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. 2022 Dec 27;28(1):226.
doi: 10.3390/molecules28010226.

High-Yield Characterization of Single Molecule Interactions with DeepTipTM Atomic Force Microscopy Probes

Daniel Corregidor  1   2 , Raquel Tabraue  1   2 , Luis Colchero  1   2 , Rafael Daza  1   2 , Manuel Elices  1   2 , Gustavo V Guinea  1   2   3   4 , José Pérez-Rigueiro  1   2   3   4
Affiliations

High-Yield Characterization of Single Molecule Interactions with DeepTipTM Atomic Force Microscopy Probes

Daniel Corregidor et al. Molecules. .

Abstract

Single molecule interactions between biotin and streptavidin were characterized with functionalized DeepTipTM probes and used as a model system to develop a comprehensive methodology for the high-yield identification and analysis of single molecular events. The procedure comprises the covalent binding of the target molecule to a surface and of the sensing molecule to the DeepTipTM probe, so that the interaction between both chemical species can be characterized by obtaining force-displacement curves in an atomic force microscope. It is shown that molecular resolution is consistently attained with a percentage of successful events higher than 90% of the total number of recorded curves, and a very low level of unspecific interactions. The combination of both features is a clear indication of the robustness and versatility of the proposed methodology.

Keywords: AFM; affinity atomic force microscopy; biotin; functionalization; streptavidin.

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Conflict of interest statement

This study was partially funded by the company Bioactive Surfaces S.L. that provided the DeepTipTM probes. DeepTipTM probes are commercially available from the company, and free trial batches of DeepTipTM probes may be available upon request to the company or after contacting the corresponding author.

Figures

Figure 1
Figure 1
Fluorescent microscopy images of DeepTipTM (left) and control probes (right). Samples were incubated with fluorescein isothiocyanate to assess the presence of amine groups at the surface. Images are composed to show both sides of the cantilevers.
Figure 2
Figure 2
SEM images and AFM topography images of the DeepTipTM probes. (A,C) functionalized tip, (B,D) non-functionalized tip (control).
Figure 3
Figure 3
Schematic representation of the silicon substrate functionalized with streptavidin and of the AFM tip functionalized with the sensor molecule (biotin). (Left): non-blocked streptavidin. (Right): sample after the streptavidin molecules are blocked with biotin-BSA.
Figure 4
Figure 4
Representative curves of each of the six groups in which the interactions were classified. A seventh group was established with those curves considered as anomalous resulting from experimental artefacts.
Figure 5
Figure 5
Histogram with the number of curves grouped by the value of the adhesion force of (A) pristine streptavidin and (B) blocked streptavidin. The range of forces that corresponds to each type of curve is indicated in the Figure.
Figure 6
Figure 6
Chart with the percentages of each group of curves in both experimental conditions without (pristine streptavidin) and with BSA (blocked streptavidin). Type 1: no interaction; Type 2: non-specific interaction; Type 3: single interaction; Type 4: multiple interactions–independent detachment; Type 5: multiple interactions–simultaneous detachment; Type 6: multiple interactions–combined independent and simultaneous detachment; Type 7: discarded F-d curves.
Figure 7
Figure 7
Difference of the percentage of curves that correspond to a given type as a function of the number of experiments for (A) experiments without BSA and (B) experiments with BSA. The % difference is calculated by subtracting the percentage of curves of a given type in an interval (i.e., curves from 51 to 100) from the arithmetic mean calculated from all the curves obtained in a given type of experiment.
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