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However, the accurate and rapid delineation of tumor margins intraoperatively is still a significant challenge. Herein, we designed a near\u2010infrared fluorescent probe DCIP\u2010ENTK by incorporating dicyanoisophorone dye with a negatively charged peptide, which can be specifically cleaved by enterokinase (ENTK, a tumor biomarker for breast cancer). In vitro experiments confirmed that the probe could quickly (&lt;10\u00a0min), specifically, and sensitively (LOD = 0.031 U\/mL) respond to ENTK. Notably, this probe was engineered as a peptide\u2010based architecture that can self\u2010assemble into a nanoprobe. This design not only provides excellent biocompatibility and in vivo stability, but also enables specific mitochondria\u2010targeting within cancer cells. Upon cleavage by ENTK, the released hydrophobic fluorophores aggregated at the enzyme\u2010rich site. This unique aggregation\u2010induced retention effect enabled long\u2010term, high\u2010contrast in situ imaging of tumor margins in tumor\u2010bearing mice and human breast cancer samples, which offers a powerful and clinically translatable tool for improving the precision of cancer surgery. Finally, we also achieved the quantitation of ENTK levels in artificial human duodenal juice with recovery rates from 93.5% to 99.8%. These results confirm the suitability of our probe for the quantitation of ENTK in real samples.<\/jats:p>","DOI":"10.1002\/adhm.71509","type":"journal-article","created":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T04:49:34Z","timestamp":1785300574000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Negatively Charged Peptides\u2010Assisted Self\u2010Assembled Mitochondria\u2010Targeting Nanoprobe for Detecting Enterokinase and Image\u2010Guided Navigation in Human Breast Cancer"],"prefix":"10.1002","volume":"15","author":[{"given":"Zhongxi","family":"Huang","sequence":"first","affiliation":[{"name":"State Key Laboratory of Flexible Electronics (LoFE) &amp; Institute of Advanced Materials (IAM) School of Flexible Electronics (Future Technologies) Nanjing Tech University (NanjingTech)  Nanjing 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