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Viscoelasticity of single macromolecules using Atomic Force Microscopy
bioRxiv - Biophysics Pub Date : 2020-05-22 , DOI: 10.1101/2020.05.21.107888
Shatruhan Singh Rajput , Surya Pratap S Deopa , Jyoti Yadav , Vikhyaat Ahlawat , Saurabh Talele , Shivprasad Patil

We measured viscoelasticity of single protein molecules using two types of Atomic Force Microscopes (AFM) which employ different detection schemes to measure the cantilever response. We used a commonly available deflection detection scheme in commercial AFMs which measures cantilever bending and a fibre-interferometer based home-built AFM which measures cantilever displacement. For both methods, the dissipation coefficient of a single macromolecule is immeasurably low. The upper bound on the dissipation coefficient is 5 X 10^(-7) kg/s whereas the entropic stiffness of single unfolded domains of protein measured using both methods is in the range of 10 mN/m. We show that in a conventional deflection detection measurement, the phase of bending signal can be a primary source of artefacts in the dissipation estimates. It is recognized that the measurement of cantilever displacement, which does not have phase lag due to hydrodynamics of the cantilever, is better suited for ensuring artefact-free measurement of viscoelasticty compared to the measurement of the cantilever bending. We confirmed that the dissipation coefficient in single macromolecules is below the detection limit of AFM by measuring dissipation in water layers confined between the tip and the substrate using similar experimental parameters. Further, we experimentally determined the limits in which the simple point-mass approximation of the cantilever works in off-resonance operation.

中文翻译:

使用原子力显微镜观察单个大分子的粘弹性

我们使用两种类型的原子力显微镜(AFM)测量了单个蛋白质分子的粘弹性,该显微镜采用不同的检测方案来测量悬臂响应。我们在商业AFM中使用了一种常见的挠度检测方案,该方案可测量悬臂弯曲,而在基于光纤干涉仪的家用AFM中则可测量悬臂位移。对于这两种方法,单个大分子的耗散系数都非常低。耗散系数的上限为5 X 10 ^(-7)kg / s,而使用这两种方法测得的单个蛋白质未折叠域的熵刚度在10 mN / m的范围内。我们表明,在常规的偏转检测测量中,弯曲信号的相位可能是损耗估计中伪像的主要来源。公认的是,与悬臂弯曲的测量相比,由于悬臂的流体动力学而没有相位滞后的悬臂位移的测量更适合于确保无假象的粘弹性测量。通过使用相似的实验参数测量尖端和底物之间的水层中的耗散,我们确认了单个大分子的耗散系数低于AFM的检测极限。此外,我们通过实验确定了在非共振操作中悬臂的简单点质量近似起作用的极限。与悬臂弯曲的测量相比,它更适合于确保无假象的粘弹性测量。我们通过使用相似的实验参数测量尖端和底物之间的水层中的耗散来确认单个大分子的耗散系数低于AFM的检测极限。此外,我们通过实验确定了在非共振操作中悬臂的简单点质量近似起作用的极限。与悬臂弯曲的测量相比,它更适合于确保无假象的粘弹性测量。我们通过使用相似的实验参数测量尖端和底物之间的水层中的耗散来确认单个大分子的耗散系数低于AFM的检测极限。此外,我们通过实验确定了在非共振操作中悬臂的简单点质量近似起作用的极限。
更新日期:2020-05-22
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