Chemiluminescent Nanoflower with Inherent Oxygen Vacancies for Coreactant-Free and Label-Free Immunoassay of pTau181

The detection of phosphorylated tau (pTau181), a key biomarker for Alzheimer's disease, remains challenging due to its ultralow physiological concentration. Chemiluminescent sandwich immunoassays provide high sensitivity, but their accuracy is compromised by truncated tau fragments that lack the secondary antibody epitope as well as by the instability of peroxide-based coreactants. Here, we developed a nanoflower with inherent oxygen vacancies, synthesized through a one-pot method, involving 8-amino-5-chloro-2,3-dihydro-7-phenylpyrido[3,4-d]pyridazine-1,4-dione (L012), dicarboxylic ferrocene (Fc), and HAuCl₄, named L012-Au-Fc nanoflower (LAF-NF). As a self-sufficient chemiluminescent microreactor, LAF-NF can efficiently catalyze adsorbed dissolved oxygen to generate strong, stable chemiluminescent emission without peroxide coreactants, achieving a 161-fold enhancement in the intensity over the L012 system. On this basis, a coreactant-free and label-free immunoassay for pTau181 using LAF-NF as the nanointerface was designed. The LAF-NF sensor achieved an ultrasensitive detection limit of 2.9 fg/mL and demonstrated excellent selectivity over total tau, theoretically eliminating immunoassay errors caused by epitope loss. This work offers a powerful platform for early Alzheimer's disease diagnostics.