Fluorescence-based biosensing with DNA nanostructures and DNA origami: from programmable architectures to quantitative readouts

DNA nanotechnology and DNA origami have emerged as promising platforms for biosensing by exploiting the programmability of DNA sequences and structural self-assembly. Diverse DNA nanostructures, including three-way junctions (TWJs), dendritic assemblies, hydrogels, and tetrahedral DNA nanostructures (TDNs), provide structurally defined frameworks for the organization of functional components. DNA origami further enables highly programmable and precise spatial positioning by folding long DNA scaffolds with multiple staple strands. Their high biocompatibility and nanoscale spatial addressability allow the controlled organization of fluorophores, quenchers, aptamers, nanoparticles, and other functional components, which is particularly advantageous for fluorescence-based sensing strategies that rely on precise spatial control of these components. In this review, we first introduce the fundamental principles of fluorescence-based biosensing, including Förster resonance energy transfer (FRET) and plasmon-enhanced fluorescence (PEF). Then categorize DNA nanostructure-based fluorescence biosensors according to their structural architectures and sensing mechanisms. DNA origami-based platforms are subsequently classified based on their signal transduction strategies. Representative studies are discussed in terms of their design principles and sensing performance. We further critically discuss the remaining challenges, including structural stability, scalability, signal reproducibility, and clinical translation, and provide perspectives on the future development of next-generation fluorescence biosensing platforms enabled by programmable DNA architectures.