Engineering Biomimetic Prussian Blue-Based Hollow Microreactors for Spatially Confined Cascade Catalysis with Cofactor Regeneration

Abstract Cofactor-dependent biocatalytic processes are often constrained by the high cost of cofactors, inefficient regeneration strategies, and limited integration of chemical and enzymatic steps. Here, we report a polyamine-assisted biomimetic layer-by-layer (LbL) strategy for fabricating hollow silica-Prussian blue (SiO2–PB) microreactors that enable spatially confined cascade catalysis and cofactor recycling. Calcium carbonate (CaCO3) serves as a sacrificial template, while a thin coating of SiO2 nanoparticles stabilizes the structure, prevents the vaterite-to-calcite phase transition, and supports the formation of stable hollow architectures. The resulting microreactors exhibit enhanced H2O2-mediated NADH (reduced nicotinamide adenine dinucleotide) oxidation relative to bare PB nanoparticles. The kinetic analysis reveals approximately 5.8-fold higher catalytic efficiency, further demonstrating the beneficial effect of the hollow architecture on PB-mediated catalysis. X-ray photoelectron spectroscopic (XPS) analysis further supports changes in the Fe chemical environment associated with catalytic operation. Encapsulation of aldehyde dehydrogenase (ALDH) establishes a tandem nanozyme-enzyme cascade, wherein in situ-generated NAD+ drives aldehyde oxidation and regenerates NADH. The hollow microreactors achieve a 4-fold enhancement in NADH regeneration compared with their solid counterparts, reaching ∼90% regeneration with acetaldehyde. The higher apparent cofactor total turnover number (TTN) and turnover frequency (TOF) values further demonstrate enhanced cofactor cycling efficiency in the hollow microreactor system. Confocal microscopy confirms ALDH localization mainly within the microreactor shell, and NMR analysis verifies selective product formation. Structural stability and enzyme/metal leaching studies further demonstrate the integrity of the microreactors and retention of their catalytic components. This biomimetic bioinorganic platform provides an efficient strategy for cofactor recycling and the development of artificial metabolic systems.