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

“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.”

2.4. Biotin–Streptavidin Interaction Measured with the AFM

In order to assess the efficiency of the DeepTipTM probes to perform experiments that imply molecular interactions, the biotin-streptavidin system was used as model. A schematic representation of the system is presented in 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.
Streptavidin-coated silicon substrates were prepared as explained below, and experiments proceeded by recording 250 F-d curves with a single tip on a substrate. Supplementary Figure S1 Subsequently, the sample was incubated with biotin–albumin to block the binding sites of streptavidin, and 250 F-d additional curves were recorded in the blocked condition with the same tip. Consequently, each couple tip-substrate was used to obtain 500 curves. Since the obtaining of each curve takes approximately 15 s, the recording of the 250 curves of each batch is completed in a time of approximately one hour. The experiment was duplicated, so that the results presented below were obtained from two different DeepTipTM probes on two different silicon substrates.
All the curves were analysed to obtain both the adhesion force and the number of interactions (peaks). These parameters were used to classify the recorded curves into six different groups as illustrated in 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.
The curves were classified by the following two criteria: (i) the maximum value of the adhesion force and (ii) the number of peaks present in the curve. Based on these two criteria, the following six groups were defined: (1) no interaction curves, in which no adhesion events are observed, (2) non-specific interaction curves, with low adhesion (upper limit of the adhesion force F = 200 pN), (3) single interaction curves, with a high adhesion event (upper limit of the adhesion force F = 650 pN), (4) multiple interactions–independent detachment curves, with several consecutive high adhesion events (upper limit of the adhesion force F = 650 pN), (5) multiple interactions–simultaneous detachment curves, with a very high adhesion event (upper limit of the adhesion force F = 2000 pN), and (6) multiple interactions–combined independent and simultaneous detachment curves, with a combination of multiple simultaneous and consecutive very high adhesion events (upper limit of the adhesion force F = 2000 pN). In addition, a seventh group that includes those curves that were discarded as experimental artefacts was also established. A representative curve of each one of the six groups is shown in Figure 4. It is worth indicating that establishing a quantitative criterium for the classification of the F-d curves allows the automatization of the procedure, so that a much larger number of curves can be conveniently classified in future works. However, the classification following this criterium of the curves indicated below proceeded manually, since the implementation of a fully automatized process will require a series of validation steps that were outside of the initial scope of this work.
Figure 5 shows the number of curves for each value of the adhesion force, and the distribution of relative frequencies of each type of curves under the two experimental conditions considered (pristine streptavidin and blocked streptavidin) is shown in Figure 6.
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. 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.
In order to assess the robustness of the procedure and, especially, of the repetitive interaction between the functionalized tip and the substrate, the distribution of the curves in the seven types with respect to the order of each curve along the temporal series is shown in Figure 7Figure 7 presents the difference in the percentage of a given type of curve in an interval of 50 consecutive curves along the temporal series and the arithmetic mean calculated for that type, considering the whole set of curves of a given experiment, either in the pristine condition or after incubation with biotin-BSA. In this case, the absence of any defined trend along the temporal series with respect to the number of curves belonging to a given group points to the robustness of the procedure, since it precludes any significant degradation of the tip as a result of the repetitive interaction with the sample.
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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