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Accurate quantification of labile Cu2+ in the brain extracellular microenvironment is critical for elucidating its pathological roles and developing AD diagnostic tools. Herein, we report a dual-mode sensing platform integrating \u201cturn-on\u201d fluorescence and ratiometric electrochemistry for sensitive and selective Cu2+ quantification, based on a resorufin-picolinate conjugate (RP) probe and a carbon nanotube-modified carbon fiber microelectrode (CFME\/CNT). Resorufin (Re) acts as both the fluorescent reporter and electrochemical redox center, while picolinate serves as the Cu2+-specific recognition moiety. Cu2+ coordinates with picolinate to catalyze the ester hydrolysis of RP, releasing free Re that triggers a fluorescence enhancement at 594 nm and a ratiometric electrochemical response, corresponding to the oxidation currents of free Re and intact RP, respectively. The RP probe exhibits high selectivity for Cu2+ over other metal ions, with limits of detection of 19 nM (fluorescence) and 7.2 nM (electrochemistry). The dual-mode platform was successfully applied to the accurate quantification of endogenous labile Cu2+ in cerebral cortex dialysate from AD mice and age-matched wild-type (WT) mice, and noninvasive in vivo fluorescence imaging further confirmed the elevated Cu2+ level in living AD mouse brains. Quantitative results revealed that the labile Cu2+ concentration in AD mouse cerebral cortex dialysate was significantly higher than that in WT mice. The two detection modes yielded highly consistent results, validating the reliability of the dual-mode platform in complex biological matrices. This dual-mode strategy synergizes the merits of fluorescence and ratiometric electrochemistry, providing a robust and selective tool for investigating AD pathogenesis related to Cu2+ dyshomeostasis and holds great potential for clinical translational applications in AD diagnosis.<\/jats:p>","DOI":"10.1021\/acs.analchem.6c03646","type":"journal-article","created":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T05:21:12Z","timestamp":1785129672000},"page":"23335-23345","source":"Crossref","is-referenced-by-count":4,"title":["Dual-Mode Turn-On Fluorescent and Ratiometric Electrochemical\nDetection of Cu2\n                    <b>+<\/b>\n                    in Alzheimer\u2019s Disease\nMouse Brains Based on a Single Resorufin\u2013Picolinate Probe"],"prefix":"10.1021","volume":"98","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8358-8326","authenticated-orcid":true,"given":"Hui","family":"Dong","sequence":"first","affiliation":[{"name":"Shangqiu Normal University , , ,","place":["Shangqiu, China, 476000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoyu","family":"Lv","sequence":"additional","affiliation":[{"name":"Shangqiu Normal University , , ,","place":["Shangqiu, China, 476000"]},{"name":"Zhengzhou University , , ,","place":["Zhengzhou, China, 450001"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yujia","family":"Qian","sequence":"additional","affiliation":[{"name":"Shangqiu Normal University , , ,","place":["Shangqiu, China, 476000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aixin","family":"Chang","sequence":"additional","affiliation":[{"name":"Shangqiu Normal University , , ,","place":["Shangqiu, China, 476000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Menghao","family":"Cheng","sequence":"additional","affiliation":[{"name":"Shangqiu Normal University , , ,","place":["Shangqiu, China, 476000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xue","family":"Yang","sequence":"additional","affiliation":[{"name":"Shangqiu Normal University , , ,","place":["Shangqiu, China, 476000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiahui","family":"Ding","sequence":"additional","affiliation":[{"name":"Shangqiu Normal University , , ,","place":["Shangqiu, China, 476000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zimo","family":"Yang","sequence":"additional","affiliation":[{"name":"Shangqiu Normal University , , ,","place":["Shangqiu, China, 476000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanli","family":"Zhou","sequence":"additional","affiliation":[{"name":"Shangqiu Normal University , , ,","place":["Shangqiu, China, 476000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2656-4878","authenticated-orcid":true,"given":"Yintang","family":"Zhang","sequence":"additional","affiliation":[{"name":"Shangqiu Normal University , , ,","place":["Shangqiu, China, 476000"]},{"name":"Zhengzhou University , , ,","place":["Zhengzhou, China, 450001"]},{"name":"Xinxiang University , , ,","place":["Xinxiang, China, 453000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3946-0656","authenticated-orcid":true,"given":"Zhaohui","family":"Li","sequence":"additional","affiliation":[{"name":"Zhengzhou University , , ,","place":["Zhengzhou, China, 450001"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maotian","family":"Xu","sequence":"additional","affiliation":[{"name":"Shangqiu Normal University , , ,","place":["Shangqiu, China, 476000"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,7,25]]},"reference":[{"key":"2026081109093802800_cit1","doi-asserted-by":"publisher","first-page":"9259","DOI":"10.3390\/ijms21239259","article-title":"Copper Dyshomeostasis\nin Neurodegenerative Diseases\u2500Therapeutic Implications. A","volume":"21","author":"Gromadzka","year":"2020","journal-title":"Int. J. Mol. Sci."},{"issue":"6","key":"2026081109093802800_cit2","doi-asserted-by":"publisher","first-page":"1995","DOI":"10.1021\/cr040410w","article-title":"Copper Homeostasis\nand Neurodegenerative Disorders (Alzheimer\u2019s, Prion, and Parkinson\u2019s\nDiseases and Amyotrophic Lateral Sclerosis)","volume":"106","author":"Gaggelli","year":"2006","journal-title":"Chem. Rev."},{"issue":"10","key":"2026081109093802800_cit3","doi-asserted-by":"publisher","first-page":"5193","DOI":"10.1021\/cr300009x","article-title":"Bioinorganic\nChemistry of Alzheimer\u2019s Disease","volume":"112","author":"Kepp","year":"2012","journal-title":"Chem.\nRev"},{"issue":"11","key":"2026081109093802800_cit4","doi-asserted-by":"publisher","first-page":"1146","DOI":"10.1021\/ar200014u","article-title":"The Heterogeneous Nature of Cu2+ Interactions with\nAlzheimer\u2019s Amyloid-\u03b2 Peptide","volume":"44","author":"Drew","year":"2011","journal-title":"Acc. Chem. Res."},{"issue":"17","key":"2026081109093802800_cit5","doi-asserted-by":"publisher","first-page":"1851","DOI":"10.1021\/ac00192a014","article-title":"Determination of iron,\ncobalt, copper, zinc, rubidium, molybdenum, and cesium in human serum\nby inductively coupled plasma mass spectrometry","volume":"61","author":"Vanhoe","year":"1989","journal-title":"Anal. Chem."},{"issue":"17","key":"2026081109093802800_cit6","doi-asserted-by":"publisher","first-page":"4319","DOI":"10.1021\/ac010319h","article-title":"Direct and Simultaneous Determination of Copper, Chromium,\nAluminum, and Manganese in Urine with a Multielement Graphite Furnace\nAtomic Absorption Spectrometer","volume":"73","author":"Lin","year":"2001","journal-title":"Anal. Chem."},{"issue":"11","key":"2026081109093802800_cit7","doi-asserted-by":"publisher","first-page":"7106","DOI":"10.1021\/acs.chemrev.3c00776","article-title":"Fluorescent Probes for\nDisease Diagnosis","volume":"124","author":"Wang","year":"2024","journal-title":"Chem.\nRev."},{"issue":"5","key":"2026081109093802800_cit8","doi-asserted-by":"publisher","first-page":"2173","DOI":"10.1039\/D0QM01008A","article-title":"A brief review\non novel pyrene based fluorometric and colorimetric chemosensors for\nthe detection of Cu2+","volume":"5","author":"Kowser","year":"2021","journal-title":"Mater. Chem. Front."},{"issue":"9","key":"2026081109093802800_cit9","doi-asserted-by":"publisher","first-page":"5846","DOI":"10.1021\/acs.chemrev.3c00819","article-title":"Small-Molecule\nFluorescent Probes for Binding- and Activity-Based Sensing of Redox-Active\nBiological Metals","volume":"124","author":"Grover","year":"2024","journal-title":"Chem. Rev."},{"key":"2026081109093802800_cit10","doi-asserted-by":"publisher","first-page":"117085","DOI":"10.1016\/j.bios.2024.117085","article-title":"A neoteric\nsmartphone-assisted colorimetric and fluorometric dual-mode probe\nbased on anthraquinone derived Schiff base for wide-range pH and Cu2+\/S2\u2013\ndetermination in bioimaging","volume":"271","author":"Zhao","year":"2025","journal-title":"Biosens. Bioelectron."},{"issue":"25","key":"2026081109093802800_cit11","doi-asserted-by":"publisher","first-page":"13422","DOI":"10.1021\/acs.analchem.5c01800","article-title":"Water-Induced\nStructural Instability in Zinc Coordination Polymers for Dual-Channel\nFluorescent Sensing of Cu2+ and Cysteine","volume":"97","author":"Li","year":"2025","journal-title":"Anal.\nChem."},{"issue":"6","key":"2026081109093802800_cit12","doi-asserted-by":"publisher","first-page":"2858","DOI":"10.1021\/acssensors.3c02549","article-title":"Near-Infrared Ratiometric Fluorescent Probe for Detecting\nEndogenous Cu(2+) in the Brain","volume":"9","author":"Zhu","year":"2024","journal-title":"ACS Sens."},{"issue":"20","key":"2026081109093802800_cit13","doi-asserted-by":"publisher","first-page":"5005","DOI":"10.1021\/acs.jpcb.5c01431","article-title":"Intramolecular\nCharge Transfer-Induced Fluorescent Probe for the Sensitive Detection\nof Copper Tripeptide","volume":"129","author":"Dkhar","year":"2025","journal-title":"J. Phys. Chem. B"},{"issue":"25","key":"2026081109093802800_cit14","doi-asserted-by":"publisher","first-page":"13256","DOI":"10.1021\/acs.langmuir.4c01436","article-title":"\u201cOn\u2013On\u2013Off\u201d\nRecyclable Fluorescence Battery for Direct and Selective Detection\nof Glyphosate and Cu2+","volume":"40","author":"Zhang","year":"2024","journal-title":"Langmuir"},{"issue":"1","key":"2026081109093802800_cit15","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202413605","article-title":"Nonconventional Fluorescent Non-Isocyanate\nPolyurethane Foams for Multipurpose Sensing Applications","volume":"64","author":"Mahapatra","year":"2024","journal-title":"Angew. Chem., Int. Ed."},{"key":"2026081109093802800_cit16","doi-asserted-by":"publisher","first-page":"114848","DOI":"10.1016\/j.bios.2022.114848","article-title":"Carbon dot based sensing platform for real-time imaging Cu2+ distribution\nin plants and environment","volume":"219","author":"Lin","year":"2023","journal-title":"Biosens. Bioelectron."},{"issue":"11","key":"2026081109093802800_cit17","doi-asserted-by":"publisher","first-page":"13848","DOI":"10.1021\/acsami.1c23199","article-title":"Fluorescence and Colorimetric\nDual-Mode Ratiometric Sensor Based\non Zr\u2013Tetraphenylporphyrin Tetrasulfonic Acid Hydrate Metal\u2013Organic\nFrameworks for Visual Detection of Copper Ions","volume":"14","author":"Hou","year":"2022","journal-title":"ACS Appl. Mater. Interfaces"},{"issue":"4","key":"2026081109093802800_cit18","doi-asserted-by":"publisher","first-page":"1982","DOI":"10.1021\/acs.inorgchem.1c03141","article-title":"Aza-Crown-Based Macrocyclic Probe Design for\n\u201cPET-off\u201d Multi-Cu2+ Responsive and \u201cCHEF-on\u201d\nMulti-Zn2+ Sensor: Application in Biological Cell Imaging and Theoretical\nStudies","volume":"61","author":"Ghorai","year":"2022","journal-title":"Inorg. Chem."},{"key":"2026081109093802800_cit19","doi-asserted-by":"publisher","first-page":"113174","DOI":"10.1016\/j.bios.2021.113174","article-title":"Development of a method for the detection\nof Cu2+ in the environment\nand live cells using a synthesized spider web-like fluorescent probe","volume":"182","author":"Zhang","year":"2021","journal-title":"Biosens. Bioelectron."},{"issue":"38","key":"2026081109093802800_cit20","doi-asserted-by":"publisher","first-page":"12922","DOI":"10.1021\/acssuschemeng.1c03830","article-title":"Ultrahigh Efficient FRET Ratiometric\nFluorescence Biosensor for Visual Detection of Alkaline Phosphatase\nActivity and Its Inhibitor","volume":"9","author":"Zhan","year":"2021","journal-title":"ACS Sustainable\nChem. Eng."},{"issue":"21","key":"2026081109093802800_cit21","doi-asserted-by":"publisher","first-page":"8234","DOI":"10.1021\/acs.orglett.0c02814","article-title":"Switching\nof C\u2013C\nand C\u2013N Coupling\/Cleavage for Hypersensitive Detection of Cu2+\nby a Catalytically Mediated 2-Aminoimidazolyl-Tailored Six-Membered\nRhodamine Probe","volume":"22","author":"Yang","year":"2020","journal-title":"Org. Lett."},{"issue":"24","key":"2026081109093802800_cit22","doi-asserted-by":"publisher","first-page":"16066","DOI":"10.1021\/acs.analchem.0c03691","article-title":"Universal Platform\nfor Ratiometric Sensing Based on Catalytically\nInduced Inner-Filter Effect by Cu2+","volume":"92","author":"Fan","year":"2020","journal-title":"Anal. Chem."},{"issue":"8","key":"2026081109093802800_cit23","doi-asserted-by":"publisher","first-page":"2438","DOI":"10.1021\/acssensors.0c00619","article-title":"Visible and Reversible Restrict of Molecular Configuration\nby Copper Ion and Pyrophosphate","volume":"5","author":"Chang","year":"2020","journal-title":"ACS Sens."},{"issue":"7","key":"2026081109093802800_cit24","doi-asserted-by":"publisher","first-page":"9333","DOI":"10.1021\/acssuschemeng.8b01647","article-title":"Ratiometric Detection of Cu2+ Using\na Luminol-Tb-GMP Nanoprobe with High Sensitivity and Selectivity","volume":"6","author":"Tong","year":"2018","journal-title":"ACS Sustainable Chem. Eng."},{"issue":"15","key":"2026081109093802800_cit25","doi-asserted-by":"publisher","first-page":"8785","DOI":"10.1021\/acs.analchem.7b05268","article-title":"Highly Sensitive Fluorescence\nImaging of Zn2+ and Cu2+ in Living\nCells with Signal Amplification Based on Functional DNA Self-Assembly","volume":"90","author":"Si","year":"2018","journal-title":"Anal. Chem."},{"issue":"5","key":"2026081109093802800_cit26","doi-asserted-by":"publisher","first-page":"1016","DOI":"10.1021\/acssensors.8b00158","article-title":"Dual-Analyte Fluorescent Sensor Based\non [5]Helicene Derivative with Super Large Stokes Shift for the Selective\nDeterminations of Cu2+ or Zn2+ in Buffer Solutions and Its Application\nin a Living Cell","volume":"3","author":"Sakunkaewkasem","year":"2018","journal-title":"ACS Sens."},{"issue":"10","key":"2026081109093802800_cit27","doi-asserted-by":"publisher","first-page":"5333","DOI":"10.1021\/acs.analchem.5b00628","article-title":"Single Probe for Imaging\nand Biosensing of pH, Cu2+Ions, and pH\/Cu2+in Live Cells with Ratiometric\nFluorescence Signals","volume":"87","author":"Han","year":"2015","journal-title":"Anal. Chem."},{"issue":"12","key":"2026081109093802800_cit28","doi-asserted-by":"publisher","first-page":"2962","DOI":"10.1021\/acs.organomet.5b00285","article-title":"Rhodamine\u2013Ferrocene\nConjugate Chemosensor for Selectively Sensing Copper(II) with Multisignals:\nChromaticity, Fluorescence, and Electrochemistry and Its Application\nin Living Cell Imaging","volume":"34","author":"Fang","year":"2015","journal-title":"Organometallics"},{"issue":"13","key":"2026081109093802800_cit29","doi-asserted-by":"publisher","first-page":"6655","DOI":"10.1021\/ic500313m","article-title":"Differentially Selective Chemosensor with Fluorescence\nOff\u2013On Responses on Cu2+ and Zn2+ Ions in Aqueous Media and\nApplications in Pyrophosphate Sensing, Live Cell Imaging, and Cytotoxicity","volume":"53","author":"Anbu","year":"2014","journal-title":"Inorg. Chem."},{"issue":"9","key":"2026081109093802800_cit30","doi-asserted-by":"publisher","first-page":"6562","DOI":"10.1021\/am501476w","article-title":"Highly Selective\nProbe Detects Cu2+ and Endogenous NO Gas in Living Cell","volume":"6","author":"Muthuraj","year":"2014","journal-title":"ACS Appl. Mater. Interfaces"},{"issue":"7","key":"2026081109093802800_cit31","doi-asserted-by":"publisher","first-page":"7985","DOI":"10.1021\/acsomega.8b00748","article-title":"On\/Off Fluorescent Chemosensor for\nSelective Detection of Divalent Iron and Copper Ions: Molecular Logic\nOperation and Protein Binding","volume":"3","author":"Tamil Selvan","year":"2018","journal-title":"ACS Omega"},{"issue":"22","key":"2026081109093802800_cit32","doi-asserted-by":"publisher","first-page":"3533","DOI":"10.1021\/ja00905a001","article-title":"Hard and\nSoft Acids and Bases","volume":"85","author":"Pearson","year":"1963","journal-title":"J. Am. Chem. Soc."},{"issue":"62","key":"2026081109093802800_cit33","doi-asserted-by":"publisher","first-page":"7685","DOI":"10.1039\/D1CC02233D","article-title":"AIE + ESIPT\nactivity-based NIR Cu2+ sensor with dye participated binding strategy","volume":"57","author":"Jiang","year":"2021","journal-title":"Chem. Commun."},{"issue":"32","key":"2026081109093802800_cit34","doi-asserted-by":"publisher","first-page":"13082","DOI":"10.1039\/D4SC03445G","article-title":"A novel NIR fluorescent\nprobe for copper(ii) imaging in Parkinson\u2019s disease mouse brain","volume":"15","author":"Chen","year":"2024","journal-title":"Chem. Sci."},{"key":"2026081109093802800_cit35","doi-asserted-by":"publisher","first-page":"137258","DOI":"10.1016\/j.snb.2025.137258","article-title":"Open-layered\nMBenes: Comparisons\nof different wet etching techniques and electrochemical detection\nof heavy metal ions with high sensitivity & selectivity","volume":"429","author":"Wei","year":"2025","journal-title":"Sens. Actuators, B"},{"issue":"5","key":"2026081109093802800_cit36","doi-asserted-by":"publisher","first-page":"2317","DOI":"10.1021\/acssensors.4c00633","article-title":"Harnessing Graphdiyne for Selective Cu2+ Detection:\nA Promising Tool for Parkinson\u2019s Disease Diagnostics and Pathogenesis","volume":"9","author":"Jiang","year":"2024","journal-title":"ACS Sens."},{"issue":"1","key":"2026081109093802800_cit37","doi-asserted-by":"publisher","first-page":"127","DOI":"10.1021\/acs.analchem.3c03337","article-title":"Electrochemical Nanopipette Sensor for In Vitro\/In Vivo Detection\nof Cu2+ Ions","volume":"96","author":"Timoshenko","year":"2024","journal-title":"Anal. Chem."},{"issue":"24","key":"2026081109093802800_cit38","doi-asserted-by":"publisher","first-page":"9508","DOI":"10.1021\/acs.inorgchem.3c00865","article-title":"Sensitive\nCurrent Sensor Based on\na Lanthanide Framework with Lewis Basic Bipyridyl Sites for Cu2+ Detection","volume":"62","author":"Liang","year":"2023","journal-title":"Inorg. Chem."},{"issue":"18","key":"2026081109093802800_cit39","doi-asserted-by":"publisher","first-page":"12006","DOI":"10.1021\/acs.analchem.9b02945","article-title":"Embedded Au\nNanoparticles-Based Ratiometric\nElectrochemical Sensing Strategy for Sensitive and Reliable Detection\nof Copper Ions","volume":"91","author":"Wang","year":"2019","journal-title":"Anal. Chem."},{"issue":"1","key":"2026081109093802800_cit40","doi-asserted-by":"publisher","first-page":"888","DOI":"10.1021\/acs.analchem.8b03764","article-title":"Graphene Aerogel-Metal-Organic\nFramework-Based\nElectrochemical Method for Simultaneous Detection of Multiple Heavy-Metal\nIons","volume":"91","author":"Lu","year":"2019","journal-title":"Anal. Chem."},{"key":"2026081109093802800_cit41","doi-asserted-by":"publisher","first-page":"111","DOI":"10.1016\/j.bios.2019.03.014","article-title":"On-line regeneration\nof electrochemical biosensor for in vivo repetitive\nmeasurements of striatum Cu2+ under global cerebral ischemia\/reperfusion\nevents","volume":"135","author":"Gu","year":"2019","journal-title":"Biosens. Bioelectron."},{"issue":"7","key":"2026081109093802800_cit42","doi-asserted-by":"publisher","first-page":"4328","DOI":"10.1021\/acs.analchem.7b02315","article-title":"High Electrochemical\nSensitivity of TiO(2- x) Nanosheets and an Electron-Induced Mutual\nInterference Effect toward Heavy Metal Ions Demonstrated Using X-ray\nAbsorption Fine Structure Spectra","volume":"90","author":"Zhou","year":"2018","journal-title":"Anal. Chem."},{"issue":"19","key":"2026081109093802800_cit43","doi-asserted-by":"publisher","first-page":"11439","DOI":"10.1021\/acs.analchem.8b02555","article-title":"Click Chemistry Reaction-Triggered 3D DNA Walking Machine for Sensitive\nElectrochemical Detection of Copper Ion","volume":"90","author":"Qing","year":"2018","journal-title":"Anal.\nChem."},{"issue":"14","key":"2026081109093802800_cit44","doi-asserted-by":"publisher","first-page":"6962","DOI":"10.1021\/acs.analchem.6b00825","article-title":"Selective\nDetection of Copper(II) Using Ionophore-Grafted Carbon-Fiber Microelectrodes","volume":"88","author":"Yang","year":"2016","journal-title":"Anal. 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