A DNA origami plasmonic sensor with environment-independent read-out

Abstract

DNA origami is a promising technology for its reproducibility, flexibility, scalability and biocompatibility. Among the several potential applications, DNA origami has been proposed as a tool for drug delivery and as a contrast agent, since a conformational change upon specific target interaction may be used to release a drug or produce a physical signal, respectively. However, its conformation should be robust with respect to the properties of the medium in which either the recognition or the read-out take place, such as pressure, viscosity and any other unspecific interaction other than the desired target recognition. Here we report on the read-out robustness of a tetragonal DNA-origami/gold-nanoparticle hybrid structure able to change its configuration, which is transduced in a change of its plasmonic properties, upon interaction with a specific DNA target. We investigated its response when analyzed in three different media: aqueous solution, solid support and viscous gel. We show that, once a conformational variation is produced, it remains unaffected by the subsequent physical interactions with the environment.

Keywords: DNA origami; gold nanoparticle; molecular detection; plasmonic sensor.

Figure 1

Design, synthesis and characterization of the tetragonal DNA origami sensor. (a) Nomenclature of the tetrahedron geometry: the blue facet is the base; the green facets are NP facets; the red striped facet is probe facet; the red dashed corners are probe struts. (b) DNA origami design: 20 nm gold nanoparticles are placed in the center of the AuNP facets so that at rest the center-to-center interparticle distance is 30 nm; a ssDNA probe links the probe struts across the probe facet. The central section of the probe struts is designed in order to have a variable flexibility. In this experiment two probe struts are used: one flexible with three single strands DNA and one double strand segment (3ss) and one with zero single strand and four double strand segments (0ss).The hybridization of a molecular target, an hairpin ssDNA strand, with the probe strand induces DNA origami squeezing, reducing the interparticle gap. (c) Agarose gel electrophoresis of 0ss and 3ss tetrahedrons decorated with AuNP before (lane 1 for 0ss, lane 3 for 3ss) and after probe-target actuation (lane 2 for 0ss and lane 4 for 3ss); the dotted boxes underline the well-folded DNA origami structures in UV/Vis mode. The excess of free staples strands is not visible in the picture because after 2 h it ran beyond the gel boundary. The AuNP band, even if coated with ssDNA, cannot be highlighted by the UV lamp because the fluorescence caused by the intercalation of GelRed requires the presence of double strand DNA. (d) SEM picture of freshly prepared DNA origami tetrahedron without AuNP on carbon TEM grid, white circles are crystallized salt residues. (e) Contact mode AFM topography in air on mica of a 2D DNA origami structures without AuNP designed and synthesized specifically to allow AFM investigations. (f) Cryo-EM image of the hybrid AuNP-DNA origami particles embedded in vitrified ice.

Figure 2

SAXS measurements performed in liquid on tetrahedron 0ss and 3ss before and after the probe-target actuation; samples were not purified from excess of AuNP. (a) Scattering pattern of all samples and the DNA-functionalized AuNP reference sample: all the traces mostly superimpose suggesting that the largest contribution to the signal comes from single AuNP form factor. (b) In order to extract the particle-particle interference, the structure factor has been derived by dividing the pattern with the AuNP signal (gold-yellow line). The structure factor of the different tetrahedrons highlights the presence of differences among the samples. The dashed box indicates the region of interest in which we expect to see a change in the structural factor as described in the main text. (c) The absolute value of the difference between the trace of the no target structure (dotted curves) and the traces of target structures (black and red curves) showed that the two samples are peaked at different q, corresponding to different interparticle distances: 25 nm for 3ss and 30 nm for 0ss. These different peak positions demonstrate that the reduction of the interparticle distance from the initial value of 30 to 25 nm is effectively detected only with the more flexible tetrahedron.

Figure 3

LSPR properties of AuNP-DNA origami hybrid stucture. (a) AuNP LSPR analysis in solution. Spectra from AuNP suspended in BSPP solution (black), DNA coated AuNP (red), 0ss (green) and 3ss (blue) origami are displayed. The LSPR peak of DNA-covered AuNP is red-shifted with respect to AuNP in BSPP solution due to an increase of dielectric permittivity of the surrounding medium. A further red-shift for the 0ss and the 3ss is caused by a weak plasmonic coupling between two AuNP connected to the tetrahedral DNA origami. A larger broadening of the 3ss curve with respect to the 0ss curve can be explained by the higher flexibility of 3ss DNA origami structure leading to a less tight interparticle distance. (b) AuNP LSPR analysis in agarose gel, after purification. Spectra from 0ss structures with (red) and without (black) target. No spectral shift is observed. (c) AuNP LSPR analysis in agarose gel, after purification. Spectra from 3ss structures with (red) and without (black) target. A significant red-shift is observed attributed to the interparticle distance reduction due to the probe-target hybridization.

Figure 4

SEM analysis of the interparticle distance. (a) Statistical distribution of interparticle distance (center-to-center) of 3ss dimers before (black) and after (red) the hybridization of target. The interparticle gaps were evaluated measuring the brightness profile of the SEM pictures through SEM SUPRA software tool. Before hybridization the distribution is broad with a bimodal character peaked at 27 and 33 nm. After target hybridization, the distribution become monomodal peaked at 23–24 nm. (b) and (c) Representative SEM image of not purified DNA origami samples. Well-folded 3ss tetrahedron decorated with two gold nanoparticles are easily identified and distinguishable from the free AuNP also present in the solution. In the inset a representative profile used to evaluate the interparticle gap of 3ss before (b) and after (c) probe-target actuation.