No abstract

The adsorption of bovine serum albumin (BSA) on fused silica at neutral pH was investigated at the single-molecule level by total internal reflection fluorescence microscopy. Dye-labeled BSA molecules that adsorbed on the quartz surface lit up as discrete, fluorescent dots which eventually disappeared upon desorption. Movies of these events offered unprecedented details for kinetics modeling. The results suggested that 99.3% of the BSA was not sticky, and even if adsorbed, it would desorb in minutes. In contrast, the remaining 0.7% was not only sticky, but would anchor in due course. Such population heterogeneity, otherwise masked in ensemble measurements, sheds new light on our understanding of protein adsorption. The methodology is also generally applicable to the studies of macromolecules at interfaces.

We spectroscopically determined the temperature and electron density of the plasma plumes produced by pulsed-laser ablation of aqueous solutions containing sodium, lithium, and rubidium. With the use of a Nd:YAG laser at 532 nm and fluence of 3 J/cm2, the plasma produced was hot (low eV range) and extensively ionized, with electron density in the 1018 cm−3 range. Analyte line signals were initially masked by intense plasma continuum emissions and would only emerge briefly above the background when the plume temperature dropped below 1 eV during the course of its very rapid cooling. Since ionization was thermally induced, the intense plasma flash was inevitable. In contrast, 193-nm laser ablation at similar fluence generated plasmas of much lower (<1 eV) temperature but comparable electron density. Plasma continuum emissions were relatively weak, and the signal-to-background ratio was a thousand times better. Consequently, this “cold” plasma was ideal for sampling biologically important elements such as sodium, potassium, and calcium.

Binding kinetics of nuclear receptors and their specific ligands was measured using polarization anisotropy complemented with total internal reflection fluorescence. Binding affinities of tethered full length human estrogen receptor alpha (ERalpha) with 17beta-estradiol, diethylstilbestrol, raloxifene, 4-hydroxytamoxifen, tamoxifen, and genistein were measured to be 100 (as reference), 100, 35, 21, 8, and 1.5, respectively. They agreed with published results. For the first time, rate constants were measured, and off rates were 1.5, 1.5, 1.3, 1.6, 1.7, and 2.3 x 10(-3) s(-1) while on rates were 11, 10, 3.3, 2.4, 1.0, and 0.26 x 10(5) M(-1)s(-1), respectively. For the antiestrogen drugs, their comparable off-rates correlated well with their equally similar potency. Eleven ginsenosides were screened as potential ligands. None were found to bind to ERalpha, but Rb1(S) and 20(S)-Rg3 were shown to bind to peroxisome proliferator-activated receptor gamma. The latter finding corroborated strongly with the therapeutic effects of ginsenosides on diabetic mice observed in a separate study. Our method would complement surface plasmon resonance assay for small ligands in the mass range of tens to hundreds of Daltons.

Highly sensitive analysis of aqueous lead carbonate colloids was demonstrated by two-pulse laser-induced atomic fluorescence. The first laser pulse at 1064 nm ablated the sample solution to create an expanding plume. The colloids, being heavier, trailed behind and became concentrated. They were then intercepted by an ArF laser pulse that induced prompt atomic fluorescence at 405.8 nm from the lead atoms. The detection limit for lead was 0.24 ppb. Tap water was analyzed, and lead emissions were clearly observed. Time-resolved spectroscopy revealed that the efficient 193-nm excitation of the analytes was more universal than expected. That was confirmed by the successful application of the technique to colloids and alloys other than lead.