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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.<\/jats:p>","DOI":"10.1021\/acsnano.6c04121","type":"journal-article","created":{"date-parts":[[2026,7,22]],"date-time":"2026-07-22T02:24:13Z","timestamp":1784687053000},"page":"21201-21214","source":"Crossref","is-referenced-by-count":1,"title":["DNA-FLASH\u2500a\nDNAzyme Walker-Based Nanosensor\nfor Digital Biosensing at the Point of Care"],"prefix":"10.1021","volume":"20","author":[{"given":"Seppe","family":"Driesen","sequence":"first","affiliation":[{"name":"KU Leuven , , ,","place":["Leuven, Belgium, 3001"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dries","family":"Vloemans","sequence":"additional","affiliation":[{"name":"KU Leuven , , ,","place":["Leuven, Belgium, 3001"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0642-6892","authenticated-orcid":true,"given":"Gangamallaiah","family":"Velpula","sequence":"additional","affiliation":[{"name":"KU Leuven , , ,","place":["Leuven, Belgium, 3001"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ce\u0301line Van","family":"Leemput","sequence":"additional","affiliation":[{"name":"KU Leuven , , ,","place":["Leuven, Belgium, 3001"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mirjam","family":"Ku\u0308mmerlin","sequence":"additional","affiliation":[{"name":"University of Oxford , , ,","place":["Oxford, U.K., OX1 3QU"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Achillefs N.","family":"Kapanidis","sequence":"additional","affiliation":[{"name":"University of Oxford , , ,","place":["Oxford, U.K., OX1 3QU"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cla\u0301udio","family":"Pinheiro","sequence":"additional","affiliation":[{"name":"Ghent University , , ,","place":["Ghent, Belgium, 9000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"An","family":"Hendrix","sequence":"additional","affiliation":[{"name":"Ghent University , , ,","place":["Ghent, Belgium, 9000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0909-9292","authenticated-orcid":true,"given":"Steven","family":"De Feyter","sequence":"additional","affiliation":[{"name":"KU Leuven , , ,","place":["Leuven, Belgium, 3001"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Karen","family":"Leirs","sequence":"additional","affiliation":[{"name":"KU Leuven , , ,","place":["Leuven, Belgium, 3001"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8143-6794","authenticated-orcid":true,"given":"Jeroen","family":"Lammertyn","sequence":"additional","affiliation":[{"name":"KU Leuven , , ,","place":["Leuven, Belgium, 3001"]},{"name":"KU Leuven , , ,","place":["Leuven, Belgium, 3000"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,7,21]]},"reference":[{"issue":"1","key":"2026080409275429200_cit1","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1042\/EBC20150001","article-title":"Introduction to Biosensors","volume":"60","author":"Bhalla","year":"2016","journal-title":"Essays\nBiochem."},{"issue":"3","key":"2026080409275429200_cit2","doi-asserted-by":"publisher","first-page":"183","DOI":"10.3390\/bios12030183","article-title":"DNA-Based Biosensors for the Biochemical\nAnalysis: A Review","volume":"12","author":"Hua","year":"2022","journal-title":"Biosensors (Basel)."},{"issue":"1","key":"2026080409275429200_cit3","doi-asserted-by":"publisher","first-page":"44","DOI":"10.1021\/acs.analchem.8b05009","article-title":"DNA-Based Scaffolds\nfor Sensing Applications","volume":"91","author":"Ranallo","year":"2019","journal-title":"Anal. 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