Histidine–cysteine–copper hybrid nanoflowers as active site-inspired laccase mimics for the colorimetric detection of phenolic compounds in PDMS microfluidic devices

Although laccases are essential multicopper oxidases in biosensing and bioremediation, their widespread use is hindered by issues such as instability and high cost. In this study, drawing inspiration from the active site structure of laccase, we developed hybrid nanoflowers through the coordination of copper ions with histidine-cysteine dipeptides (HC–Cu NFs) as highly robust laccase-mimicking nanozymes. The HC–Cu NFs possessed higher laccase-like activity compared to free laccase, yielding an approximately five-fold higher Vmax with a similar Km at the same mass concentration. Additionally, HC–Cu NFs exhibited exceptional stability across diverse environments, including a broad range of pH, temperature, salinity, and extended storage periods. Notably, HC-Cu NFs catalyzed the decolorization of phenolic dyes more rapidly than natural laccase. Utilizing these beneficial features, HC–Cu NFs were incorporated in a polydimethylsiloxane-based microfluidic device, enabling the on-site colorimetric determination of phenolic compounds, such as p-aminophenol, catechol, and hydroquinone, with outstanding sensitivity. By injecting only 20 μL of the sample solution through the inlet of the device, the sample solution rapidly moves to the detection zones where the HC-Cu NFs are retained, followed by catalytic oxidation to further react with 4-aminoantipyrine to produce a colored adduct. Using the microfluidic device, the target phenolic compounds were successfully determined within 20 min at ambient conditions using an image acquired with a smartphone with the ImageJ software. This study unequivocally demonstrates that active site-mimicking dipeptide-incorporated hybrid nanoflowers have the potential to serve as nanozymes, to replace natural laccase. This lays the groundwork for the further advancement of hybrid nanoflower-type nanozymes and their applications, particularly in point-of-care testing environments.