“Seeing and Counting” Individual Antigens Captured on a Microarrayed Spot with Force-Based Atomic Force Microscopy

Abstract

The mapping capability of atomic force microscopy (AFM) enabled us to see captured prostate-specific antigens (PSAs) on a spot microarrayed with the corresponding antibody and count the number of the antigens in a submicrometer area. To enhance the reliability and the reproducibility of the approach, a third-generation dendron was employed for the surface treatment. The specific force between the captured PSA and the detection antibody (5A6) was measured after cross-linking, and the mean unbinding force was 56 +/- 2 pN. At 100 fM, there were 12 captured antigens in 4.32 x 10(4) nm(2), and the number was dependent upon the concentration. A larger hydrodynamic distance (8 +/- 2 nm) of the immunocomplex resulted in a cluster of pixels corresponding to the single complex in a map recorded over a selected area with a positional interval of 3 nm, and this feature helped to discriminate between pixels of the specific interaction and the nonspecific ones. The results indicate that the approach can be applicable to the quantitative analysis of the antigen in a sample and imply that it can be extended to a sample of very low copy numbers as long as the size of the microarrayed spot is reduced.

Direct quantitative analysis of HCV RNA by atomic force microscopy without labeling or amplification

“…In previous studies, we demonstrated that with surface control the 1:1 interaction between DNAs could be enhanced (36), the individual PSAs [prostate-specific antigens] captured on a surface could be counted (40), and that Pax6 mRNA expressed in a mouse embryonic tissue could be imaged (41). As the first step, the capture probe DNA (black), with an amine group at its 3′ end, was covalently linked to the apex of the dendron immobilized on the slide, whereas the detection DNA (green), with an amine group at its 5′ end, was covalently linked to the apex of the dendron immobilized on the AFM tip.…”

The Power of Assemblies at Interfaces: Nanosensor Platforms Based on Synthetic Receptor Membranes

“…Interestingly, these unique abilities obtained from the synthetic receptor membranes were manipulated by controlling the highly-ordered structures of the assemblies (i.e., the intermolecular distance of receptor moieties), suggesting that the sensing ability of the assembly-functionalized sensors can be easily fine-tuned without additional complicated synthesized materials. In fact, the relationship between the molecular recognition ability and lateral spacing of the molecular assembly at the sensing interfaces has been investigated by some groups [94][95][96]. From the perspective of materials science, it is interesting that the fact that the sensing features of molecules follow the configuration of molecular assemblies.…”

Cytosolic targeting factor AKR2A captures chloroplast outer membrane-localized client proteins at the ribosome during translation

“…Force mapping measures key parameters including cluster size, stretching distance, unbinding force value and unbinding probability, and these parameters can be used to confirm the interaction type [28][29][30][31] . Adhesion force maps were obtained for a 300 nm  300 nm area, and the unbinding force was recorded at 15-min intervals with five measurements at each pixel.…”