Concurrent atomic force spectroscopy

Carolina Pimenta-Lopes,Carmen Suay-Corredera,Jorge Alegre-Cebollada,David Sánchez-Ortiz,Diana Velázquez-Carreras

doi:10.1038/s42005-019-0192-y

Carolina Pimenta-Lopes, Carmen Suay-Corredera + Show 3 more

Open Access

https://doi.org/10.1038/s42005-019-0192-y

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Abstract

Force-spectroscopy by atomic force microscopy (AFM) is the technique of choice to measure mechanical properties of molecules, cells, tissues and materials at the nano and micro scales. However, unavoidable calibration errors of AFM probes make it cumbersome to quantify modulation of mechanics. Here, we show that concurrent AFM force measurements enable relative mechanical characterization with an accuracy that is independent of calibration uncertainty, even when averaging data from multiple, independent experiments. Compared to traditional AFM, we estimate that concurrent strategies can measure differences in protein mechanical unfolding forces with a 6-fold improvement in accuracy or a 30-fold increase in throughput. Prompted by our results, we demonstrate widely applicable orthogonal fingerprinting strategies for concurrent single-molecule nanomechanical profiling of proteins.

Highlights

Force-spectroscopy by atomic force microscopy (AFM) is the technique of choice to measure mechanical properties of molecules, cells, tissues and materials at the nano and micro scales
We find that the relative improvement in relative standard deviation (RSD) achieved by concurrent over traditional atomic force spectroscopy increases with the number of unfolding events per experiment, and remains fairly constant at increasing number of experiments (Fig. 2g)
We address a main limitation of force spectroscopy by AFM that arises from uncertain calibration of cantilevers

Summary

Introduction

Force-spectroscopy by atomic force microscopy (AFM) is the technique of choice to measure mechanical properties of molecules, cells, tissues and materials at the nano and micro scales. Averaging results from several experiments obtained with different cantilevers can reduce the systematic error, since individual calibration errors are more probable to be averaged out as more experiments are included in the analysis[20] The drawback of this method, which we refer to as traditional atomic force spectroscopy, is a considerable loss of throughput of the technique. Concurrent mechanical characterization of several proteins in a single experiment has been achieved using microfluidics, on-chip protein patterning, and force spectroscopy measurements in custom-built atomic force/total internal reflection fluorescence microscopes[22,23]. This advanced technology is not available to most AFM users, and the extent of improvement in performance by concurrent AFM remains unexplored

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