Identification of Individual Target Molecules Using Antibody-Decorated DeepTipTM Atomic-Force Microscopy Probes

Abstract

A versatile and robust procedure is developed that allows the identification of individual target molecules using antibodies bound to a DeepTip^TM functionalized atomic-force microscopy probe. The model system used for the validation of this process consists of a biotinylated anti-lactate dehydrogenase antibody immobilized on a streptavidin-decorated AFM probe. Lactate dehydrogenase (LDH) is employed as target molecule and covalently immobilized on functionalized MicroDeck^TM substrates. The interaction between sensor and target molecules is explored by recording force-displacement (F-z) curves with an atomic-force microscope. F-z curves that correspond to the genuine sensor-target molecule interaction are identified based on the following three criteria: (i) number of peaks, (ii) value of the adhesion force, and (iii) presence or absence of the elastomeric trait. The application of these criteria leads to establishing seven groups, ranging from no interaction to multiple sensor-target molecule interactions, for which force-displacement curves are classified. The possibility of recording consistently single-molecule interaction events between an antibody and its specific antigen, in combination with the high proportion of successful interaction events obtained, increases remarkably the possibilities offered by affinity atomic-force microscopy for the characterization of biological and biomimetic systems from the molecular to the tissue scales.

Keywords: affinity atomic-force microscopy; antibody; antigen; functionalization; single-molecule resolution.

Figure 1

Florescence images of MicroDeck^TM surfaces: (a) Control substrate functionalized with amine groups. (b) Control substrate decorated with aldehyde groups. (c) Substrate decorated with thiol groups after incubation of a sulfo-LC-SPDP-decorated sample with TCEP. (d) Bar chart comparing the mean fluorescence intensities of the decorated and control samples. Statistical significance: p < 0.01 (**).

Figure 2

Florescence images of DeepTip^TM probes: (a) Probes decorated with an anti-LDH antibody and incubated with a fluorescent anti-anti-LDH antibody. (b) Control probes not decorated with an anti-LDH antibody, but incubated with a fluorescent anti-anti-LDH antibody. The broken lines correspond to the silhouette of the cantilevers (c) Bar chart comparing the mean fluorescent intensity of the decorated and non-decorated samples. Statistical significance: p < 0.01 (**).

Figure 3

Comparison of the mean 340 nm absorbance variation of non-decorated (physical adsorption) and LDH-decorated surfaces. Statistical significance: p < 0.05 (*), p < 0.01 (**).

Figure 4

Representative force–displacement (F–z) curves obtained from the interaction between anti-LDH antibodies and LDH. In this figure, six types are shown: (i) no−interaction curve; (ii) elastomeric curve; (iii) non−elastomeric curve; (iv) elastomeric curve—two peaks; (v) double-peak curve—combined; (vi) three- or four-peak curve. An additional 7% of the curves were discarded due to them exhibiting an abnormal shape. Compression forces are positive and positive displacement corresponds to the retraction of the AFM probe from the substrate.

Figure 5

Percentages of each type of force–distance curve. (Type 1: 47%—no-interaction curves; Type 2: 10%—elastomeric curves; Type 3: 26%—non-elastomeric curves; Type 4: 2%—elastomeric–two-peak curves; Type 5: 7%—combined curves; Type 6: 1%—curves with threefold or fourfold elastomeric peaks; Type 7: 7%—discarded curves).

Figure 6

Statistical distribution of the force–distance curves corresponding to elastomeric and non-elastomeric peaks as a function of the measured adhesion force.