Synthesis of Prussian Blue nanoparticles in water/alcohol mixtures

Prussian Blue, a blue coordination polymer, emerges as a promising candidate in the realm of biomedicine. Its nanoparticles, known as catalytic labels or nanozymes, exhibit remarkable peroxidase-like properties and serve as effective antioxidants. Unsurprisingly, the demand for synthesizing Prussian Blue nanoparticles with customizable sizes is on the rise. In this study, we unveil a novel approach to synthesizing Prussian Blue nanoparticles. In this work, the synthesis of Prussian Blue nanoparticles by reducing an equimolar mixture of FeCl3 and K3[Fe(CN)6] with hydrogen peroxide in different water-alcohol mixtures was demonstrated for the first time. Alcohols with a lower dielectric constant (propanol-1, isopropyl alcohol, and tert-butanol) contribute to an increase in nanoparticle size, particularly at mole fractions of 0.02-0.05 and beyond. Conversely, alcohols with a higher dielectric constant (ethanol, methanol, ethylene glycol, and propylene glycol, excluding glycerol) demonstrate the ability to decrease nanoparticle size at mole fractions of 0.2-0.26 and higher. Building upon these findings, we present a scalable and reproducible method for preparing small Prussian Blue nanoparticles, measuring 30-40 nm, with enhanced peroxidase-like activity using 79.2% ethylene glycol as a solvent. The proposed mechanism behind the effect of ethylene glycol involves the limitation of both growth and secondary aggregation of Prussian Blue nanoparticles. These synthesized nanoparticles prove their efficiency as catalytic labels in a model vertical flow immunoassay designed to detect antibodies against SARS-CoV-2.