Sequential site engineering of an Fe-N₄ oxidase-like nanozyme for colorimetric detection of alkaline phosphatase

Oxidase-like nanozymes use dissolved O₂ as the terminal electron acceptor and do not require exogenous H₂O₂, offering methodological advantages; however, conventional "one-pot carbonization" often causes concurrent carbonization and active-site formation, making the coordination environment difficult to control. Here, a sequential "pre-coordinate-polymerize-pyrolyze" site-engineering route was developed. On carbon black, OPD-Fe³⁺ pre-coordination and in situ oxidative polymerization were carried out, followed by thermal treatment and purification, by which Fe-N moieties were immobilized within the shell region of the material to afford highly dispersed Fe-N_x moieties predominantly featuring Fe-N₄-like coordination (Fe-N₄@CB^d). Multiscale characterization (XRD/XPS/XANES/EXAFS) consistently showed that iron resided predominantly in a first coordination sphere dominated by Fe-N (coordination number CN ≈ 3.7; R ≈ 2.01 Å), with no resolvable Fe-Fe scattering and only minor, non-dominant Fe-containing phases. ROS-trapping and time-dependent EPR measurements further confirmed continuous O₂ activation with the generation of O₂•^-, •OH, and ¹O₂ in the Fe-N₄@CB^d/O₂ system. DFT calculations additionally supported Fe-N₄-like Fe-N_x sites as the most plausible dominant active structures for O₂ activation. Under a unified TMB/O₂ system, steady-state enzyme kinetics exhibited outstanding performance, with V_max = 351 nmol L^-1 s^-1 and K_m = 0.56 mM, which are competitive among reports using the same substrate/readout. An H₂O₂-free colorimetric platform for alkaline phosphatase (ALP) was constructed on this material, achieving a low limit of detection and good linearity; acceptable accuracy and precision were verified by standard additions in diluted serum. This sequential strategy predefines the Fe-N neighborhood and topology at the precursor stage, reduces uncertainty in site formation, and avoids metal-organic framework (MOF)/hard-template/ionic-liquid multi-step procedures, thereby providing a strong basis for reproducible Fe-N₄-like/Fe-N_x oxidase-like nanozyme preparation and streamlined, robust bioanalysis.