The bottom-up manufacturing of DNA origami structures allows for precise control of DNA-based nanostructures’ shape, size, and functionality, making it a powerful tool in nanotechnology. Single-molecule fluorescence measurements, including Forster resonance energy transfer (FRET), are often used to visualise the nanoscale movements of these structures. However, FRET is prone to crosstalk when labelled structures are within a 10 nm range of each other. DNA origami immobilisation within nanoarrays can minimise the crosstalk, since their spatial spacing would be beyond the effective FRET range.

This research aimed to generate nanoarrays with at least 5x5 binding spots and develop a method for filling up this nanoarray with at least 50% DNA origami structures without altering their dynamics. Using nanosphere lithography (NSL), a technique in which the nanospheres self-assemble into a hexagonal closed-packed (HCP) structured monolayer mask, with 300, 600, and 800 nm nanospheres, two types of nanoarrays were fabricated. (1) Oxygen plasma exposure of the nanosphere of the mask, followed by gold sputtering,
resulted in binding spots with a diameter as small as 191.1 nm for biotin-BSA surface functionalization. (2) Heat treatment of the nanosphere mask, reducing the hole size between nanospheres to a minimum of 60 nm. Annealing resulted in regularly spaced small gold islands with a diameter of ∼173.2 nm for thiol-modified DNA immobilization. Both array types were able to create 5x5 binding spot areas confidently.

Fluorescent experiments were performed to analyse the nanoarray occupancy and Holliday Junctions (HJ) dynamics. Within the reduced sphere size via oxygen plasma treatment array, ∼4.3% of 700 analysed traces were HJs, while only ∼0.7% showed their characteristic switching states. Therefore, the proposed method did not reach the occupancy goal of 50%, but the HJ dynamics were preserved. Two traces within the array were compared with baseline traces and gave t-values of 0.59 and 8.73, indicating no significant change in dynamics. 

The novelty of using gold sputtering to create nanoarrays while placing a dynamic DNA origami structure (HJ) led to functional DNA behaviour, as verified by their dynamics, but more research is required to optimise further the results found.