Determination of protein surface excess on a liquid/solid interface by single-molecule counting

Abstract

Determination of protein surface excess is an important way of evaluating the properties of biomaterials and the characteristics of biosensors. A single-molecule counting method is presented that uses a standard fluorescence microscope to measure coverage of a liquid/solid interface by adsorbed proteins. The extremely low surface excess of lysozyme and bovine serum albumin (BSA), in a bulk concentration range from 0.3 nmol L(-1) (0.02 microg mL(-1)) to 3 nmol L(-1) (0.2 microg mL(-1)), were measured by recording the counts of spatially isolated single molecules on either hydrophilic (glass) or hydrophobic (polydimethylsiloxane, PDMS) surfaces at different pH. The differences observed in amounts of adsorbed proteins under different experimental conditions can be qualitatively explained by the combined interactions of electrostatic and hydrophobic forces. This, in turn, implies that single-molecule counting is an effective way of measuring surface coverage at a liquid/solid interface.

In silico modeling of the pore region of a KCNQ4 missense mutant from a patient with hearing loss

“…The electrostatic potential energy for two charges in protein or lipid membrane interiors is therefore between 16 and 40 times greater than that in water. Consequently, the long-range electrostatic repulsive force between the positively charged His270 and a K + might possibly impede K + passage through the channel [ 30 ], whereas the force on K + would be smaller in the extracellular region.…”

Polymerase Chain Reaction and Immunoassay−Matrix Assisted Laser Desorption Mass Spectrometry Using Tag-Mass Technology: New Tools to Break Down Quantification Limits and Multiplexes

“…Although, various strategies of assays and different detections were proposed in the last 10 years, currently, much detection are performed by on fluorescence measurements which also prove to be very sensitive. Fluorescence assays can be performed according very different strategies, including beads grafted with antibodies, microarrays − with fluorescence detection, or by classical ImmunoSorbent Assays with fluorescence detection (FLISA). Fluorescence is much more sensitive than UV detection and present a limit of detection ranging from hundreds of picograms per milliliter up to 10 pg/mL according to the strategy used.…”

Molecular Modelling-Based Investigations of a Mutant Protein in Patients with Hearing Loss

“…The electrostatic potential energy for two charges in protein or lipid membrane interiors is therefore between 16 and 40 times greater than that in water. Consequently, the long-range electrostatic repulsive force between positively charged His270 and a K + ion might possibly impede passage of K + through the channel [47], whereas the force on K + would be smaller in the extracellular region.…”