Using dissolved O₂ as the cathodic co-reactant of three-dimensional (3D) g-C₃N₄ is a convenient method to improve the electrochemiluminescence (ECL) signal, but it still suffers the disadvantages of limited luminous efficiency of 3D g-C₃N₄ and low content, low reactivity, and instability of dissolved O₂. Here, N vacancy with high density was first introduced into the structure of 3D g-C₃N₄ (3D g-C₃N₄-NV), which could conveniently realize multipath ECL improvement by simultaneously solving the above shortcomings effectively. Specifically, N vacancy could change the electronic structure of 3D g-C₃N₄ to broaden its band gap, increase fluorescence (FL) lifetime, and accelerate electron transfer rate, obviously improving the luminous efficiency of 3D g-C₃N₄. Meanwhile, N vacancy made the excitation potential of 3D g-C₃N₄-NV to shift from -1.3 to -0.6 V, effectively weakening the electrode passivation. Moreover, the adsorption capacity of 3D g-C₃N₄-NV was obviously enhanced, which could make the dissolved O₂ enrich around 3D g-C₃N₄-NV. And massive active NV sites of 3D g-C₃N₄-NV could promote O₂ to more efficiently convert to reactive oxygen species (ROS) that were key intermediates in ECL generation. Using the newly proposed 3D g-C₃N₄-NV-dissolved O₂ system as an ECL emitter, an ultrasensitive target conversion biosensor was constructed for miRNA-222 detection. The fabricated ECL biosensor exhibited satisfactory analytical performance for miRNA-222 with a detection limit of 16.6 aM. The strategy achieved multipath ECL improvement by introducing high-density N vacancy simply in the 3D structure of g-C₃N₄ and could open a new horizon for developing a high-performance ECL system.

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1	Engineering	4	4	104
2	Chemistry	3	3	67
3	Biomedical Engineering	2	2	70
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