4 (4), 2077-2087Microfluidic Mixing and the Formation of Nanoscale Lipid Vesicles
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History
- Published In Issue January 01, 2010
- Article ASAPDecember 01, 2009
- Received: July 29, 2009
Accepted: November 9, 2009
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