Adsorption Kinetics of Bovine Serum Albumin on Fused Silica:  Population Heterogeneities Revealed by Single-Molecule Fluorescence Microscopy

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.

Adsorption of bovine serum albumin on fused silica: Elucidation of protein–protein interactions by single-molecule fluorescence microscopy

“…Desorption was analyzed by tracking each adsorbate until it desorbed. Details of the methodology were explained previously [10]. In contrast to the case of neutral pH when early settlers were found to stick more firmly than late comers [10], the adsorbates here behaved homogeneously regardless of when they adsorbed.…”

“…The experimental details and the TIRFM setup were described in our previous report [10]. Briefly, stock solutions of BSA and stock solutions of Cy3B-labeled BSA were prepared in the standard way (see Supplementary Material).…”

“…Total internal reflection occurred at the interface of the fused silica and water. Both the quartz slide and the cover glass were cleaned most thoroughly with a KOH protocol [10]. The thickness of the liquid layer inside the cell was 30 ± 5 m. The penetration depth of the evanescent wave was about 150 nm.…”

“…One can then dial up the protein concentrations to let the last two factors take effect. We recently reported our study of BSA adsorption on fused silica at neutral pH, at the level of single molecules using total-internal-reflection fluorescence microscopy (TIRFM) [10]. Adsorbates were microns apart and interactions among them were negligible.…”

Biosensing in a microelectrofluidic system using optical whispering-gallery mode spectroscopy

“…13,14 To validate the current simulation, the adsorption of BSA at two different concentrations (20 pM and 500 pM) onto a silica micro resonator at pH 6.6 in the absence of external electrical field is simulated and compared with the experimental results by Yeung et al 28,31 The parameter values used in the simulation were given in Table I of Yeung et al through a simple Langmuir model. 28 The surface saturation concentration are calculated as 0.448 Â 10 À13 and 5.418 Â 10 À12 mol/m 2 for 20 pM and 500 pM, respectively.…”