Enzymatic time−temperature indicator with cysteine-loaded chitosan microspheres/silver nanoparticles

A time−temperature indicator (TTI) based on acid−base reaction was developed by applying a new pH dye composed of cysteine-loaded chitosan (Cys-CS) microspheres and silver nanoparticles (AgNPs). It was hypothesized that cysteine released by the disintegration of Cys-CS microspheres at a critical pH would cause AgNPs to aggregate, leading to color change. Cys-CS microspheres were produced as water-in-oil (paraffin oil, MCT oil, soybean oil) emulsions according to the KOH addition method. An enzymatic TTI was made using glucose oxidase, glucose, and catalase. Only paraffin oil produced Cys-CS microspheres (average diameter, 335 ± 100 µm), whereas the others did not, probably due to saponification with KOH. FTIR analysis confirmed that cysteine was encapsulated in the microspheres. The microspheres disintegrated at pH 6.18 in a titration test. The TTI pH gradually decreased and showed a sudden color change at pH 6.10, which was similar to the critical pH of microsphere disintegration.