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Here, we show that ligand\u2010defined interfacial charge regulation, achieved using weakly ionized ascorbic acid (AA) ligands, markedly lowers the kinetic barrier for DNA chemisorption on AuNPs. Under near\u2010neutral conditions, this interface enables rapid, sequence\u2010general functionalization of AuNPs with both thiolated and non\u2010thiolated oligonucleotides through simple vortex mixing, even for DNA containing only a single terminal adenine. The AA\u2010regulated interface also overcomes the long\u2010standing incompatibility between \u2010butanol dehydration and non\u2010thiolated DNA, enabling sequence\u2010general SNA formation under dehydration conditions across multiple nanoparticle systems. Mechanistic studies indicate that AA reduces interfacial electrostatic repulsion while promoting hydrogen\u2010bond\u2010assisted surface interactions, and base\u2010substitution experiments identify the adenine N6\u2010amino group as a critical site for chemisorption. The resulting ordered DNA corona generates uniform plasmonic nanogaps, enabling reproducible, label\u2010free SERS with single\u2010base resolution for nucleotide discrimination and cytosine methylation analysis. This work establishes a general interfacial design strategy for constructing uniform plasmonically active nucleic acid nanostructures under mild conditions.<\/jats:p>","DOI":"10.1002\/advs.75804","type":"journal-article","created":{"date-parts":[[2026,6,9]],"date-time":"2026-06-09T07:42:12Z","timestamp":1780990932000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Ligand\u2010Defined Interfacial Chemistry Enables Instant and Sequence\u2010General DNA Chemisorption on Gold Nanoparticles Toward Label\u2010Free SERS With Single\u2010Base Resolution"],"prefix":"10.1002","volume":"13","author":[{"given":"Guangping","family":"Li","sequence":"first","affiliation":[{"name":"Joint Research Center for Food Derived Functional Factors and Synthetic Biology of IHM, Anhui Provincial International Science and Technology Cooperation Base for Major Metabolic Diseases and Nutritional Interventions, China Light Industry Key Laboratory of Meat Microbial Control and Utilization, School of Food and Biological Engineering Ministry of Education Hefei University of Technology Hefei P. 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