Nanozyme-Linked Immunosorbent Assays: A Kinetic Perspective

Nanozymes with peroxidase-like (POD) activity are increasingly utilized as functional replacements for horseradish peroxidase in various assays. In particular, their application in enzyme-linked immunosorbent assays (ELISA) has led to the development of nanozyme-linked immunosorbent assays (NLISA). NLISA follow the well-established ELISA procedure and have been reported for a wide range of nanozymes and analytes. However, most developments overlook the fundamental differences between enzyme and nanozyme catalysis, often resulting in nonoptimal protocols in a kinetically limited regime. Herein, using core@shell Au@Pt and Au@Pd POD-like nanozymes, we demonstrate significant differences in the Michaelis-Menten constant depending on the shell thickness. Furthermore, for the first time, we report the unusually high stability of POD-like activity at ultralow pH values (down to minus 0.56). This unique feature enabled us to propose new strategies for terminating the catalytic reaction. In summary, we show that consideration of the distinct catalytic properties of nanozymes enables the development of NLISA protocols with up to an order of magnitude higher sensitivity and minimized background.