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

Oxidase-like nanozymes use dissolved O 2 as the terminal electron acceptor and do not require exogenous H 2 O 2 , 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-Fe 3+ 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–N 4 -like coordination (Fe–N 4 @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 O 2 activation with the generation of O 2 • - , •OH, and 1 O 2 in the Fe–N 4 @CB d /O 2 system. DFT calculations additionally supported Fe–N 4 -like Fe–N x sites as the most plausible dominant active structures for O 2 activation. Under a unified TMB/O 2 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 H 2 O 2 -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–N 4 -like/Fe–N x oxidase-like nanozyme preparation and streamlined, robust bioanalysis.