Recent Progress in Molecular Recognition Imaging of Protein Systems at the Nanoscale Level ^†

Identification of proteins has received considerable attention in recent years due to the increasing interest in resolving individual biomolecules under physiologically relevant conditions. In this framework, atomic force microscopy (AFM) has shown great potential to acquire a variety of biomolecular physico-chemical properties at the single molecule level [1]. Particularly, force spectroscopy based on AFM (AFM-FS) allows the intermolecular interactions between two biomolecules to be determined, requiring one to be covalently immobilized on a flat surface and the other linked to the AFM tip. Previous work was developed in this field by simultaneous topography and recognition imaging (TREC) [2] and tuning-fork-based transverse dynamic force microscopy (TDFM) [3], although both methods lack quantitive information. To overcome the aforementioned limitations, force–volume (F-V) [4] emerged as promising alternative, but the extremely large data acquisition times can lead to drifting effects during the image recording. Here, we present the intermittent jumping force mode (JM) as suitable approach to gathering quantitative high-resolution force maps at local areas of the scanned sample with fast-acquisition times. Using this mode, by applying very low forces under respulsive regime conditions, simultaneous maps of topography and specific rupture forces corresponding to the unbinding of the protein:ligand complexes are obtained. Two different protein systems are employed to illustrate the capabilities of the built-up methodological improvements. First, the flavoenzyme system formed between flavodoxin NADP⁺ reductase (FNR) and its redox partners, ferredoxin and flavodoxin [5,6], and second, the strongest non-covalent complexes observed in nature between avidin and streptavidin and biotin [7]. In the first case, the results were optimized when an oriented immobilization procedure was designed. In the second work, discrimination between avidin and streptavidin molecules in a hybrid sample was achieved with a unique sensor ligand. The most relevant scientific outcomes can serve as a proof-of-principle stage to design diagnostic devices with an ultra-sensitivity detection signal for drug screening applications.