Smartphone-Assisted Label-Free Bicolor Colorimetric Assay for on-site Detection of 5-Fluorouracil

5-Fluorouracil (5-FU) is a widely used anticancer drug, and its production and biological metabolic process have serious impacts on the ecological environment. Therefore, developing a platform capable of instantly monitoring 5-FU is critical to address this issue. In this study, we successfully developed folic acid (FA)-functionalized ultrasmall copper-based nanozymes (Cu-FA), which exhibit good dispersibility and peroxidase-like activity (POD-like) in a neutral environment. We further proposed a strategy for bicolor colorimetric detection of urease over a wide detection range by utilizing Cu-FA in synergy with phenolphthalein (PP) in water. This strategy effectively circumvents the acid–base buffering effect and the inconvenience of buffer replacement encountered in conventional assays, thereby improving the accuracy and sensitivity of the results. A label-free bicolor colorimetric sensor was developed for the detection of 5-FU by employing a dual-enzyme cascade reaction involving urease and Cu-FA+PP. Furthermore, the sensor was validated for detection of 5-FU residues in environmental samples in a portable manner. This method effectively bridges the gap left by traditional colorimetric techniques for 5-FU detection and holds promise for applications in environmental monitoring, which is expected to play a significant role in drug production and ecological protection.