Simple test system for single molecule recognition force microscopy

Christian K. Riener, Cordula M. Stroh, Andreas Ebner, Christian Klampfl, Alex A. Gall, Christoph Romanin, Yuri L. Lyubchenko, Peter Hinterdorfer, Hermann J. Gruber

Research output: Contribution to journalArticlepeer-review

168 Scopus citations


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 6nm 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, PEG800 diamine was glutarylated, the mono-adduct NH2-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 (∼40pN at 1.4nN/s loading rate), unbinding length (<13nm), the characteristic nonlinear force-distance relation of the PEG linker, and by specific block with excess of free d-biotin. The convenience of the test system allowed to evaluate, and compare, different methods and conditions of tip aminofunctionalization with respect to specific binding and nonspecific adhesion. It is concluded that this system is well suited as calibration or start-up kit for single molecule recognition force microscopy.

Original languageEnglish (US)
Pages (from-to)59-75
Number of pages17
JournalAnalytica Chimica Acta
Issue number1
StatePublished - Mar 5 2003
Externally publishedYes


  • Aminosilane
  • Atomic force microscopy
  • Avidin
  • Biotin
  • Ethanolamine
  • Silicon nitride

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Environmental Chemistry
  • Spectroscopy


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