Protein-templated synthesis of noble metal nanoclusters, particularly gold (Au) and silver (Ag) with sizes below 2 nm, represents a cutting-edge approach in nanomaterial fabrication. These ultrasmall nanoclusters exhibit molecule-like properties due to quantum confinement effects, distinguishing them from larger plasmonic nanoparticles. The atomic precision achieved through biomolecular templating enables fine control over their optical and electronic characteristics, making them highly valuable for biosensing applications.

Proteins serve as ideal scaffolds for synthesizing Au/Ag nanoclusters due to their well-defined three-dimensional structures and diverse functional groups. The amino acid residues, particularly cysteine, histidine, and tyrosine, act as coordination sites for metal ions, facilitating nucleation and growth. For example, bovine serum albumin (BSA) has been widely employed to synthesize fluorescent Au nanoclusters (AuNCs). The synthesis typically involves reducing Au³⁺ or Ag⁺ ions in the presence of the protein under mild conditions, often using biocompatible reducing agents like ascorbic acid or sodium borohydride. The protein not only stabilizes the nanoclusters but also dictates their size and atomic arrangement, leading to discrete energy levels and strong photoluminescence.

The fluorescence of protein-templated Au/Ag nanoclusters arises from electronic transitions between quantized energy states, a phenomenon governed by their sub-2 nm size. Unlike larger nanoparticles that exhibit surface plasmon resonance, these nanoclusters display excitation-dependent or excitation-independent emission, depending on their precise atomic configuration. For instance, Au₂₅(SR)₁₈⁻ (where SR represents a thiolate ligand) exhibits a distinct emission peak around 700 nm due to intra-band transitions. Similarly, Ag₉ nanoclusters stabilized by peptides emit in the visible range, with quantum yields varying between 5% and 40%, depending on the protecting ligand environment. The exact emission mechanism involves a combination of metal-centered (sp→sp) and ligand-to-metal charge transfer (LMCT) transitions, influenced by the protein's structural constraints.

Biosensing applications of protein-templated Au/Ag nanoclusters leverage their high fluorescence, biocompatibility, and sensitivity to environmental changes. Their small size allows for minimal steric hindrance when interacting with biomolecules, making them ideal probes for detecting ions, small molecules, and proteins. For example, Hg²⁺ detection has been achieved using BSA-AuNCs, where Hg²⁺ quenches fluorescence due to metallophilic Au-Hg interactions with detection limits as low as 0.5 nM. Similarly, Ag nanoclusters synthesized with DNA or peptides have been employed for selective detection of glutathione, leveraging thiol-induced fluorescence enhancement.

Protein-templated nanoclusters also serve as efficient sensors for enzymatic activity. The fluorescence response can be modulated by enzymatic cleavage of the protein scaffold or by changes in local pH induced by enzyme reactions. Trypsin, for instance, degrades BSA-AuNCs, leading to fluorescence quenching, which enables real-time monitoring of protease activity. Additionally, these nanoclusters have been integrated into immunoassays, where their emission signals correlate with antigen-antibody binding events, providing a label-free alternative to conventional fluorescent dyes.

The atomic precision of protein-templated synthesis allows for systematic tuning of nanocluster properties by modifying the protein template or synthesis conditions. Site-directed mutagenesis of proteins can introduce specific metal-binding sites, further enhancing control over cluster size and luminescence. For example, replacing non-coordinating amino acids with cysteine in a protein template can increase the stability of Ag₈ clusters, resulting in brighter emission. Similarly, adjusting the pH during synthesis influences the reduction kinetics, leading to variations in cluster nuclearity and optical properties.

Despite their advantages, challenges remain in scaling up production and ensuring batch-to-batch consistency. The sensitivity of protein structure to environmental conditions necessitates stringent control over synthesis parameters. However, advances in bioconjugation techniques and computational modeling of protein-metal interactions are addressing these limitations, paving the way for broader adoption in diagnostics and bioimaging.

In summary, protein-templated Au/Ag nanoclusters combine atomic precision with exceptional fluorescence properties, making them powerful tools for biosensing. Their synthesis leverages natural biomolecular scaffolds, while their optical behavior is governed by quantum confinement and ligand interactions. As research progresses, these nanoclusters are expected to play an increasingly prominent role in analytical chemistry, medical diagnostics, and molecular imaging.