Rapid Mass Spectrometric Identification of the Radical Intermediates in Nanozyme-Catalyzed Reactions Using a Tip-Based Microreactor

The capture and identification of reactive intermediates, especially the short-lived radical species, play essential roles in elucidating the reaction mechanisms in nanozyme catalysis. To permit the direct characterization of the short-lived intermediates at the solid-liquid catalytic interface with electrospray ionization mass spectrometry (ESI-MS), this work develops a tip-based dual-function nanozyme microreactor/ion emitter. This innovative device was fabricated by decorating a thin-layer nanozyme deposit (<i>e.g</i>., the iron oxide-based nanozyme with peroxidase-like activity) into the inner surface of a microsized glass capillary. Unlike the conventional bottom-up wet-chemistry approaches, the immobilization of the nanozyme was proceeded via a top-down and thermal oxidation method. This procedure enables a clog-free production of the desired thin-layer nanozyme into the front tip of a tapered capillary, with a length of only a few microns. By employing a nanozyme-decorated ion emitter, we achieved the direct detection of various radical intermediates in the peroxidase-like nanozyme-catalyzed chromogenic reactions. Beyond the classical chromogenic reactions, the practicability of the integrative device was further characterized by probing a widely employed fluorogenic reaction, <i>i.e</i>., the deacetylation conversion of Amplex Red (AR) to resorufin. Notably, a radical intermediate (<i>i.e</i>., AR^•+) was successfully detected, providing direct evidence supporting the previously speculated radical-mediated reaction mechanism. This work established an effective and convenient method to implement cutting-edge nanozyme catalysis with powerful <i>in situ</i> mass spectrometry. We believe it holds promise for broader application to the in-depth explorations of diverse nanozyme-catalyzed reactions.