Viscoelasticity of single biopolymers using Atomic Force Microscopy

Abstract

Viscoelasticity of single protein molecules is essential to fully understand their dynamic properties and functions. It is also believed that the initial collapse in protein folding is governed by their viscoelasticity. Dynamic atomic force microscopy is recognised as a powerful tool for direct measurement of viscoelasticity in the single molecules. This method had also been vastly applied to understand the dynamic properties of the nanoconfined liquids. The estimated viscoelasticity using this method is always debated due to the complex dynamic behaviour of AFM cantilever-beam in liquid environment. In order to resolve this issue, understanding the cantilever dynamics under the influence of interaction force in liquid environment is essential. We have done a comprehensive work to precisely determine the interaction viscoelasticity using amplitude-modulation atomic force microscopy (AM-AFM). Single protein molecule (titin I278) has been chosen as a model system for the study. We have applied two types of AFMs- slope detection based (commercial) and displacement detection based (interferometer based home-built) AFM. Two types of cantilever excitation mechanisms have been used- acoustic excitation (cantileverbase is excited using dither piezo) and magnetic excitation (cantilever-tip is excited using the magnetic excitation). Experiments were performed at truly off-resonance regime to avoid the complexities arising at on-resonance operation. Data has been analyzed using two mathematical models- continuous-beam (CB) and point-mass (PM) model. The experiments performed using different AFMs and using different cantilever excitation schemes and data analyzed using different models have been compared and an unified understanding have been tried building up to understand the AM-AFM measurements and its outcomes. We found that there are various sources which can introduce errors/artefacts in final results such as offset in the cantilever initial phase, inappropriate choice of operation frequency and mathematical model for data analysis etc. We propose methodology to perform AM-AFM measurements in order to get accurate results. We also propose the validity limit of widely used point-mass (PM) model. Our work is not only applicable for single protein molecule measurements but all the biopolymers and nano-scale systems which viscoelasticity can be probed using dynamic atomic force microscopy technique.