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.

Minor group human rhinoviruses (HRVs) attach to members of the low-density lipoprotein receptor family and are internalized via receptor-mediated endocytosis. The attachment of HRV2 to the cell surface, the first step in infection, was characterized at the singlemolecule level by atomic force spectroscopy. Sequential binding of multiple receptors was evident from recordings of characteristic quantized force spectra, which suggests that multiple receptors bound to the virus in a timely manner. Unbinding forces required to detach the virus from the cell membrane increased within a time frame of several hundred milliseconds. The number of receptors involved in virus binding was determined, and estimates for on-rate, off-rate, and equilibrium binding constant of the interaction between HRV2 and plasma membrane-anchored receptors were obtained.force spectroscopy ͉ molecular recognition ͉ single virus binding ͉ very low density lipoprotein receptor ͉ picornavirus H uman rhinoviruses (HRVs), members of the Picornaviridae family, are the most frequent cause of colds. Their icosahedral capsid (30 nm in diameter) is built from 60 copies each of 4 viral proteins VP1, VP2, VP3, and VP4 that surround the RNA genome. Of the 99 so far characterized serotypes, 12 (the minor receptor group) bind low-density lipoprotein receptor (LDLR), very-LDLR (VLDLR), and LDLR-related protein (LRP) (1, 2). This receptor family functions in endocytosis and signal transduction recognizing a variety of ligands (3). LDLR and VLDLR possess 5 domains (4), including an N-terminal ligand-binding domain composed of 7 (LDLR; L1-L7) and 8 (VLDLR; V1-V8) modules, a region similar to the EGFprecursor and a ␤-propeller with YWTD motifs that is implicated in low pH-induced release of the ligands in endosomes (5). Adjacent to the plasma membrane is a domain carrying O-linked oligosaccharides followed by the transmembrane anchor and the carboxyl terminus carrying a NPXY clathrin localization signal. The ligand binding modules are Ϸ40 amino acid residues in length. They are stabilized by a Ca ion and 6 highly conserved cysteines forming disulfide bridges (6). Differences in the types and numbers of repeats allow for recognition of a large variety of structurally and functionally diverse ligands.For infection, HRV2 attaches to LDLR and/or LRP at the cell membrane. It can be released with EDTA immediately after attachment to the cell but within some minutes becomes tightly bound and not dissociable (7). This finding was taken to indicate either recruitment of multiple receptors, thus enforcing an initial bond with a single receptor, and/or engulfment within membranes as the virus enters in clathrin coated vesicles (8). Subsequently, it presumably dissociates from its receptors upon arrival in the mildly acidic milieu (pH 6.5-6.0) of early endosomes (9); finally, the virus is delivered to endosomal carrier vesicles and late endosomes from where its RNA genome is released into the cytosol.Performing single-molecule force spectroscopy with an atomic force microscop...

The measuring tip of an atomic force microscope (AFM) can be upgraded to a specific biosensor by attaching one or a few biomolecules to the apex of the tip. The biofunctionalized tip is then used to map cognate target molecules on a sample surface or to study biophysical parameters of interaction with the target molecules. The functionality of tip-bound sensor molecules is greatly enhanced if they are linked via a thin, flexible polymer chain. In a typical scheme of tip functionalization, reactive groups are first generated on the tip surface, a bifunctional cross-linker is then attached with one of its two reactive ends, and finally the probe molecule of interest is coupled to the free end of the cross-linker. Unfortunately, the most popular functional group generated on the tip surface is the amino group, while at the same time, the only useful coupling functions of many biomolecules (such as antibodies) are also NH2 groups. In the past, various tricks or detours were applied to minimize the undesired bivalent reaction of bifunctional linkers with adjacent NH2 groups on the tip surface. In the present study, an uncompromising solution to this problem was found with the help of a new cross-linker (“acetal-PEG-NHS”) which possesses one activated carboxyl group and one acetal-protected benzaldehyde function. The activated carboxyl ensures rapid unilateral attachment to the amino-functionalized tip, and only then is the terminal acetal group converted into the amino-reactive benzaldehyde function by mild treatment (1% citric acid, 1–10 min) which does not harm the AFM tip. As an exception, AFM tips with magnetic coating become demagnetized in 1% citric acid. This problem was solved by deprotecting the acetal group before coupling the PEG linker to the AFM tip. Bivalent binding of the corresponding linker (“aldehyde-PEG-NHS”) to adjacent NH2 groups on the tip was largely suppressed by high linker concentrations. In this way, magnetic AFM tips could be functionalized with an ethylene diamine derivative of ATP which showed specific interaction with mitochondrial uncoupling protein 1 (UCP1) that had been purified and reconstituted in a mica-supported planar lipid bilayer.

Recent developments in single molecule force spectroscopy have allowed investigating the interaction between two redox partners, Azurin and Cytochrome C 551. Azurin has been directly chemisorbed on a gold electrode whereas cytochrome c has been linked to the atomic force microscopy tip by means of a heterobifunctional flexible cross-linker. When recording force-distance cycles, molecular recognition events could be observed, displaying unbinding forces of approximately 95 pN for an applied loading rate of 10 nN/s. The specificity of molecular recognition was confirmed by the significant decrease of unbinding probability observed in control block experiments performed adding free azurin solution in the fluid cell. In addition, the complex dissociation kinetics has been here investigated by monitoring the unbinding forces as a function of the loading rate: the thermal off-rate was estimated to be approximately 14 s(-1), much higher than values commonly estimated for complexes more stable than electron transfer complexes. Results here discussed represent the first studies on molecular recognition between two redox partners by atomic force microscopy.