Concurrent atomic force spectroscopy

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.J.A.-C. acknowledges funding from the Ministerio de Ciencia, Innovación y Universidades (MCNU) through grants BIO2014-54768-P, BIO2017-83640-P (AEI/FEDER, UE), and RYC-2014-16604, the European Research Area Network on Cardiovascular Diseases (ERA-CVD/ISCIII, MINOTAUR, AC16/00045), the Comunidad de Madrid (P2018/NMT-4443), and the CNIC-Severo Ochoa intramural grant program (03-2016 IGP). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), MCNU and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). C.P.-L. was a recipient of CNIC Master Fellowship. C.S.-C. is the recipient of an FPI predoctoral fellowship BES-2016-076638. We thank Natalia Vicente for excellent technical support (through grant PEJ16/MED/TL-1593 from Comunidad de Madrid).S