{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T17:06:30Z","timestamp":1788973590053,"version":"build-2803163510"},"reference-count":43,"publisher":"American Chemical Society (ACS)","issue":"35","license":[{"start":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T00:00:00Z","timestamp":1787702400000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T00:00:00Z","timestamp":1787702400000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T00:00:00Z","timestamp":1787702400000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"name":"Start-up Research Fund of Southeast University","award":["RF1028624037"],"award-info":[{"award-number":["RF1028624037"]}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China (NSFC)","doi-asserted-by":"crossref","award":["22374019"],"award-info":[{"award-number":["22374019"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,9]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Accurate spatiotemporal single-cell quantification of intracellular enzyme activity is critical for understanding cellular heterogeneity and disease screening. However, current methods relying on single-signal modalities lack precision, and spatiotemporal multimodal strategies remain unexplored. Here, we develop a spatiotemporal sequential fluorescence\u2013electrochemistry bimodal platform for both spatial mapping and precise quantitation of asparaginyl endopeptidase (AEP) activity in living single cells. A dual-aggregation-induced emission (AIE) probe, pyTPE-CBT, is delivered into single cells via a \u03b8-nanopipette, yielding an 11.18-fold fluorescence enhancement to visualize AEP spatial distribution. Guided by fluorescence imaging, the nanopipette\u2019s detection channel is positioned at target subcellular regions. AEP-mediated cleavage of probe molecules alters charge density and effective channel size, modulating the ionic current for quantitative detection with a limit of detection (LOD) of 3.83 ng\/mL. It overcomes the drawbacks of traditional single-mode methods and offers a powerful tool for studying enzyme heterogeneity, subcellular localization, and dynamic enzymatic activity in single cells.<\/jats:p>","DOI":"10.1021\/acs.nanolett.6c02449","type":"journal-article","created":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T07:52:40Z","timestamp":1788249160000},"page":"11714-11720","source":"Crossref","is-referenced-by-count":0,"title":["Spatiotemporal\nSequential Fluorescence and Electrochemistry\nBimodal Quantitation of Enzyme Activity in a Living Cell"],"prefix":"10.1021","volume":"26","author":[{"given":"Weiwei","family":"Liu","sequence":"first","affiliation":[{"name":"Southeast University , , ,","place":["Nanjing, China, 211189"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lingling","family":"Xu","sequence":"additional","affiliation":[{"name":"Yangzhou University , , ,","place":["Yangzhou, China, 2250012"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5552-1298","authenticated-orcid":true,"given":"Wenjun","family":"Zhan","sequence":"additional","affiliation":[{"name":"Southeast University , , ,","place":["Nanjing, China, 211189"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4686-5291","authenticated-orcid":true,"given":"Songqin","family":"Liu","sequence":"additional","affiliation":[{"name":"Southeast University , , ,","place":["Nanjing, China, 211189"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0549-5420","authenticated-orcid":true,"given":"Yafeng","family":"Wu","sequence":"additional","affiliation":[{"name":"Southeast University , , ,","place":["Nanjing, China, 211189"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,26]]},"reference":[{"key":"2026090911315912100_cit1","doi-asserted-by":"publisher","first-page":"377","DOI":"10.1038\/s41586-023-06816-9","article-title":"Dictionary of Immune\nResponses to Cytokines at Single-Cell\nResolution","volume":"625","author":"Cui","year":"2024","journal-title":"Nature"},{"key":"2026090911315912100_cit2","doi-asserted-by":"publisher","first-page":"1481","DOI":"10.1038\/s41592-024-02305-7","article-title":"Large-Scale Foundation Model on Single-Cell\nTranscriptomics","volume":"21","author":"Hao","year":"2024","journal-title":"Nat. Methods"},{"key":"2026090911315912100_cit3","doi-asserted-by":"publisher","first-page":"1474","DOI":"10.1038\/s41590-024-01883-0","article-title":"High-Dimensional\nSingle-Cell Analysis of Human Natural Killer Cell Heterogeneity","volume":"25","author":"Rebuffet","year":"2024","journal-title":"Nat. Immunol."},{"key":"2026090911315912100_cit4","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202421658","article-title":"Stimuli-Responsive\nDNA Nanomachines for Intracellular Targeted Electrochemiluminescence\nImaging in Single Cells","volume":"64","author":"Li","year":"2025","journal-title":"Angew. Chem., Int.\nEd."},{"key":"2026090911315912100_cit5","doi-asserted-by":"publisher","first-page":"34870","DOI":"10.1021\/jacs.4c14190","article-title":"Spatially Quantitative Imaging of\nEnzyme Activity in a Living Cell","volume":"146","author":"Wang","year":"2024","journal-title":"J. Am. Chem.\nSoc."},{"key":"2026090911315912100_cit6","doi-asserted-by":"publisher","first-page":"2189","DOI":"10.1038\/nprot.2017.091","article-title":"Synthesis of a HyCoSuL Peptide Substrate\nLibrary to Dissect Protease Substrate Specificity","volume":"12","author":"Poreba","year":"2017","journal-title":"Nat. Protoc."},{"key":"2026090911315912100_cit7","doi-asserted-by":"publisher","DOI":"10.1002\/advs.202409477","article-title":"Sox6 and\nALDH1A1 Truncation by Asparagine Endopeptidase Defines Selective Neuronal\nVulnerability in Parkinson\u2019s Disease","volume":"12","author":"Nie","year":"2025","journal-title":"Adv. Sci."},{"key":"2026090911315912100_cit8","doi-asserted-by":"publisher","DOI":"10.1016\/j.trac.2022.116605","article-title":"Emerging ELISA derived technologies\nfor in vitro diagnostics","volume":"152","author":"Peng","year":"2022","journal-title":"TrAC, Trends Anal.\nChem."},{"key":"2026090911315912100_cit9","doi-asserted-by":"publisher","first-page":"2882","DOI":"10.1038\/s41596-022-00736-0","article-title":"Measuring\nkey human carbohydrate digestive enzyme activities using high-performance\nanion-exchange chromatography with pulsed amperometric detection","volume":"17","author":"Barber","year":"2022","journal-title":"Nat. Protoc."},{"key":"2026090911315912100_cit10","doi-asserted-by":"publisher","first-page":"1178","DOI":"10.1038\/s41589-021-00874-8","article-title":"Activity-Based Rna-Modifying Enzyme\nProbing Reveals\nDUS3L-Mediated Dihydrouridylation","volume":"17","author":"Dai","year":"2021","journal-title":"Nat. Chem.\nBiol."},{"key":"2026090911315912100_cit11","doi-asserted-by":"publisher","first-page":"803","DOI":"10.1038\/s41566-025-01707-z","article-title":"Theory, Innovations and Applications\nof Stimulated Raman Scattering Microscopy","volume":"19","author":"Min","year":"2025","journal-title":"Nat.\nPhotonics"},{"key":"2026090911315912100_cit12","doi-asserted-by":"publisher","first-page":"7145","DOI":"10.1021\/acsnano.2c08855","article-title":"An Alkaline\nPhosphatase-Responsive Aggregation-Induced\nEmission Photosensitizer for Selective Imaging and Photodynamic Therapy\nof Cancer Cells","volume":"17","author":"Lam","year":"2023","journal-title":"ACS Nano"},{"key":"2026090911315912100_cit13","doi-asserted-by":"publisher","first-page":"12305","DOI":"10.1021\/acsnano.2c03219","article-title":"Protease-Responsive Peptide-Conjugated Mitochondrial-Targeting\nAIEgens for Selective Imaging and Inhibition of SARS-CoV-2-Infected\nCells","volume":"16","author":"Cheng","year":"2022","journal-title":"ACS Nano"},{"key":"2026090911315912100_cit14","doi-asserted-by":"publisher","first-page":"1115","DOI":"10.1038\/s41563-024-01918-9","article-title":"Stretchable\nIonic\u2013Electronic Bilayer Hydrogel\nElectronics Enable in Situ Detection of Solid-State Epidermal Biomarkers","volume":"23","author":"Arwani","year":"2024","journal-title":"Nat. Mater."},{"key":"2026090911315912100_cit15","doi-asserted-by":"publisher","first-page":"4889","DOI":"10.1038\/s41467-025-59564-x","article-title":"Depletion-Dependent Activity-Based Protein Profiling\nUsing SWATH\/DIA-MS Detects Serine Hydrolase Lipid Remodeling in Lung\nAdenocarcinoma Progression","volume":"16","author":"Sajic","year":"2025","journal-title":"Nat. Commun."},{"key":"2026090911315912100_cit16","doi-asserted-by":"publisher","first-page":"890","DOI":"10.1038\/s41592-025-02644-z","article-title":"Raman Imaging\nas a Window into Cellular Complexity: A Future Perspective","volume":"22","author":"Fujita","year":"2025","journal-title":"Nat. Methods"},{"key":"2026090911315912100_cit17","doi-asserted-by":"publisher","first-page":"3808","DOI":"10.1038\/s41467-025-59030-8","article-title":"Photon-Counting Raman\nSpectroscopy at a MHz Spectral Rate for Biochemical Imaging of an\nEntire Organism","volume":"16","author":"Li","year":"2025","journal-title":"Nat. Commun."},{"key":"2026090911315912100_cit18","doi-asserted-by":"publisher","first-page":"7532","DOI":"10.1021\/jacs.0c00771","article-title":"Real-Time Imaging and Simultaneous\nQuantification of Mitochondrial H2O2 and ATP\nin Neurons with a Single Two-Photon Fluorescence-Lifetime-Based Probe","volume":"142","author":"Wu","year":"2020","journal-title":"J. Am. Chem. Soc."},{"key":"2026090911315912100_cit19","doi-asserted-by":"publisher","first-page":"7094","DOI":"10.1021\/acs.analchem.5c07917","article-title":"Ultrasensitive electrochemiluminescence detection of\ndual tumor markers on living cell surface with a closed bipolar electrode\narray chip","volume":"98","author":"Tian","year":"2026","journal-title":"Ana. Chem."},{"key":"2026090911315912100_cit20","doi-asserted-by":"publisher","first-page":"7403","DOI":"10.1021\/acssensors.5c01182","article-title":"Visual\nelectrofluorochromic detection\nof cancer cells via a bipolar electrode array chip with a COF@Au@Fc\/FA\nsignal probe","volume":"10","author":"Tian","year":"2025","journal-title":"ACS Sens."},{"key":"2026090911315912100_cit21","doi-asserted-by":"publisher","first-page":"281","DOI":"10.1038\/s41551-023-01009-1","article-title":"Activatable\nNear-Infrared Probes\nfor the Detection of Specific Populations of Tumour-Infiltrating Leukocytes\nin Vivo and in Urine","volume":"7","author":"He","year":"2023","journal-title":"Nat. Biomed. Eng."},{"key":"2026090911315912100_cit22","doi-asserted-by":"publisher","first-page":"17689","DOI":"10.1021\/jacs.3c04027","article-title":"Activation of Pyroptosis\nUsing AIEgen-Based\nsp2 Carbon-Linked Covalent Organic Frameworks","volume":"145","author":"Zhang","year":"2023","journal-title":"J. Am. Chem. 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Chem."},{"key":"2026090911315912100_cit29","doi-asserted-by":"publisher","first-page":"12404","DOI":"10.1021\/acs.analchem.4c01901","article-title":"Monitoring\nEnzymatic Reaction Kinetics and Activity Assays in Confined Nanospace","volume":"96","author":"Yang","year":"2024","journal-title":"Anal. Chem."},{"key":"2026090911315912100_cit30","doi-asserted-by":"publisher","first-page":"17558","DOI":"10.1021\/jacs.2c06962","article-title":"Electrochemical\nMolecule Trap-Based Sensing of Low-Abundance Enzymes in One Living\nCell","volume":"144","author":"Pan","year":"2022","journal-title":"J. Am. Chem. Soc."},{"key":"2026090911315912100_cit31","doi-asserted-by":"publisher","first-page":"54","DOI":"10.1038\/nchem.480","article-title":"A Biocompatible\nCondensation Reaction for Controlled\nAssembly of Nanostructures in Living Cells","volume":"2","author":"Liang","year":"2010","journal-title":"Nat. Chem."},{"key":"2026090911315912100_cit32","doi-asserted-by":"publisher","first-page":"10331","DOI":"10.1021\/jacs.9b03649","article-title":"Activatable NIR Fluorescence\/MRI\nBimodal Probes for in Vivo Imaging by Enzyme-Mediated Fluorogenic\nReaction and Self-Assembly","volume":"141","author":"Yan","year":"2019","journal-title":"J. Am. Chem. Soc."},{"key":"2026090911315912100_cit33","doi-asserted-by":"publisher","DOI":"10.1002\/adma.202307679","article-title":"Peptide-Driven Proton Sponge Nano-Assembly\nfor Imaging and Triggering\nLysosome-Regulated Immunogenic Cancer Cell Death","volume":"36","author":"He","year":"2024","journal-title":"Adv. Mater."},{"key":"2026090911315912100_cit34","doi-asserted-by":"publisher","first-page":"22776","DOI":"10.1021\/jacs.3c08627","article-title":"More Is Better: Dual-Acceptor Engineering for Constructing\nSecond Near-Infrared Aggregation-Induced Emission Luminogens to Boost\nMultimodal Phototheranostics","volume":"145","author":"Yang","year":"2023","journal-title":"J. Am. Chem. Soc."},{"key":"2026090911315912100_cit35","doi-asserted-by":"publisher","first-page":"2010","DOI":"10.1021\/jacs.4c15150","article-title":"The Midas Touch by\nIridium: A Second Near-Infrared Aggregation-Induced\nEmission-Active Metallo-Agent for Exceptional Phototheranostics of\nBreast Cancer","volume":"147","author":"You","year":"2025","journal-title":"J. Am. Chem. Soc."},{"key":"2026090911315912100_cit36","doi-asserted-by":"publisher","first-page":"11089","DOI":"10.1021\/jacs.4c16835","article-title":"Anodized Aluminum\nOxide Membrane Ionic Memristors","volume":"147","author":"Baram","year":"2025","journal-title":"J. Am. Chem.\nSoc."},{"key":"2026090911315912100_cit37","doi-asserted-by":"publisher","DOI":"10.1002\/adfm.202001566","article-title":"Furin-Instructed Intracellular\nGold Nanoparticle Aggregation for Tumor Photothermal Therapy","volume":"30","author":"Chen","year":"2020","journal-title":"Adv. Funct. Mater."},{"key":"2026090911315912100_cit38","doi-asserted-by":"publisher","first-page":"11897","DOI":"10.1021\/jacs.2c05743","article-title":"Trident Molecule with\nNanobrush-Nanoparticle-Nanofiber Transition Property Spatially Suppresses\nTumor Metastasis","volume":"144","author":"Gao","year":"2022","journal-title":"J. Am. Chem. Soc."},{"key":"2026090911315912100_cit39","doi-asserted-by":"publisher","first-page":"3691","DOI":"10.1021\/jacs.8b13497","article-title":"Rectification\nof Concentration Polarization in Mesopores Leads to High Conductance\nIonic Diodes and High Performance Osmotic Power","volume":"141","author":"Lin","year":"2019","journal-title":"J. Am. Chem. Soc."},{"key":"2026090911315912100_cit40","doi-asserted-by":"publisher","first-page":"432","DOI":"10.1038\/s41565-024-01823-x","article-title":"ON-OFF nanopores for optical control\nof transmembrane ionic communication","volume":"20","author":"Wang","year":"2025","journal-title":"Nat. Nanotechnol."},{"key":"2026090911315912100_cit41","doi-asserted-by":"publisher","first-page":"13183","DOI":"10.1021\/jacs.4c00922","article-title":"Negative Differential\nResistance in Conical Nanopore Iontronic Memristors","volume":"146","author":"Yang","year":"2024","journal-title":"J. Am. Chem. Soc."},{"key":"2026090911315912100_cit42","doi-asserted-by":"publisher","first-page":"13244","DOI":"10.1002\/anie.202014798","article-title":"An Integrated Electrochemical Nanodevice\nfor Intracellular RNA Collection and Detection in Single Living Cell","volume":"60","author":"Wang","year":"2021","journal-title":"Angew. Chem. Int. Ed."},{"key":"2026090911315912100_cit43","doi-asserted-by":"publisher","first-page":"74","DOI":"10.1186\/s13046-025-03334-6","article-title":"Radiotherapy\nresistance driven by Asparagine endopeptidase through ATR pathway\nmodulation in breast cancer","volume":"44","author":"Morillo-Huesca","year":"2025","journal-title":"J. Exp. Clin. Cancer\nRes."}],"container-title":["Nano Letters"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.nanolett.6c02449","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/nalefd\/article-pdf\/26\/35\/11714\/67412988\/acs.nanolett.6c02449.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/nalefd\/article-pdf\/26\/35\/11714\/67412988\/acs.nanolett.6c02449.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T16:06:32Z","timestamp":1788969992000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/nalefd\/article\/26\/35\/11714\/5329332\/Spatiotemporal-Sequential-Fluorescence-and"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,26]]},"references-count":43,"journal-issue":{"issue":"35","published-online":{"date-parts":[[2026,8,26]]},"published-print":{"date-parts":[[2026,9,9]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.nanolett.6c02449","relation":{},"ISSN":["1530-6984","1530-6992"],"issn-type":[{"value":"1530-6984","type":"print"},{"value":"1530-6992","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,26]]}}}