Antibody Linking to Atomic Force Microscope Tips via Disulfide Bond Formation

Institute of Biophysics and Institute of Organic Chemistry, J. Kepler University, Altenberger Str. 69, A-4040 Linz, Austria.
Bioconjugate Chemistry (Impact Factor: 4.51). 11/2006; 17(6):1473-81. DOI: 10.1021/bc060252a
Source: PubMed


Covalent binding of bioligands to atomic force microscope (AFM) tips converts them into monomolecular biosensors by which cognate receptors can be localized on the sample surface and fine details of ligand-receptor interaction can be studied. Tethering of the bioligand to the AFM tip via a approximately 6 nm long, flexible poly(ethylene glycol) linker (PEG) allows the bioligand to freely reorient and to rapidly "scan" a large surface area while the tip is at or near the sample surface. In the standard coupling scheme, amino groups are first generated on the AFM tip. In the second step, these amino groups react with the amino-reactive ends of heterobifunctional PEG linkers. In the third step, the 2-pyridyl-S-S groups on the free ends of the PEG chains react with protein thiol groups to give stable disulfide bonds. In the present study, this standard coupling scheme has been critically examined, using biotinylated IgG with free thiols as the bioligand. AFM tips with PEG-tethered biotin-IgG were specifically recognized by avidin molecules that had been adsorbed to mica surfaces. The unbinding force distribution showed three maxima that reflected simultaneous unbinding of 1, 2, or 3 IgG-linked biotin residues from the avidin monolayer. The coupling scheme was well-reproduced on amino-functionalized silicon nitride chips, and the number of covalently bound biotin-IgG per microm2 was estimated by the amount of specifically bound ExtrAvidin-peroxidase conjugate. Coupling was evidently via disulfide bonds, since only biotin-IgG with free thiol groups was bound to the chips. The mechanism of protein thiol coupling to 2-pyridyl-S-S-PEG linkers on AFM tips was further examined by staging the coupling step in bulk solution and monitoring turnover by release of 2-pyridyl-SH which tautomerizes to 2-thiopyridone and absorbs light at 343 nm. These experiments predicted 10(3)-fold slower rates for the disulfide coupling step than actually observed on AFM tips and silicon nitride chips. The discrepancy was reconciled by assuming 10(3)-fold enrichment of protein on AFM tips via preadsorption, as is known to occur on comparable inorganic surfaces.

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    • "Then, the amino-functionalized tip has to be bridged to the biomolecule of interest. This can be achieved through the use of heterobifunctionalized polyethylene glycol (PEG; Kamruzzahan et al., 2006; Ebner et al., 2008; Wildling et al., 2011) or, as we decided in our study, through the use of an aldehyde–phosphorus dendrimer, as we previously described (Jauvert et al., 2012). This strategy developed in our team in 2012, and already used for probing the surface of live bacteria (Formosa et al., 2012), consists in making " dendritips " by reacting amino-functionalized AFM tips with dendrimers, therefore leading to dendrimer-activated tips. "
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    ABSTRACT: Single-molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the β2-adrenergic receptor (β2-AR), a G protein-coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of β2-AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.
    Journal of Molecular Recognition 02/2015; 28(1). DOI:10.1002/jmr.2407 · 2.15 Impact Factor
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    • "Differences in experiments may derive from the choice of AFM equipment (commercial [23], custom built [6] and hybrid setups [5]), substrates (primarily agarose beads [2,6] versus flat substrates; silicon [32], glass [33], gold [5,34]), cantilevers, calibration techniques [35–38] and the varied methods used to analyse the large amounts of data [39,40]. Most significantly however, the surface attachment protocols for coupling the biomolecules of interest to tips and substrates vary greatly between studies [2,5,10,11,34,41] (for a detailed review see [42]). The use of covalent bonds versus physisorption mediated attachment, type of passivation of surfaces employed to minimise non-specific interactions and the use of poly(ethylene glycol) (PEG) linkers [43] which improve ligand-receptor mobility, are all extremely important in these studies. "
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    ABSTRACT: Single molecule force spectroscopy is a technique that can be used to probe the interaction force between individual biomolecular species. We focus our attention on the tip and sample coupling chemistry, which is crucial to these experiments. We utilised a novel approach of mixed self-assembled monolayers of alkanethiols in conjunction with a heterobifunctional crosslinker. The effectiveness of the protocol is demonstrated by probing the biotin-avidin interaction. We measured unbinding forces comparable to previously reported values measured at similar loading rates. Specificity tests also demonstrated a significant decrease in recognition after blocking with free avidin.
    International Journal of Molecular Sciences 12/2012; 13(10):13521-41. DOI:10.3390/ijms131013521 · 2.86 Impact Factor
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    • "Such functionalized probes are then used to scan the sample and map the corresponding target antigen. During the topography scan, the binding of the antibody with its antigen will be detected by the modification of the cantilever oscillation, generating a parallel image of antigen-antibody interaction hits (Hinterdorfer & Dufrene, 2006; Kamruzzahan et al, 2006; Raab et al, 1999; Stroh et al, 2004). The success of the recognition depends on the choice of this specific antibody and the quality of its coating. "
    Atomic Force Microscopy Investigations into Biology - From Cell to Protein, 03/2012; , ISBN: 978-953-51-0114-7
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