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Force spectroscopic detection of peptide cleavage by thrombin exploiting biotin–streptavidin interactions in a bio-sensing context

Author:
Li, Jingfeng, Li, Qing, Potthoff, Sebastian, Wei, Gang, Lucio, Colombi Ciacchi
Source:
Analytical methods 2019 v.11 no.8 pp. 1102-1110
ISSN:
1759-9679
Subject:
ligands, spectroscopy, thrombin
Abstract:
Atomic-force-microscopy-based single-molecule force spectroscopy (AFM-SMFS) has become an important technique as the basis of novel, label-free biosensing strategies. In this work, we explored the possibility of using AFM-SMFS to detect the specific peptide cleavage activity by thrombin proteases. To achieve this aim, an oligopeptide with a sequence that is recognized and cleaved by thrombin was spanned between an AFM tip and a solid surface via a PEG-based linker system including the stable biotin–streptavidin receptor/ligand complex. We found that force spectral signatures associated with the biotin–streptavidin unbinding in the absence of thrombin can be clearly distinguished from the signatures of spectra collected after thrombin-induced peptide cleavage. However, this is possible only in a well-defined window of peak force and tip–sample separation distance values, in which specific streptavidin–biotin interactions do not overlap with non-specific interactions among all the other system components. The results were employed to engineer a sensing architecture capable of detecting the presence of thrombin at concentrations higher than about 0.2 μM.
Agid:
6314149