Fluorescent nanodiamonds are biocompatible, infinitely photostable, and fluoresce in a variety of wavelengths, including the near-infrared (NIR) region. These characteristics suggest transformative applications in areas ranging from pure biological research to biomedical imaging, where protein-conjugated nanodiamonds could be targeted to specific tissues in living patients or even structures within individual cells. In order to reach this potential, however, the coupling of specific targeting proteins to nanodiamond surfaces must be simplified to a point where rapid nanodiamond prototyping can be carried out by personnel with little or no chemical conjugation expertise. To address this need, we propose the development of simple ?kits? for the production of novel protein-conjugated fluorescent nanodiamonds, where the end user provides the recombinant targeting protein and specifies the nanodiamond core for conjugation. The kits would consist of expression plasmids for production of a tagged version of the end user?s target protein, along with nanodiamond ?blanks? for quick and simple conjugation. The conjugation reactions will be mediated by either a naturally split trans- splicing intein, or the Sortase A enzyme. Each of these enzymatic coupling reactions allow uniform and highly oriented binding of the target protein to the nanodiamond surface via either N-terminal (intein) or C-terminal (Sortase A) single- point attachment. Thus, the tagged target protein, provided by the end user using their recombinant expression host of choice, and nanodiamond blank will allow researchers with little skill in chemical protein conjugation to rapidly generate highly customized nanodiamond prototypes for their desired applications. In our Phase 1 work, we will develop two core enzymatic coupling technologies based on the GOS-TerL split intein (for N-terminal protein attachment), and the Staphylococcus aureus Sortase A transpeptidase (for C-terminal protein attachment). In each case, a short synthetic peptide will be produced and chemically coupled to the nanodiamond surface using conventional methods, while a complimentary peptide will be genetically fused to the desired target protein using the provided expression plasmids. Both the intein and Sortase reactions take place spontaneously under ambient conditions, allowing highly specific coupling via simple mixing of the purified target protein and nanodiamond blank. To demonstrate these methods, we will conjugate Green Fluorescent Protein (GFP) and ?-lactamase onto the surface of a suitable nanodiamond. These two well- characterized model proteins have been selected due to the availability of simple activity assays, which will facilitate the characterization and optimization of the coupling chemistries. In the case of GFP, we will use confocal microscopy to demonstrate co-localization of the protein to the nanodiamond surface, while ?-lactamase will allow demonstration of the target protein retaining enzymatic activity after immobilization. In addition, the coupling of both model proteins will be quantified using a variety of conventional immunological and biochemical assay methods. The generation of at least three successfully coupled target protein configurations will be considered a successful demonstration of the Phase 1 goals. In future work, we will further develop the kit components and nanodiamond blank production process, and demonstrate additional applications of the system using FRET and more complex targeting and drug delivery strategies.
We describe enzymatic processes for attaching proteins to fluorescent nanodiamonds for bioimaging applications. Enzyme mediated ligations are simple, fast, efficient and require little chemical conjugation experience by the researcher. The resulting conjugates will have uniform orientations and site specific linkages to the nanodiamond surface. These are much needed improvements for the development of novel bioimaging agents and will enable the development of a host of tools for the diagnosis and treatment of disease.
