Covalent Attachment of Single Molecules for AFM-based Force Spectroscopy

Abstract

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and surfaces. For a true single molecule experiment, covalent attachment of the probe molecule is essential because only then can hundreds of force-extension traces with one and the same single molecule be obtained. Many traces are in turn necessary to prove that a single molecule alone is probed. Additionally, passivation is crucial for preventing unwanted interactions between the single probe molecule and the AFM cantilever tip as well as between the AFM cantilever tip and the underlying surface. The functionalization protocol presented here is reliable and can easily be applied to a variety of polymers. Characteristic single molecule events (i.e., stretches and plateaus) are detected in the forceextension traces. From these events, physical parameters such as stretching force, desorption force and desorption length can be obtained. This is particularly important for the precise investigation of stimuli-responsive systems at the single molecule level. As exemplary systems poly(ethylene glycol) (PEG), poly(N-isopropylacrylamide) (PNiPAM) and polystyrene (PS) are stretched and desorbed from SiO x (for PEG and PNiPAM) and from hydrophobic self-assembled monolayer surfaces (for PS) in aqueous environment.

Adsorbing DNA to mica by cations: Influence of the valency and ion type

“…41 nm was used to bind the DNA to the AFM cantilever tip. 67,68 AFM Cantilever Tip Functionalization Si 3 N 4 AFM cantilevers, namely, MLCT-BIO-DC (force constants: 0.01 N/m -0.1 N/m, Bruker AFM probes, Camarillo, CA, USA) were used for all measurements. First, the cantilevers were activated with oxygen plasma (40%, 0.1 mbar, 2.0 min, Diener Electronics, Ebhausen, Germany) to gain hydroxyl groups on the surface of the cantilever tips.…”

“…The spring constant of the cantilever was determined via the thermal noise method. 68,69 The measurement parameters were defined as follows: force distance: 1 µm; pulling velocity for NaCl: 0.01 µms −1 , 0.1 µms −1 , 1 µms −1 and 10 µms −1 , pulling velocity for all other experiments: 1.00 µms −1 ; contact force: 500 pN; sampling rate: 0.5-16.7 kHz; contact time towards the surface: 0-1 s. Force-extension curves were recorded in a grid-like manner with 10•10 points covering 20 • 20 µm 2 (force maps) as well as in the continuous mode at different surface spots. At least two force maps were obtained per cantilever, pulling velocity and contact time towards the surface.…”

“…For data evaluation, a self-programmed evaluation software based on Igor Pro (Wavemetrics, Portland, OR, USA) was used. The forceextension curves were corrected for drift 67 and classified according to their shapes into plateau and stretching (including multiple stretches). 39 In each force-extension curve the last detachment event, either a plateau or a peak at the end of a stretching event, was taken to determine the detachment force.…”