Rational design of a 3D DNA origami cube as an ideal signal carrier for glass nanopore-based biosensors

Enhancing the signal-to-noise ratio (SNR) has long been a focus of research in the development of nanopore sensors. Herein, a 3D DNA origami cube with adjustable rigidity that exhibits exceptional selectivity and an ultra-high SNR exceeding 100 is designed and optimized. The assembly of this 3D DNA origami cube relies on a single scaffold strand derived from target DNA/RNA, as well as multiple staple strands. This construction process ensures that the cube only forms in the presence of the target DNA/RNA, thereby providing a highly sensitive and specific detection strategy. Without any intermediate steps, DNA amplicons amplified from a plasmid containing the S gene of the Omicron variant of SARS-CoV-2 were successfully transformed into 3D DNA origami cubes in the presence of staple strands in a one-pot reaction process. Nanopore counting of 3D DNA origami cubes, instead of the direct detection of the target gene, significantly ameliorates the interference effect from non-target subjects possessing a similar size and charge. This approach features an ultra-low detection limit of 50 aM, with a broad detection range from 50 aM to 50 pM, even in commercial buffers that contain large amounts of enzymes, stable proteins, and other non-target DNA sequences. Moreover, the DNA origami cube has a thermal stability of up to 70 °C, which allows it to be used in a wide range of scenarios, including harsh conditions. We aim to extend this approach to detect many other targets and to integrate it into broader diagnostic toolkits. Nevertheless, given space constraints, not all possible applications could be thoroughly explored within this study.