Carbon Nanotube AFM tips for biotechnology – BIONANOTIP

Biosensors are bioanalytical and biomedical devices aimed at measuring precisely the quantity and/or the function of a target molecular complex (ligand-receptor). Nanobiotechnology aims at developing more reliable devices with an increasing sensitivity, robustness, and a decreasing cost. A key step in the development of biosensors is the covalent attachment of biomolecules to solid surfaces. The success of this step implies that the attached molecule (usually the receptor) retains its native conformation and its cognate function. According to the structure-function relationship, it is assumed that when a receptor is functional its native structure is preserved.
Consequently, the goal of BIONANOTIP is to develop novel techniques for assembling biosensor surfaces, grafting receptors on these surfaces, and demonstrating that these receptors retained their activity. The strategy used for the biosensor surface is self-assembled monolayers (SAMs). SAMs are highly-ordered two-dimensional structures that form spontaneously on a variety of solid surfaces. They provide a convenient route to tailor their surface properties such as wettability, corrosion resistance, and biocompatibility. In the BIONANOTIP project, the immobilization step of biomolecules onto SAMs surface, will be realized at ISM. This Laboratory has already reported the synthesis of silylated coupling agents which have been used to tailor new SAMs for biosensors applications. Moreover theses modified surfaces are readily imaged by atomic force microscopy (AFM).
AFM is a powerful tool able to image sample surfaces at a nanometer scale as well as to measure biomolecular interactions in the range of piconewton forces (10-12 N). Owing to the exceptionally good signal-to-noise ratio (S/N), AFM is the only technique able to image single molecules at high resolution in physiological conditions. However, to improve spatial resolution, it was shown a few years ago that carbon nanotubes (CNTs) could be manually attached to a commercial tip or grown on an AFM tip by chemical vapor deposition.
CNTs are ultra-light weight, chemically stable, and possess an apex diameter of the order of 1 nm for single wall CNT which allows the improvement of lateral resolution. Native CNTs will be used to characterize SAMs and to perform topographical images in the BIONANOTIP project; results will be compared to those obtained with classical silicon-based tips. CNTs will be functionalized at CBMN with a biomolecule (usually a ligand) to perform the functional test of receptors grafted on SAMs. The long term strategy developed in our approach is to use a single probe, the biofunctionalized CNT, to perform the topographical analysis of SAMs and to perform the functional test of grafted receptor. The challenge of one of the tasks will be to selectively functionalize CNTs at the nanotube apex by an original dipping procedure.
The efficiency of the CNT functionalization will be tested at the LIRM by dynamic force spectroscopy (DFS). Due its piconewton force sensitivity, DFS has became a widely used technique for investigating several force-related molecular mechanism such as polymer stretching, cell adhesion, cell-receptor recognition, protein folding/unfolding, antibody-antigen recognition, providing new insights into their structure-function relationships, even in physiological and pathological contexts. DFS allows the characterization of the energy landscape of the rupture of interaction between a ligand and a receptor. Current formalisms allow the identification of the presence of single or multiple parallel bonds in a ligand-receptor interaction. This specificity of DFS will be used to characterize the energy landscape of the interaction between grafted receptors on SAMs and attached ligand on CNTs. In particular, the well-characterize avidin-biotin system will be used to confirm the presence of single ligands at the apex of CNTs.