Quantitative time-resolved measurement of membrane protein–ligand interactions using microcantilever array sensors

Abstract

Membrane proteins are central to many biological processes, and the interactions between transmembrane protein receptors and their ligands are of fundamental importance in medical research. However, measuring and characterizing these interactions is challenging. Here we report that sensors based on arrays of resonating microcantilevers can measure such interactions under physiological conditions. A protein receptor--the FhuA receptor of Escherichia coli--is crystallized in liposomes, and the proteoliposomes then immobilized on the chemically activated gold-coated surface of the sensor by ink-jet spotting in a humid environment, thus keeping the receptors functional. Quantitative mass-binding measurements of the bacterial virus T5 at subpicomolar concentrations are performed. These experiments demonstrate the potential of resonating microcantilevers for the specific, label-free and time-resolved detection of membrane protein-ligand interactions in a micro-array format.

An optimized measurement chamber for cantilever array measurements in liquid incorporating an automated sample handling system

“…A biological assay was introduced to compare the results obtained with the new optimised measurement chamber to previous results where mass changes upon biological interaction were evaluated [36,3,11,39,37,5,22,16,15,12,21]. The response of four sensors functionalised with biotin-terminated self-assembled monolayers, a ligand interacting firmly with the protein receptor streptavidin, was utilized.…”

“…Within the latter category, a large subset of sensing applications exists in the fields of proteomics [1][2][3][4][5], genomics [6][7][8] and microbiology [9][10][11][12][13][14][15], amongst others. A crucial component in achieving biologically relevant metrics in bio-sensing is the environment in which the test is performed.…”

Nanoscience and Technology

“…36 However, many biomolecular detection nanotechnologies, such as nanowire 37 or nanocantilever 38 biosensors, are simply too expensive for wide-scale use.…”

Effect of Conformational Entropy on the Nanomechanics of Microcantilever-Based Single-Stranded DNA Sensors

“…Such devices can either be static-mode (bending) sensors or dynamic-mode (resonance frequency variation) sensors. The low-cost sensors exhibit fast response, high sensitivity and suitability for parallelization into arrays and allow for a wide range of analyses, such as gene mutation [1], DNA hybridization [4] and protein-ligand interactions [5].…”