We electrokinetically characterize properties of single 42-nm polystyrene nanoparticles (NP) in nanofluidic channels imaged with frustrated total internal reflection fluorescence microscopy (fTIRFM). Specifically, we demonstrate fTIRFM of individual NPs in nanofluidic channels shallower than the evanescent field and use the resultant illumination field to gain insight into the behavior and electrokinetic properties of individual NP transport in channels. We find that the electrophoretic mobility of nanoparticles in 100-nm channels is lower than in larger channels or in bulk, presumably due to hindrance effects. Furthermore, we notice a non-intuitive increase in mobility with buffer concentration, which we attribute to electric double layer interactions. Finally, since the evanescent field intensity decreases with distance from the channel wall, we use the measured fluorescence intensity to report probable transverse distributions of free-solution 42-nm polystyrene fluorescent particles. Our method promises to be useful for characterizing nanoscale molecules for many applications in drug discovery, bioanalytics, nanoparticle synthesis, viral targeting, and the basic science of understanding nanoparticle behavior.
Micro- and nanofluidic lab-on-chip technology offers the unique capability of high-resolution separation, identification, and manipulation of biomolecules with broad applications in chemistry, biology, and medicine. In this work, we probe the effects of ionic strength on separation of ss- and dsDNA within 1 micron and 100 nm-deep glass channels. Separation behavior of DNA is influenced by a number of parameters, including ionic strength, melting temperature, strand length, strand conformation, and channel size. Specifically, we find a shift in the observed mobility of 10-bp (base pair) dsDNA for different ionic strengths due to changes in kinetic parameters, underlying the importance of these considerations when working with short DNA. For 50-base DNA, the electrophoretic mobility difference between ss- and dsDNA increases as the ionic strength increases due to changes in conformation of the ssDNA. Finally, we find that decreasing channel size decreases the absolute electrophoretic mobility of 10- and 20-bp ss- and dsDNA, due to both hydrodyamic confinement and electric double layer (EDL) interactions. We hypothesize that about 4% mobility reduction is due to hydrodynamic confinement, which is observed at all ionic strengths, and further reduction is due to EDL interactions between the DNA and the channel walls, only observed at low ionic strengths.
Capillary electrophoresis (CE) is a powerful analytical tool for performing separations and characterizing properties of charged species. For reacting species during a CE separation, local concentrations change leading to nonequilibrium conditions. Interpreting experimental data with such nonequilibrium reactive species is nontrivial due to the large number of variables involved in the system. In this work we develop a COMSOL multiphysics-based numerical model to simulate the electrokinetic mass transport of short interacting ssDNAs in microchip capillary electrophoresis. We probe the importance of the dissociation constant, K(D), and the concentration of DNA on the resulting observed mobility of the dsDNA peak, μ(w), by using a full sweep of parametric simulations. We find that the observed mobility is strongly dependent on the DNA concentration and K(D), as well as ssDNA concentration, and develop a relation with which to understand this dependence. Furthermore, we present experimental microchip capillary electrophoresis measurements of interacting 10 base ssDNA and its complement with changes in buffer ionic strength, DNA concentration, and DNA sequence to vary the system equilibria. We then compare our results to thermodynamically calculated K(D) values.
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