Rapid determination of sucrose and glucose in microbial fermentation and fruit juice samples using engineered multi-enzyme biosensing microchip

•An engineered tri-enzymatic system was designed for accurate recognizing sucrose and glucose.•The multi-enzyme system was coupled with PB nanocubes microchip to construct a sucrose biosensor.•This biosensor is of an ultrawide linearity in analysis of the real fermentation and juice samples. Amperometric biosensor utilizing prussian blue (PB) nanocubes microchip for rapid and accurate determination of sucrose and glucose contents in the fermentation broth and juice samples is described. Using a chitosan embedding immobilization strategy, this multi-enzyme system consisting of engineered invertase/mutarotase/glucose oxidase was co-immobilized on a PB nanocubes screen-printed microchip to construct a high-performance and portable sucrose biosensor. Under work potential of −0.05 V, the optimized biosensor showed a fast response time of 20 s and wide linear range from 0.5 to 4.5 mM (R2 = 0.998) with a detection limit of 0.02 mM sucrose. Besides, the biosensor showed a strong anti-interference and operational stability with a low RSD (