Nanoparticles are nanometer-scale materials capable of interacting with biomolecules on various size scales from small molecules to oligonucleotides to proteins. Their unique ability to distribute throughout the body and internalize into cells has led to their recent use as signal transducers for analyte sensing and therapeutic cargo carriers. Current techniques for attaching biomolecular cargo to nanoparticles typically rely on affinity between the two species, especially noncovalent adsorption via hydrophobic interactions. Proteins in particular have complex tertiary structures that must be maintained for biosensing and delivery applications. This is difficult to achieve when attaching proteins to nanoparticles by noncovalent adsorption due to the propensity of proteins to denature and desorb in complex biological environments such as blood plasma. Additionally, the large variability among proteins and their affinity for nanoparticles makes it difficult to predict the successful generation of potential protein-nanoparticle systems. Control of the interactions between nanoparticles and proteins is thus crucial to the successful development of tools to understand and interface with biological systems.
Towards this end, exploring protein conjugation to single-walled carbon nanotubes (SWCNTs) could advance the study of nanoparticle-protein conjugates. SWCNTs have shown utility as biosensing signal transducers due to their intrinsic, photostable, and minimally attenuated fluorescence emission in biological environments. Furthermore, their small size and large surface area make them ideal vehicles for cellular cargo delivery. Several noncovalent protein-SWCNT constructs for sensing and delivery have been developed but are susceptible to instability and loss of function when applied in biological systems. Covalent conjugation of proteins to SWCNTs could mitigate these effects and produce stable constructs to monitor disease progression and deliver therapeutics.
This dissertation presents the development of an adaptable platform for the covalent attachment of proteins to SWCNTs for analyte sensing and protein delivery. We first optimized the platform and workflow by attaching a model enzyme, Horseradish Peroxidase, to SWCNTs for hydrogen peroxide (H2O2) sensing. We find that it is possible to maintain both intrinsic SWCNT fluorescence and peroxidase enzymatic activity via triazine-thiol-SMCC crosslinker chemistry, which are key to subsequent function as a sensor. The resulting sensor shows sensitive, stable, and repeatable fluorescence modulation when exposed to H2O2 in solution and immobilized on glass. After demonstrating the utility of the platform, we have applied this methodology to covalently conjugate the CRISPR-Cas9 ribonucleoprotein to SWCNTs for delivery to cells for targeted gene editing. Using an in vitro DNA cleavage assay, we verified that Cas9 activity is maintained after SWCNT conjugation and dependent on crosslinker:protein ratio. This also suggests that the covalent conjugation platform is adaptable to other proteins, but reaction optimization should be completed every time to elucidate the optimal conditions for each protein.
The findings presented in this dissertation review the creation and optimization of a platform for the development of covalent protein-SWCNT constructs for increased stability and function in biological environments. Prioritizing covalent attachment of recognition elements and functional groups will lead to nanoparticle-based sensors and delivery vehicles with enhanced efficiency compared to previous efforts. Future directions of inquiry are also discussed and guidelines for efficiently exploring these new directions are summarized within.
