Comparative analysis of natural and recombinant CMd from barley malt in the synthesis of gold nanoclusters: The influence of polyphenol binding

Protein-templated gold nanoclusters (Au NCs) are promising fluorescent nanomaterials owing to their biocompatibility, stability, and eco-friendly synthesis. CMd, a cysteine-rich subunit of the barley α-amylase/trypsin inhibitor tetramer, is well-suited as a protein template due to its high thiol content. However, native CMd (nCMd) interacts with polyphenolic compounds, potentially altering its structure and function. To enable direct comparison, recombinant CMd (rCMd) was heterologously expressed in E. coli . Spectroscopic analyses (UV–Vis, fluorescence, CD, and FTIR) revealed pronounced structural differences: nCMd displayed extensive polyphenol binding and markedly lower surface hydrophobicity. Both proteins were further employed as templates for gold nanoclusters (Au NCs) synthesis. rCMd-Au NCs exhibited a higher fluorescence quantum yield (∼8.2 %) and enabled ultrasensitive Hg 2+ detection with a detection limit of 2.95 nM (3σ). The rCMd-Au NCs probe also achieved reliable visual detection of Hg 2+ in Chinese herbal medicines, including Astragalus and Codonopsis . Overall, this study demonstrates the impact of polyphenol binding on CMd structural and functional properties and expands the application potential of CMd-based systems in bioanalytical sensing.