Functionalization of Probe Tips and Supports for Single-Molecule Recognition Force Microscopy

Abstract

The measuring tip of a force microscope can be converted into a monomolecular sensorif one or few "ligand" molecules are attached to the apex of the tip while maintainingligand function. Functionalized tips are used to study fine details of receptor-ligand interactionby force spectroscopy or to map cognate "receptor" molecules on the sample surface. Thereceptor (or target) molecules can be present on the surface of a biological specimen; alternatively,soluble target molecules must be immobilized on ultraflat supports. This review describes the methodsof tip functionalization, as well as target molecule immobilization. Silicon nitride tips, siliconchips, and mica have usually been functionalized in three steps: (1) aminofunctionalization,(2) crosslinker attachment, and (3) ligand/receptor coupling, whereby numerous crosslinkersare available to couple widely different ligand molecules. Gold-covered tips and/or supports haveusually been coated with a self-assembled monolayer, on top of which the ligand/receptor moleculehas been coupled either directly or via a crosslinker molecule. Apart from these general strategies,many simplified methods have been used for tip and/or support functionalization, even single-stepmethods such as adsorption or chemisorption being very efficient under suitable circumstances. Allmethods are described with the same explicitness and critical parameters are discussed. In conclusion,this review should help to find suitable methods for specific problems of tip and support functionalization.

How Antibody Surface Coverage on Nanoparticles Determines the Activity and Kinetics of Antigen Capturing for Biosensing

“…These parameters may be quantified in capture assays using biomolecules that bind to specific parts of the immobilized antibody molecules, e.g., secondary antibodies, protein G, protein A. Furthermore, the molecular picture may be clarified using techniques with singlemolecule resolution, e.g., by recognition imaging atomic force microscopy 37 or force induced dissociation experiments. 38 The present and future studies on the fundamental aspects of antibody activity will play an important role in reaching the maximum levels of sensitivity, specificity, and kinetics in future diagnostic and therapeutic technologies based on antibodyfunctionalized particles.…”

Force spectroscopy of barnase–barstar single molecule interaction

“…Covalent attachment of proteins onto the tip and sample surfaces without special linkers was used. (Currently we are repeating this and some other force spectroscopy experiments using benzaldehyde-PEG6-NHS linkers (Ebner et al, 2008), these results will be published elsewhere). Very briefly, the procedure of tip/sample functionalization was as follows.…”

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

“…Another advantage of single molecule experiments is that they can be modeled by means of molecular dynamics simulations 24,25 . The cantilever functionalization protocol described above is a modified version of procedures published previously [26][27][28] . After activation and silanization, the AFM cantilever is functionalized with a mixture of two different types of polyethylene-glycol (PEG) molecules (linker molecules).…”