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.
"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. "
"Differences in experiments may derive from the choice of AFM equipment (commercial , custom built  and hybrid setups ), substrates (primarily agarose beads [2,6] versus flat substrates; silicon , glass , 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 ). 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  which improve ligand-receptor mobility, are all extremely important in these studies. "
[Show abstract][Hide abstract] 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
"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. "
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