title: A new, simple method for linking of antibodies to atomic force microscopy tips.
abstract: Functionalization of atomic force microscope (AFM) tips with bioligands converts them into monomolecular biosensors which can detect complementary receptor molecules on the sample surface. Flexible PEG tethers are preferred because the bioligand can freely reorient and locally palpate the sample surface while the AFM tip is moved along. In a well-established coupling scheme [Hinterdorfer et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 3477-3481], a heterobifunctional PEG linker is used to tether thiol-containing bioligands to amino-functionalized AFM tips. Since antibodies contain no free thiol residues, prederivatization with N-succinimidyl 3-(acetylthio)propionate (SATP) is needed which causes a relatively high demand for antibody. The present study offers a convenient alternative with minimal protein consumption (e.g., 5 microg of protein in 50 microL of buffer) and no prederivatization, using a new heterobifunctional cross-linker that has two different amino-reactive functions. One end is an activated carboxyl (N-hydroxysuccinimide ester) which is much faster to react with the amino groups of the tips than the benzaldehyde function on its other end. The reactivity of the latter is sufficient, however, to covalently bind lysine residues of proteins via Schiff base formation. The method has been critically examined, using biotinylated IgG as bioligand on the tip and mica-bound avidin as complementary receptor. These experiments were well reproduced on amino-functionalized silicon nitride chips where the number of specifically bound IgG molecules (approximately 2000 per microm2) was estimated from the amount of specifically bound ExtrAvidin-peroxidase conjugate. For a bioscientific application, human rhinovirus particles were tethered to the tip, very-low-density lipoprotein receptor fragments were tethered to mica, and the specific interaction was studied by force microscopy.
authors.affiliation: Institute of Biophysics, J. Kepler University, Altenberger Strasse 69, A-4040 Linz, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Ebner
authors.forename: Linda
authors.initials: L
authors.lastname: Wildling
authors.forename: A S M
authors.initials: AS
authors.lastname: Kamruzzahan
authors.forename: Christian
authors.initials: C
authors.lastname: Rankl
authors.forename: Jürgen
authors.initials: J
authors.lastname: Wruss
authors.forename: Christoph D
authors.initials: CD
authors.lastname: Hahn
authors.forename: Martin
authors.initials: M
authors.lastname: Hölzl
authors.forename: Rong
authors.initials: R
authors.lastname: Zhu
authors.forename: Ferry
authors.initials: F
authors.lastname: Kienberger
authors.forename: Dieter
authors.initials: D
authors.lastname: Blaas
authors.forename: Peter
authors.initials: P
authors.lastname: Hinterdorfer
authors.forename: Hermann J
authors.initials: HJ
authors.lastname: Gruber
mesh_terms: D000447:Aldehydes / Q000737:chemistry; D000538:Aluminum Silicates / Q000737:chemistry; D000906:Antibodies / Q000737:chemistry*; D001360:Avidin / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry; D018625:Microscopy, Atomic Force; D011092:Polyethylene Glycols / Q000737:chemistry; D011422:Propionates / Q000737:chemistry; D011973:Receptors, LDL / Q000378:metabolism; D012229:Rhinovirus; D013388:Succinimides / Q000737:chemistry; D014771:Virion / Q000378:metabolism
chemical_list: D000447:Aldehydes; D000538:Aluminum Silicates; D000906:Antibodies; D007074:Immunoglobulin G; D011422:Propionates; D011973:Receptors, LDL; D013388:Succinimides; C036053:VLDL receptor; D001360:Avidin; D011092:Polyethylene Glycols; D001710:Biotin; C001426:N-hydroxysuccinimide; C011934:mica