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Herein, we report a self-locking DNAzyme (SLOD) biosensor that suppresses spontaneous activation through a thermodynamically stabilized intramolecular inactive conformation. Unlike conventional hairpin DNAzymes that rely on direct target-induced activation and often exhibit limited capability for single-base discrimination, the proposed SLOD platform operates through a decoupled mechanism in which single-base mutation recognition is performed through flap endonuclease 1 (FEN1), whereas DNAzyme activation is driven by polymerase-generated competitive unlocking strands. This design separates mutation discrimination from signal generation, thereby improving specificity against wild-type background. The platform enabled quantitative detection of EGFR and KRAS mutations at mutant allele fractions as low as 0.1%\u20130.5%. A dual-color format was further established for the simultaneous detection of EGFR L858R and T790M in clinical samples. In a cohort of 35 clinical samples, the SLOD platform achieved an overall qualitative concordance of 97.1% (34\/35) with the ARMS-PCR. In addition, the programmability of the biosensor was demonstrated through the AND, OR, and INHIBIT logic operations. This work establishes a low-background, single-base-discriminating, and modular DNAzyme biosensor platform for single-base mutation detection and clinically relevant nucleic acid sensing.<\/jats:p>","DOI":"10.1021\/acs.analchem.6c04170","type":"journal-article","created":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T23:02:27Z","timestamp":1790204547000},"page":"29042-29051","source":"Crossref","is-referenced-by-count":0,"title":["A Self-Locking\nDNAzyme Platform for Highly Specific\nSensing of Low-Abundance Single-Base Mutations"],"prefix":"10.1021","volume":"98","author":[{"given":"Yuzhao","family":"Wang","sequence":"first","affiliation":[{"name":"China Pharmaceutical University , , , ,","place":["Nanjing, P. R. 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