Bioinspired nanozyme for portable immunoassay of allergenic proteins based on A smartphone

Inspired by the structure of the catalytically active center of natural laccase, a novel laccase mimics (named LM nanozymes) with a superior catalytic activity was successfully prepared by using glutathione (GSH) and copper (II) chloride as precursors via a facile hydrothermal method. The catalytically active center structure of LM nanozymes was revealed, which was constructed based on the numerous copper (Ⅰ) and copper (II) coordinating with thiol/amino group. The possible catalytic mechanism of LM nanozymes was also proposed. Similar to natural laccase, the prepared LM nanozymes can catalyze the oxidative coupling reaction between 2,4-dichlorophenol (2,4-DP) and 4-aminoantipyrine (4-AP) to produce an obvious red product. Compared with natural laccase, the LM nanozymes showed many outstanding advantages such as robust stability, lower cost, stronger catalytic activity and substrate affinity. Based on its excellent performances, LM nanozymes were employed as a powerful alternative to the natural enzyme in a traditional enzyme-linked immunosorbent assay (ELISA) to establish a nanozyme-based ELISA towards alpha-lactalbumin (allergenic protein). Impressively, a high-throughput and portable detection method was established by the integration of the nanozyme-based ELISA with a smartphone. The portable detection strategy achieved a limit of detection as low as 0.056 ng/mL with high specificity, and also showed excellent applicability in food sample analysis. This work not only enriches the diversities of nanozymes, but also broadens the promising applications of nanozymes in the biosensing area.