Ratiometric fluorescent detection of protease activity in foods based on microwave-assisted synthesized casein-directed gold nanoclusters

Developing a versatile probe for activity detection of protease is of significant importance due to its crucial roles in food processing, disease diagnosis, and pharmaceuticals. However, the sensitivity of probe towards interfering agents especially in complex samples hindered its development. Here, casein-directed gold nanoclusters (casein-AuNCs) were fabricated in a household microwave oven in 30 s, which displayed strong red emission with a large Stokes shift of 310 nm and high storage stability. Conjugation of fluorescein isothiocyanate (FITC) with casein-AuNCs enabled the ratiometric fluorescent detection of protease activity. Mechanistic investigation confirmed that upon protease hydrolysis, aggregation of AuNCs occurred, leading to fluorescence decay. This sensor exhibited good linearity and sensitivity for protease activity detection over a wide pH range, with detection limits of 0.29 U/mL and good anti-interference performance in real sample analysis.