Simple test system for single molecule recognition force microscopy

• Published in: Analytica chimica acta 2003-03, Vol.479 (1), p.59-75
• Main Authors: Riener, Christian K., Stroh, Cordula M., Ebner, Andreas, Klampfl, Christian, Gall, Alex A., Romanin, Christoph, Lyubchenko, Yuri L., Hinterdorfer, Peter, Gruber, Hermann J.
• Format: Article
• Language: English
• Subjects: Aminosilane; Atomic force microscopy; Avidin; Biotin; Ethanolamine; Silicon nitride
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/S0003-2670(02)01373-9

We have established an easy-to-use test system for detecting receptor–ligand interactions on the single molecule level using atomic force microscopy (AFM). For this, avidin–biotin, probably the best characterized receptor–ligand pair, was chosen. AFM sensors were prepared containing tethered biotin molecules at sufficiently low surface concentrations appropriate for single molecule studies. A biotin tether, consisting of a 6 nm poly(ethylene glycol) (PEG) chain and a functional succinimide group at the other end, was newly synthesized and covalently coupled to amine-functionalized AFM tips. In particular, PEG 800 diamine was glutarylated, the mono-adduct NH 2–PEG–COOH was isolated by ion exchange chromatography and reacted with biotin succinimidylester to give biotin–PEG–COOH which was then activated as N-hydroxysuccinimide (NHS) ester to give the biotin–PEG–NHS conjugate which was coupled to the aminofunctionalized AFM tip. The motional freedom provided by PEG allows for free rotation of the biotin molecule on the AFM sensor and for specific binding to avidin which had been adsorbed to mica surfaces via electrostatic interactions. Specific avidin–biotin recognition events were discriminated from nonspecific tip–mica adhesion by their typical unbinding force (∼40 pN at 1.4 nN/s loading rate), unbinding length (