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Here, focusing on reactive oxygen species (ROS) as pivotal signaling mediators, we developed defect\u2010programmed DNA origami ROS sensors (DOSs) for portable urinalysis of localized oxidative stress. Using triangular DNA origami (DO) nanostructures as two\u2010dimensional synthetic soft crystals, we programmed the number of discontinuity defects between adjacent staple strands and established a positive correlation between defect number and ROS\u2010triggered degradation kinetics. To transform this programmable degradation into a diagnostic function, we then engineered DOSs via orthogonal assembly of targeting and signaling modules onto DO. In a murine model of acute liver injury (ALI), DOSs selectively accumulated in the liver and underwent ROS\u2010triggered fragmentation into renal\u2010clearable debris, converting hepatic ROS levels into quantifiable urinary signals. Notably, this transformation efficiency depended positively on defect number in DOSs, enabling portable urinalysis that detected ALI onset at least 4\u00a0h earlier than conventional alanine aminotransferase (ALT) testing, with a maximum area under the curve of 0.94. This defect\u2010engineering strategy establishes a generalizable platform for early, noninvasive diagnosis of ROS\u2010related diseases.<\/jats:p>","DOI":"10.1002\/adma.74432","type":"journal-article","created":{"date-parts":[[2026,8,1]],"date-time":"2026-08-01T10:17:53Z","timestamp":1785579473000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Defect Engineering of DNA Origami ROS Sensors for Portable Urinalysis"],"prefix":"10.1002","volume":"38","author":[{"given":"Shuangye","family":"Zhang","sequence":"first","affiliation":[{"name":"State Key Laboratory of Synergistic Chem\u2010Bio Synthesis School of Chemistry and Chemical Engineering New Cornerstone Science Laboratory Frontiers Science Center For Transformative Molecules Zhang Jiang Institute For Advanced Study and National Center For Translational Medicine Shanghai Jiao Tong University  Shanghai China"},{"name":"Department of Chemistry The Chinese University of Hong Kong  Hong Kong China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhe","family":"Liu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Synergistic Chem\u2010Bio Synthesis School of Chemistry and Chemical Engineering New Cornerstone Science Laboratory Frontiers Science Center For Transformative Molecules Zhang Jiang Institute For Advanced Study and National Center For Translational Medicine Shanghai Jiao Tong University  Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuanhao","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Liver Surgery School of Medicine Shanghai Institute of Transplantation Renji Hospital Shanghai Jiao Tong University  Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaodong","family":"Xie","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Synergistic Chem\u2010Bio Synthesis School of Chemistry and Chemical Engineering New Cornerstone Science Laboratory Frontiers Science Center For Transformative Molecules Zhang Jiang Institute For Advanced Study and National Center For Translational Medicine Shanghai Jiao Tong University  Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mingqiang","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Synergistic Chem\u2010Bio Synthesis School of Chemistry and Chemical Engineering New Cornerstone Science Laboratory Frontiers Science Center For Transformative Molecules Zhang Jiang Institute For Advanced Study and National Center For Translational Medicine Shanghai Jiao Tong University  Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7505-2727","authenticated-orcid":false,"given":"Xiaolei","family":"Zuo","sequence":"additional","affiliation":[{"name":"Institute of Molecular Medicine Shanghai Key Laboratory for Nucleic Acids Chemistry and Nanomedicine School of Medicine Renji Hospital Shanghai Jiao Tong University  Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1166-6583","authenticated-orcid":false,"given":"Qian","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Synergistic Chem\u2010Bio Synthesis School of Chemistry and Chemical Engineering New Cornerstone Science Laboratory Frontiers Science Center For Transformative Molecules Zhang Jiang Institute For Advanced Study and National Center For Translational Medicine Shanghai Jiao Tong University  Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9087-960X","authenticated-orcid":false,"given":"Dennis K. 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