Affinity Constants of Bovine Serum Albumin for 5 nm Gold Nanoparticles (AuNPs) with ω-Functionalized Thiol Monolayers Determined by Fluorescence Spectroscopy.

A detailed understanding of the binding of serum proteins to small (dcore <10 nm) nanoparticles (NPs) is essential for the mediation of protein corona formation in next generation nanotherapeutics. While a number of studies have investigated the details of protein adsorption on large functionalized NPs, small NPs (with a particle surface area comparable in size to the protein) have not received extensive study. This study determined the affinity constant (Ka) of BSA when binding to three different functionalized 5 nm gold nanoparticles (AuNPs). AuNPs were synthesized using three ω-functionalized thiols (mercaptoethoxy-ethoxy-ethanol (MEEE), mercaptohexanoic acid (MHA), and mercaptopentyltrimethylammonium chloride (MPTMA)), giving rise to particles with three different surface charges. The binding affinity of bovine serum albumin (BSA) to the different AuNP surfaces was investigated using UV-visible absorbance spectroscopy, dynamic light scattering (DLS), and fluorescence quenching titrations. Fluorescence titrations indicated that the affinity of BSA was actually highest for small AuNPs with a negative surface charge (MHA-AuNPs). Interestingly, the positively charged MPTMA-AuNPs showed the lowest Ka for BSA, indicating that electrostatic interactions are likely not the primary driving force in binding of BSA to these small AuNPs. Ka values at 25 °C for MHA, MEEE, and MPTMA-AuNPs were 5.2 ± 0.2 × 107, 3.7 ± 0.2 × 107, and 3.3 ± 0.16 × 107 M-1 in water, respectively. Fluorescence quenching titrations performed in 100 mM NaCl resulted in lower Ka values for the charged AuNPs, while the Ka value for the MEEE-AuNPs remained unchanged. Measurement of the hydrodynamic diameter (Dh) by dynamic light scattering (DLS) suggests that adsorption of 1-2 BSA molecules is sufficient to saturate the AuNP surface. DLS and negative-stain TEM images indicate that, despite the lower observed Ka values, the binding of MPTMA-AuNPs to BSA likely induces significant protein misfolding and may lead to extensive BSA aggregation at specific BSA:AuNP molar ratios.

Fabrication and performance of a microfluidic traveling-wave electrophoresis system.

A microfluidic traveling-wave electrophoresis (TWE) system is reported that uses a locally defined traveling electric field wave within a microfluidic channel to achieve band transport and separation. Low voltages, over a range of -0.5 to +0.5 V, are used to avoid electrolysis and other detrimental redox reactions while the short distance between electrodes, ∼25 µm, provides high electric fields of ∼200 V cm(-1). It is expected that the low voltage requirements will simplify the future development of smaller portable devices. The TWE device uses four interdigitated electrode arrays: one interdigitated electrode array pair is on the top of the microchannel and the other interdigitated electrode array pair is on the microchannel bottom. The top and bottom substrates are joined by a PDMS spacer that has a nominal height of 15 µm. A pinched injection scheme is used to define a narrow sample band within an injection cross either electrokinetically or hydrodynamically. Separation of two dyes, fluorescein and FLCA, with baseline resolution is achieved in less than 3 min and separation of two proteins, insulin and casein is demonstrated. Investigation of band broadening with fluorescein reveals that sample band widths equivalent to the diffusion limit can be achieved within the microfluidic channel, yielding highly efficient separations. This low level of band broadening can be achieved with capillary electrophoresis, but is not routinely observed in microchannel electrophoresis. Sample enrichment can be achieved very easily with TWE using a device with converging electric field waves controlled by two sets of independently controlled interdigitated electrodes arrays positioned serially along the microchannel. Sample enrichment of 40-fold is achieved without heterogeneous buffer/solvent systems, sorptive, or permselective materials. While there is much room for improvement in device fabrication, and many capabilities are yet to be demonstrated, it is anticipated that the capabilities and performance demonstrated herein will enable new lab-on-a-chip processes and systems.

Traveling-wave electrophoresis for microfluidic separations.

Models and microfluidic experiments are presented of an electrophoretic separation technique in which charged particles whose mobilities exceed a tunable threshold are trapped between the crests of a longitudinal electric wave traveling through a stationary viscous fluid. The wave is created by applying periodic potentials to electrode arrays above and below a microchannel. Predicted average velocities agree with experiments and feature chaotic attractors for intermediate mobilities.