Imaging and Force Recognition of Single Molecular Behaviors Using Atomic Force Microscopy

Abstract

The advent of atomic force microscopy (AFM) has provided a powerful tool for investigating the behaviors of single native biological molecules under physiological conditions. AFM can not only image the conformational changes of single biological molecules at work with sub-nanometer resolution, but also sense the specific interactions of individual molecular pair with piconewton force sensitivity. In the past decade, the performance of AFM has been greatly improved, which makes it widely used in biology to address diverse biomedical issues. Characterizing the behaviors of single molecules by AFM provides considerable novel insights into the underlying mechanisms guiding life activities, contributing much to cell and molecular biology. In this article, we review the recent developments of AFM studies in single-molecule assay. The related techniques involved in AFM single-molecule assay were firstly presented, and then the progress in several aspects (including molecular imaging, molecular mechanics, molecular recognition, and molecular activities on cell surface) was summarized. The challenges and future directions were also discussed.

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

“…From the force curve, the maximum interaction force (called the peak force) between the AFM probe and the sample is used to control the vertical forces exerted on the sample from the AFM tip. We know that the conventional tapping mode reduces the influence of lateral forces on the sample, but the vibration frequency of the AFM probe in the conventional tapping mode is near the resonant frequency of the probe, 53 resulting in the relatively large tapping force on the sample and therefore this can easily blur the fine structures on the cell surface. In PFT, the vibration frequency of the AFM probe is much less than its resonant frequency, and thus exerts a smaller tapping force (in the range of some tens of piconewtons 52 ) on the sample.…”

“…First, PEG is covalently bound to both the tip and the antibodies. 53 The covalent bonds are at least ten times stronger ( could link unmodified antibodies onto the AFM tip without loop formation on the tip surface. Besides, this method requires a small consumption of antibodies, therefore providing a promising way for AFM tip functionalization.…”

“…Each peak corresponds to a specific section of the membrane protein. The recorded force curve is fitted by the worm-like chain (WLC) model: 53,141 FðxÞ…”

“…139,[153][154][155] We know that most proteins must fold into unique three-dimensional structures to perform their biological functions. 53 Folding and unfolding are crucial ways of regulating biological activities and targeting proteins to different cellular locations, whereas protein misfolding is often related to a wide range of diseases. 156 Hence, mechanically force unfolding single membrane proteins by AFM can help us to understand how membrane proteins fold, how they are stabilized, and how they misfold, which in turn provides novel insights into cellular processes related to diseases.…”

Nano-scientific Application of Atomic Force Microscopy in Pathology: from Molecules to Tissues

“…In addition to its sub-nanometer resolution and pico-newton force sensitivity, AFM is capable of recognizing single molecular events, compositional changes, and intercellular interactions occurring in heterogeneous biological systems during disease progression ( Fig. 1) [1,2]. Many pioneer studies have investigated the possibility of utilizing AFM as a nano-diagnostic tool to establish unbiased quantitative assessment rubrics to monitor pathological conditions [3].…”