(299c) Covalent Attachment of Horseradish Peroxidase to Single-Walled Carbon Nanotubes for Hydrogen Peroxide Detection

Hydrogen peroxide (H₂O₂), a reactive oxygen species (ROS), is critical to the regulation of mammalian cellular processes such as apoptosis and response to oxidative stress. However, unregulated cellular levels of H₂O₂ leads to erratic DNA mutation and damage, implicating it as a biomarker for diseases such as cancer, Parkinson’s, Alzheimer’s, and Huntington’s. It is thus important to develop sensitive and selective technologies to sense elevated cellular H₂O₂ levels for improved disease diagnostics. Current techniques for H₂O₂ sensing involve chemiluminescent or fluorescent dyes, but these suffer from instability in dynamic environments, photobleaching, and low spatiotemporal resolution.

Here, we developed a nanosensor for detection of H₂O₂. We base our nanosensor on single-walled carbon nanotubes (SWCNTs), owing to their intrinsic near-infrared (nIR) fluorescence, high aspect ratio, and photostability. The selectivity of our nanosensor is conferred by covalently attaching horseradish peroxidase (HRP) to the SWCNT surface, a key enzyme in the oxidative stress response pathway of the horseradish plant. We employed covalent surface modification of SWCNTs with cyanuric chloride, maintaining intrinsic nIR fluorescence¹ while priming the SWCNT surface for further modification with common protein conjugation handles such as carboxylic acid, primary amine, and thiol groups.² We functionalized HRP with maleimide groups via NHS-lysine conjugation with a linker named Sulfo-SMCC. We subsequently incubated maleimide-functionalized HRP with thiol-functionalized SWCNTs to covalently attach HRP to SWCNTs without compromising enzymatic activity.

We characterized the nanosensor’s physical and optical properties to confirm successful protein conjugation while maintaining intrinsic SWCNT fluorescence and HRP enzymatic activity. Additionally, we assessed the nanosensor’s concentration-dependent response to H₂O₂ in solution and demonstrated a limit of detection of 31 uM. Furthermore, we demonstrated the nanosensor’s selectivity for H₂O₂ by its lack of response to an analyte panel of related reactive oxygen species and other biologically relevant analytes. Finally, we immobilized the nanosensor on glass to confirm its fluorescent signal is reversible, which we can use to sense elevated peroxide levels in cell lysate from oxidatively stressed MCF-7 cells. Taken together, these results demonstrate covalent attachment of enzymes to SWCNTs with a chemical strategy that preserves both intrinsic SWCNT fluorescence and enzyme function. We anticipate this platform can be adapted to covalently attach other proteins of interest such as other biologically relevant enzymes for sensing and larger protein cargo for cellular delivery including CRISPR-Cas9.

References

1. Setaro, A. et al. Preserving π-conjugation in covalently functionalized carbon nanotubes for optoelectronic applications. Nat. Commun. 8, 14281 (2017).
2. Chio, L., Pinals, R. L., Murali, A., Goh, N. S. & Landry, M. P. Covalent Surface Modification Effects on Single‐Walled Carbon Nanotubes for Targeted Sensing and Optical Imaging. Adv. Funct. Mater. 1910556 (2020) doi:10.1002/adfm.201910556.