DNA-FLASH─a DNAzyme Walker-Based Nanosensor for Digital Biosensing at the Point of Care

The convergence of biosensing and nucleic acid (NA) nanotechnology represents an opportunity for the development of diagnostic technologies. By harnessing the programmability of nucleic acids, we can design biosensors that offer advantages in stability, scalability, versatility and sensitivity, compared to protein-based systems. In this work we introduce DNA-FLASH (DNA-based FLuorescence Amplification upon Single-target Hybridization), a DNA nanosensor concept for digital biosensing. DNA-FLASH leverages fluorescence amplification by a multicomponent NA enzyme (MNAzyme)-driven DNA walker mechanism on a DNA origami disk. Using super-resolution microscopy and single-molecule photobleaching, we demonstrate reproducible fabrication of DNA-FLASH nanosensors with 12 fluorophore-quencher substrates on a ring-shaped track, surrounding a single MNAzyme walker. This nanoarchitecture enables single-molecule detection of DNA targets down to picomolar concentrations. Through precise patterning of DNA-FLASH nanosensors in arrays on glass, we facilitate high-throughput single-molecule readout. We successfully demonstrate DNA-FLASH in human plasma samples and on an in-house developed, fully integrated, self-powered, disposable microfluidic chip, highlighting its potential use in point-of-care settings. Altogether, DNA-FLASH may support the development of next-generation biosensors capable of addressing pressing global challenges, including rapid disease detection, environmental sustainability, and personalized healthcare.