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Protein post-translational modifications (PTMs), which are chemical modifications and most often regulated by enzymes, play key roles in functional proteomics. Detection of PTM enzymes, thus, is critical in the study of cell functioning and development of diagnostic and therapeutic tools. Herein, we develop a simple peptide-templated method to direct rapid synthesis of highly fluorescent gold nanoclusters (AuNCs) and interrogate the effect of enzymatic modifications on their luminescence. A new finding is that enzymes are able to exert chemical modifications on the peptide-templated AuNCs and quench their fluorescence, which furnishes the development of a real-time and label-free sensing strategy for PTM enzymes. Two PTM enzymes, histone deacetylase 1 and protein kinase A, have been employed to demonstrate the feasibility of this enzyme-responsive fluorescent nanocluster beacon. The results reveal that the AuNCs' fluorescence can be dynamically decreased with increasing concentration of the enzymes, and subpicomolar detection limits are readily achieved for both enzymes. The developed strategy can thus offer a useful, label-free biosensor platform for the detection of protein-modifying enzymes and their inhibitors in biomedical applications.

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A novel phospholipid-graphene nanoassembly is developed based on self-assembly of phospholipids on nonoxidative graphene surfaces. The nanoassembly can be prepared easily through noncovalent hydrophobic interactions between the lipid tails and the graphene without destroying the electronic conjugation within the graphene sheet. This imparts the nanoassembly with desired electrical and optical properties with nonoxidative graphene. The phospholipid coating offers excellent biocompatibility, facile solubilization, and controlled surface modification for graphene, making the nanoassembly a useful platform for biofunctionalization of graphene. The nanoassembly is revealed to comprise a bilayer of phospholipids with a reduced graphene oxide sheet hosting in the hydrophobic interior, thus affording a unique planar mimic of the cellular membrane. By using a fluorescein-labeled phospholipid in this nanoassembly, a fluorescence biosensor is developed for activity assay of phospholipase D. The developed biosensor is demonstrated to have high sensitivity, wide dynamic range, and very low detection limit of 0.010 U/L. Moreover, because of its single-step homogeneous assay format it displays excellent robustness, improved assay simplicity and throughput, as well as intrinsic ability to real-time monitor the reaction kinetics.