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Herein, the fabrication of single\u2010layer graphene\u2010coated indium tin oxide (GITO) electrodes with exceptional electrochemical activity are reported, which can accelerate the electrochemical oxidation of tri\u2010\n                    <jats:italic>n<\/jats:italic>\n                    \u2010propylamine by 9400\u2010fold and thus significantly enhance the ECL generation from Ru(bpy)\n                    <jats:sub>3<\/jats:sub>\n                    <jats:sup>2+<\/jats:sup>\n                    \u2010labeled microbeads. Furthermore, the high transparency of the GITO electrode enables the precise characterization of ECL generation by spatially mapping the distribution of ECL signals on individual microbeads using ECL self\u2010interference spectroscopy and inverted ECL microscopy. Additionally, the GITO electrode facilitates the efficient collection of ECL signals from the electrode underneath. Comparative microscopic imaging of single microbeads in both upright and inverted configurations demonstrates that the inverted signal\u2010collection mode can remarkably reduce the optical loss and enhance the optical signal by a factor of 6, as the optical signal does not need to travel across the microbeads and bulk solution. This enhancement results in a 240% increase in the signal\u2010to\u2010noise ratio for bead\u2010based ECL immunoassays. The study offers valuable insights into ECL generation in bead\u2010based ECL systems and presents an innovative strategy for enhancing immunoassay sensitivity through the synergistic integration of single\u2010layer graphene electrodes with an inverted signal\u2010collection mode.\n                  <\/jats:p>","DOI":"10.1002\/smll.202506599","type":"journal-article","created":{"date-parts":[[2025,8,6]],"date-time":"2025-08-06T09:55:52Z","timestamp":1754474152000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Transparent Single\u2010Layer Graphene Electrodes for Deciphering and Enhancing Microbead\u2010Based Electrochemiluminescence Immunoassay"],"prefix":"10.1002","volume":"21","author":[{"given":"Jialian","family":"Ding","sequence":"first","affiliation":[{"name":"State Key Laboratory of Soil Pollution Control and Safety Zhejiang Key Laboratory of Excited\u2010State Energy Conversion and Energy Storage Department of Chemistry Zhejiang University  Hangzhou 310058 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yafeng","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Sir Run Run Shaw Hospital Zhejiang University School of Medicine  Hangzhou 310016 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leslie R.","family":"Arias\u2010Aranda","sequence":"additional","affiliation":[{"name":"Bordeaux INP, ISM, UMR 5255, ENSMAC, CNRS University of Bordeaux  Pessac 33607 France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patrick C.","family":"Chaumet","sequence":"additional","affiliation":[{"name":"Institute Fresnel, CNRS, Centrale Marseille Aix Marseille University  Marseille 13013 France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Neso","family":"Sojic","sequence":"additional","affiliation":[{"name":"Bordeaux INP, ISM, UMR 5255, ENSMAC, CNRS University of 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