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However, their development is constrained by an inherent dilemma: extending \u03c0-conjugation to narrow the energy gap inevitably compromises the fluorescence brightness in the activated state (dim on), while the strong intramolecular charge transfer (ICT) effect makes effective quenching in the locked state difficult to achieve (leaky off), leading to poor response sensitivity and a low on\/off ratio. Here, we rationally designed a xanthene\u2013cyanine hybrid NIR-II activatable platform (RC-OH), wherein activated-state brightness was enhanced by strengthening the steric hindrance to suppress intermolecular aggregation (bright on), while locked-state quenching was promoted by amplifying nonradiative decay via the inductive effect of an ortho-chlorine atom on the phenolic hydroxyl group (tight off). Leveraging the dual-optimized RC3-OH platform, we constructed RC3-Cys, RC3-ALP, and RC3-ONOO probes and validated their high on\/off ratio (up to 16-fold) and specific detection of cysteine (Cys), alkaline phosphatase (ALP) and peroxynitrite (ONOO\u2013), respectively. These results highlight RC3-OH as a versatile NIR-II platform for constructing high-sensitivity, biomarker-activatable NIR-II probes.<\/jats:p>","DOI":"10.1021\/acs.analchem.6c04533","type":"journal-article","created":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T09:56:52Z","timestamp":1788429412000},"page":"27171-27180","source":"Crossref","is-referenced-by-count":0,"title":["Rational Design\nof Hemicyanine Platform for Engineering\nHigh-Contrast Activatable NIR-II Probes"],"prefix":"10.1021","volume":"98","author":[{"given":"Xingyu","family":"Wang","sequence":"first","affiliation":[{"name":"University of Science and Technology of China , , , ,","place":["Hefei, China, 230026"]},{"name":"Chinese Academy of Sciences , , , ,","place":["Suzhou, China, 215123"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qian","family":"Chen","sequence":"additional","affiliation":[{"name":"Chinese Academy of Sciences , , , ,","place":["Suzhou, China, 215123"]},{"name":"The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Gusu School of Nanjing Medical University , , , ,","place":["Suzhou, China, 215000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tuanwei","family":"Li","sequence":"additional","affiliation":[{"name":"Chinese Academy of Sciences , , , ,","place":["Suzhou, China, 215123"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xingyu","family":"Yang","sequence":"additional","affiliation":[{"name":"University of Science and Technology of China , , , ,","place":["Hefei, China, 230026"]},{"name":"Chinese Academy of Sciences , , , ,","place":["Suzhou, China, 215123"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chunyan","family":"Li","sequence":"additional","affiliation":[{"name":"University of Science and Technology of China , , , ,","place":["Hefei, China, 230026"]},{"name":"Chinese Academy of Sciences , , , ,","place":["Suzhou, China, 215123"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2127-920X","authenticated-orcid":true,"given":"Jiang","family":"Jiang","sequence":"additional","affiliation":[{"name":"University of Science and Technology of China , , , ,","place":["Hefei, China, 230026"]},{"name":"Chinese Academy of Sciences , , , ,","place":["Suzhou, China, 215123"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6589-6328","authenticated-orcid":true,"given":"Qiangbin","family":"Wang","sequence":"additional","affiliation":[{"name":"University of Science and Technology of China , , , ,","place":["Hefei, China, 230026"]},{"name":"Chinese Academy of Sciences , , , ,","place":["Suzhou, China, 215123"]},{"name":"ShanghaiTech University , , , ,","place":["Shanghai, China, 201210"]},{"name":"University of Chinese Academy of Sciences , , , ,","place":["Beijing, China, 100049"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,1]]},"reference":[{"key":"2026091511071594800_cit1","doi-asserted-by":"publisher","first-page":"0010","DOI":"10.1038\/s41551-016-0010","article-title":"Near-Infrared Fluorophores for Biomedical\nImaging","volume":"1","author":"Hong","year":"2017","journal-title":"Nat. Biomed. Eng."},{"key":"2026091511071594800_cit2","doi-asserted-by":"publisher","first-page":"1044","DOI":"10.1039\/C3CS60237K","article-title":"Ultra-Stable\nOrganic Fluorophores for Single-Molecule Research","volume":"43","author":"Zheng","year":"2014","journal-title":"Chem. Soc. Rev."},{"key":"2026091511071594800_cit3","doi-asserted-by":"publisher","first-page":"14789","DOI":"10.1021\/jacs.0c07022","article-title":"Advanced Fluorescence\nImaging Technology in the Near-Infrared-II Window for Biomedical Applications","volume":"142","author":"Li","year":"2020","journal-title":"J. Am. Chem. Soc."},{"key":"2026091511071594800_cit4","doi-asserted-by":"publisher","first-page":"161","DOI":"10.1038\/s44222-022-00017-1","article-title":"Fluorescence\nImage-Guided Tumour Surgery","volume":"1","author":"Wang","year":"2023","journal-title":"Nat. Rev. Bioeng."},{"key":"2026091511071594800_cit5","doi-asserted-by":"publisher","DOI":"10.1002\/adom.201900917","article-title":"Recent Advances on\nActivatable NIR-II Fluorescence Probes for Biomedical Imaging","volume":"7","author":"Tang","year":"2019","journal-title":"Adv. Opt. Mater."},{"key":"2026091511071594800_cit6","doi-asserted-by":"publisher","first-page":"828","DOI":"10.1039\/D2QM01120D","article-title":"Recent Advances\nin Activatable NIR-II Organic Fluorophores for Biomedical Applications","volume":"7","author":"Xu","year":"2023","journal-title":"Mater. Chem. Front."},{"key":"2026091511071594800_cit7","doi-asserted-by":"publisher","first-page":"60","DOI":"10.1038\/s44222-022-00002-8","article-title":"Near-Infrared Luminescence High-Contrast In Vivo Biomedical Imaging","volume":"1","author":"Chen","year":"2023","journal-title":"Nat. Rev.\nBioeng."},{"key":"2026091511071594800_cit8","doi-asserted-by":"publisher","first-page":"3448","DOI":"10.1039\/D0SC04789A","article-title":"Activatable Fluorescence Sensors\nfor In Vivo Bio-Detection in the Second Near-Infrared\nWindow","volume":"12","author":"Zhao","year":"2021","journal-title":"Chem. Sci."},{"key":"2026091511071594800_cit9","doi-asserted-by":"publisher","first-page":"566","DOI":"10.1039\/D1CS00525A","article-title":"Activatable Molecular\nProbes for Fluorescence-Guided Surgery, Endoscopy and Tissue Biopsy","volume":"51","author":"Zhang","year":"2022","journal-title":"Chem. Soc. Rev."},{"key":"2026091511071594800_cit10","doi-asserted-by":"publisher","first-page":"26541","DOI":"10.1002\/ange.202109728","article-title":"ROS\/RNS and Base Dual Activatable Merocyanine-Based NIR-II Fluorescent\nMolecular Probe for In Vivo Biosensing","volume":"133","author":"Zhang","year":"2021","journal-title":"Angew. Chem., Int. Ed."},{"key":"2026091511071594800_cit11","doi-asserted-by":"publisher","first-page":"11321","DOI":"10.1021\/acs.analchem.2c02052","article-title":"H2O2-Activated NIR-II Fluorescent Probe with a Large Stokes\nShift for High-Contrast Imaging in Drug-Induced Liver Injury Mice","volume":"94","author":"Tian","year":"2022","journal-title":"Anal. Chem."},{"key":"2026091511071594800_cit12","doi-asserted-by":"publisher","first-page":"7219","DOI":"10.1002\/anie.202000947","article-title":"Tumor Microenvironment-Activated\nNIR-II Nanotheranostic System for Precise Diagnosis and Treatment\nof Peritoneal Metastasis","volume":"59","author":"Ling","year":"2020","journal-title":"Angew. Chem., Int.\nEd."},{"key":"2026091511071594800_cit13","doi-asserted-by":"publisher","DOI":"10.1002\/ange.202211409","article-title":"Engineering of Reversible\nNIR-II Redox-Responsive Fluorescent Probes for Imaging of Inflammation\nIn Vivo","volume":"134","author":"He","year":"2022","journal-title":"Angew. Chem., Int. Ed."},{"key":"2026091511071594800_cit14","doi-asserted-by":"publisher","first-page":"15757","DOI":"10.1021\/acs.analchem.9b04002","article-title":"Lighting up NIR-II\nFluorescence In Vivo: An Activable\nProbe for Noninvasive Hydroxyl Radical Imaging","volume":"91","author":"Feng","year":"2019","journal-title":"Anal. Chem."},{"key":"2026091511071594800_cit15","doi-asserted-by":"publisher","DOI":"10.1016\/j.biomaterials.2022.121956","article-title":"An Oral Ratiometric NIR-II Fluorescent\nProbe for Reliable Monitoring of Gastrointestinal Diseases In Vivo","volume":"293","author":"Li","year":"2023","journal-title":"Biomaterials"},{"key":"2026091511071594800_cit16","doi-asserted-by":"publisher","DOI":"10.1002\/smll.202008061","article-title":"NIR-II Photoacoustic Reporter for\nBiopsy-Free and Real-Time Assessment of Wilson\u2019s Disease","volume":"17","author":"Fu","year":"2021","journal-title":"Small"},{"key":"2026091511071594800_cit17","doi-asserted-by":"publisher","DOI":"10.1016\/j.nantod.2023.102027","article-title":"Activatable NIR-II Ratiometric Fluorescence\nNanoprobe for In Vivo Real-Time Dynamic Imaging of\nGSH and Its-Associated Diseases","volume":"53","author":"Bi","year":"2023","journal-title":"Nano Today"},{"key":"2026091511071594800_cit18","doi-asserted-by":"publisher","first-page":"2072","DOI":"10.1002\/ange.201812878","article-title":"Precise In Vivo Inflammation Imaging\nUsing In Situ Responsive Cross-Linking of Glutathione-Modified\nUltra-Small NIR-II Lanthanide Nanoparticles","volume":"131","author":"Zhao","year":"2019","journal-title":"Angew. Chem., Int. Ed."},{"key":"2026091511071594800_cit19","doi-asserted-by":"publisher","first-page":"1493","DOI":"10.1021\/acsmaterialslett.2c00451","article-title":"\u2033Crossbreeding\u2033 Small-Molecular\nWeight NIR-II Flavchromenes Endows Activatable Multiplexed In Vivo Imaging","volume":"4","author":"Zhang","year":"2022","journal-title":"ACS Mater. Lett."},{"key":"2026091511071594800_cit20","doi-asserted-by":"publisher","first-page":"6025","DOI":"10.7150\/thno.26607","article-title":"Hypoxia-Triggered Single\nMolecule Probe for High-Contrast NIR II\/PA Tumor Imaging and Robust\nPhotothermal Therapy","volume":"8","author":"Meng","year":"2018","journal-title":"Theranostics"},{"key":"2026091511071594800_cit21","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202201541","article-title":"NIRII-HDs: A Versatile Platform for\nDeveloping Activatable NIR-II Fluorogenic Probes for Reliable In Vivo Analyte Sensing","volume":"61","author":"Qin","year":"2022","journal-title":"Angew. Chem.,\nInt. Ed."},{"key":"2026091511071594800_cit22","doi-asserted-by":"publisher","first-page":"2144","DOI":"10.31635\/ccschem.025.202505932","article-title":"Developing Enzyme Activatable Second Near-Infrared Fluorescent Probes\nfor High-Fidelity Disease Diagnosis In Vivo","volume":"8","author":"Qin","year":"2026","journal-title":"CCS Chem."},{"key":"2026091511071594800_cit23","doi-asserted-by":"publisher","DOI":"10.1002\/smo.20240020","article-title":"\u2033Crossbreeding\u2033\nNIR-II Flavchromene for PSMA-Positive Prostate Cancer Detection and\nImage-Guided Surgery","volume":"2","author":"Huang","year":"2024","journal-title":"Smart Mol."},{"key":"2026091511071594800_cit24","doi-asserted-by":"publisher","first-page":"1795","DOI":"10.1039\/D1CS00307K","article-title":"Activity-Based NIR Fluorescent Probes\nBased on the Versatile Hemicyanine Scaffold: Design Strategy, Biomedical\nApplications, and Outlook","volume":"51","author":"Li","year":"2022","journal-title":"Chem. Soc. Rev."},{"key":"2026091511071594800_cit25","doi-asserted-by":"publisher","first-page":"6836","DOI":"10.1021\/jacs.0c11599","article-title":"Bright\nChromenylium Polymethine Dyes Enable Fast, Four-Color In Vivo Imaging with Shortwave Infrared Detection","volume":"143","author":"Cosco","year":"2021","journal-title":"J. Am. Chem. Soc."},{"key":"2026091511071594800_cit26","doi-asserted-by":"publisher","DOI":"10.1016\/j.snb.2023.133627","article-title":"Highly Selective Imaging of Intratumoral\nHydrogen Sulfide by NIR-II Emissive Fluorescent Probes","volume":"384","author":"Wang","year":"2023","journal-title":"Sens. Actuators B"},{"key":"2026091511071594800_cit27","doi-asserted-by":"publisher","first-page":"30794","DOI":"10.1021\/jacs.5c06682","article-title":"Shortwave\nInfrared Hemicyanine-6\nfor Cancer-Activated and Shaving-Free Preclinical Imaging of Lung\nMetastasis","volume":"147","author":"Yang","year":"2025","journal-title":"J. Am. Chem. Soc."},{"key":"2026091511071594800_cit28","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.ado2037","article-title":"A Firm-Push-to-Open and Light-Push-to-Lock\nStrategy for a General Chemical Platform to Develop Activatable Dual-Modality\nNIR-II Probes","volume":"10","author":"Shen","year":"2024","journal-title":"Sci. Adv."},{"key":"2026091511071594800_cit29","doi-asserted-by":"publisher","first-page":"21010","DOI":"10.1021\/jacs.2c10041","article-title":"Polymethine\nMolecular Platform for Ratiometric Fluorescent Probes in the Second\nNear-Infrared Window","volume":"144","author":"Lan","year":"2022","journal-title":"J. Am. Chem. Soc."},{"key":"2026091511071594800_cit30","doi-asserted-by":"publisher","first-page":"681","DOI":"10.1038\/nchem.2002","article-title":"A Spontaneously\nBlinking Fluorophore\nBased on Intramolecular Spirocyclization for Live-Cell Super-Resolution\nImaging","volume":"6","author":"Uno","year":"2014","journal-title":"Nat. Chem."},{"key":"2026091511071594800_cit31","doi-asserted-by":"publisher","first-page":"18860","DOI":"10.1002\/anie.202105905","article-title":"A General Approach\nto Convert Hemicyanine\nDyes into Highly Optimized Photoacoustic Scaffolds for Analyte Sensing","volume":"60","author":"Gardner","year":"2021","journal-title":"Angew. Chem., Int. Ed."},{"key":"2026091511071594800_cit32","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202400637","article-title":"Zero Intrinsic Fluorescence\nSensing-Platforms Enable Ultrasensitive Whole Blood Diagnosis and In Vivo Imaging","volume":"63","author":"Jiang","year":"2024","journal-title":"Angew. Chem., Int.\nEd."},{"key":"2026091511071594800_cit33","doi-asserted-by":"publisher","first-page":"8280","DOI":"10.1021\/jacs.4c15614","article-title":"Tracking\nthe Fate of Therapeutic Proteins Using Ratiometric Imaging of Responsive\nShortwave Infrared Probes","volume":"147","author":"Shrestha","year":"2025","journal-title":"J. Am. Chem. Soc."},{"key":"2026091511071594800_cit34","doi-asserted-by":"publisher","first-page":"94","DOI":"10.1038\/s42004-019-0194-4","article-title":"Design Strategy for\nGermanium-Rhodamine Based pH-Activatable Near-Infrared Fluorescence\nProbes Suitable for Biological Applications","volume":"2","author":"Koide","year":"2019","journal-title":"Commun. Chem."},{"key":"2026091511071594800_cit35","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202524043","article-title":"Twist and Shine:\nThe Impact of Halogen Substitution on Thiele Hydrocarbon\u2019s\nOptical Properties","volume":"65","author":"Punzi","year":"2026","journal-title":"Angew. Chem., Int. Ed."},{"key":"2026091511071594800_cit36","doi-asserted-by":"publisher","first-page":"7325","DOI":"10.1038\/s41467-023-42874-3","article-title":"The Carbonyl-Lock\nMechanism Underlying Non-Aromatic Fluorescence in Biological Matter","volume":"14","author":"Miro\u0301n","year":"2023","journal-title":"Nat. Commun."},{"key":"2026091511071594800_cit37","doi-asserted-by":"publisher","first-page":"10377","DOI":"10.1021\/acs.nanolett.1c03683","article-title":"Alkaline Phosphatase\nEnabled Fluorogenic Reaction and In Situ Coassembly\nof Near-Infrared and Radioactive Nanoparticles\nfor In Vivo Imaging","volume":"21","author":"Hu","year":"2021","journal-title":"Nano Lett."},{"key":"2026091511071594800_cit38","doi-asserted-by":"publisher","first-page":"7842","DOI":"10.1039\/D1CC02845F","article-title":"A Turn-on\nProbe for Detecting Antituberculotic Drug-Induced Liver Injury in\nMice via NIR-II Fluorescence\/Optoacoustic Imaging","volume":"57","author":"Chen","year":"2021","journal-title":"Chem. Commun."}],"container-title":["Analytical Chemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.analchem.6c04533","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/36\/27171\/68095178\/acs.analchem.6c04533.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/36\/27171\/68095178\/acs.analchem.6c04533.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T15:53:27Z","timestamp":1789487607000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/ancham\/article\/98\/36\/27171\/5395140\/Rational-Design-of-Hemicyanine-Platform-for"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,1]]},"references-count":38,"journal-issue":{"issue":"36","published-online":{"date-parts":[[2026,9,1]]},"published-print":{"date-parts":[[2026,9,15]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.analchem.6c04533","relation":{},"ISSN":["0003-2700","1520-6882"],"issn-type":[{"value":"0003-2700","type":"print"},{"value":"1520-6882","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,1]]}}}