Nanoscale Mapping Functional Sites on Nanoparticles by Points Accumulation for Imaging in Nanoscale Topography (PAINT)

Abstract

The ability of nanoparticles to selectively recognize a molecular target constitutes the key toward nanomedicine applications such as drug delivery and diagnostics. The activity of such devices is mediated by the presence of multiple copies of functional molecules on the nanostructure surface. Therefore, understanding the number and the distribution of nanoparticle functional groups is of utmost importance for the rational design of effective materials. Analytical methods are available, but to obtain quantitative information at the level of single particles and single functional sites, i. e., going beyond the ensemble, remains highly challenging. Here we introduce the use of an optical nanoscopy technique, DNA points accumulation for imaging in nanoscale topography (DNA-PAINT), to address this issue. Combining subdiffraction spatial resolution with molecular selectivity and sensitivity, DNA-PAINT provides both geometrical and functional information at the level of a single nanostructure. We show how DNA-PAINT can be used to image and quantify relevant functional proteins such as antibodies and streptavidin on nanoparticles and microparticles with nanometric accuracy in 3D and multiple colors. The generality and the applicability of our method without the need for fluorescent labeling hold great promise for the robust quantitative nanocharacterization of functional nanomaterials.

Detecting nanoscale distribution of protein pairs by proximity dependent super-resolution microscopy

“…Using i = 5 nM we obtained D, mean single = 57.1 s (Fig. 5b, see Methods in the Supporting Information) and on = 3.5 × 10 6 M −1 s −1 for conventional DNA-PAINT, in broad agreement with previously reported values 35,38 .…”

“…The very high affinity of biotin ensures an effectively permanent attachment of biotinylated S1 and S2 constructs. Streptavidin was attached to the surface of polystyrene colloidal particles, to provide a convenient imaging geometry as previously used in the context of DNA-PAINT by ourselves 34 and others 35 . Biotinylated S1 and S2 constructs were mixed thoroughly before streptavidin-coated particles were dispersed on the coverslip to ensure a stochastic attachment of S1 and S2 to the biotin-binding sites.…”

“…5a) 22 . Binding of a single docking strand on a particle was achieved by functionalizing the particles with a very dilute solution of docking strands (0.5 nM, see also Delcanale et al 35 ). Using i = 5 nM we obtained D, mean single = 57.1 s (Fig.…”

3D super-resolution microscopy performance and quantitative analysis assessment using DNA-PAINT and DNA origami test samples

“…The large size and high signal-to-noise ratio under brightfield illumination make them also an ideal candidate for sample tracking using correlation methods [62]. In fact, similar microspheres have been used as an effective and affordable way to calibrate the z response of astigmatic 3D localisation [46,63].…”

“…Now, commercial slides with 3D DNA-origami structures are available which provide a stringent test of 3D imaging performance. In addition, we [45] and others [46,47] have established DNA-PAINT samples based on surface coated microspheres which can be used for a variety of testing purposes associated with DNA-PAINT, including their use as convenient 3D structures of known shape.…”