Linking of Sensor Molecules with Amino Groups to Amino-Functionalized AFM Tips

Abstract

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.

Cleavage and Cell Adhesion Properties of Human Epithelial Cell Adhesion Molecule (HEPCAM)

“…AFM measurements were performed at 37°C in Hanks' buffered saline solution buffer using the force spectroscopy mode of a Nanowizard III (JPK Instruments, Berlin, Germany) AFM setup to detect possible single molecule interactions as detailed before (38). Briefly, Si3N4 tips of the cantilever (MLCT, Bruker, Mannheim, Germany) and freshly cleaved mica sheets (SPI Supplies, West Chester, PA) were functionalized with the same amounts of recombinant proteins or BSA using polyethylene glycol spacers (39). To measure single molecule interactions, the AFM tip was repetitively lowered to and retracted from the mica sheet.…”

Peptide-mediated desmoglein 3 crosslinking prevents pemphigus vulgaris autoantibody-induced skin blistering

“…The principles of cell-free binding studies of recombinant DSGs have been outlined earlier (8). In brief, recombinant DSG3-Fc molecules were attached to a mica substrate and to the sharp tip of soft (spring constants 0.01 and 0.03 N/m) silicon nitride cantilevers (MSCT probes; Bruker) via a flexible polyethylenglycol linker (50). Tip and mica were repeatedly brought in contact and retracted using a Bioscope AFM attached to a Nanoscope III controller (Bruker).…”

Nanoscale imaging and force probing of biomolecular systems using atomic force microscopy: from single molecules to living cells

“…However, the current protocols for reliable AFM tip functionalization are quite complex and timeconsuming. 79 Besides, sometimes the chemical synthesis of linker molecules is required, which is usually challenging in cell biology laboratories. Hence, defining simple standardized protocols and providing commercially available linkers for tip functionalization will contribute to making AFM single-molecule force recognition spectroscopy accessible for more researchers.…”

“…101 In the working medium (e.g., cell culture medium) and on the cell surface, there are many different types of biological macromolecules which may bind to the functionalized molecules on the tip and then result in tip contamination. Though the functionalized molecules attached to an AFM tip via PEG linkers can freely move to a certain extent, 79 the frequent contact between the AFM tip and the cell surface may at times compress the functionalized molecules, which may result in the denaturation of the molecules. Since detailed situations of the AFM tip are currently not visible during AFM measurements, these real-time changes on the tip (e.g., tip contamination) cannot be observed and thus potential tip contamination may influence the experiments.…”