Site-Specific Protein Conjugation onto Fluorescent Single-Walled Carbon Nanotubes

Semiconducting single-walled carbon nanotubes (SWCNTs) are among the few photostable optical emitters that are ideal for sensing, imaging, drug delivery, and monitoring of protein activity. These applications often require strategies for immobilizing proteins onto the nanotube while preserving the optical properties of the SWCNTs. Site-specific and oriented immobilization strategies, in particular, offer advantages for improving sensor and optical signaling responses. In this study, we demonstrate site-specific protein immobilization of a model of enhanced yellow fluorescent protein with a single engineered cysteine residue, using either single-stranded DNA or a pyrene-containing linker to interact with the SWCNT surface. Protein expression and bioconjugation were characterized using a combination of gel electrophoresis, absorbance, fluorescence, mass spectrometry, and circular dichroism measurements. The results confirm successful protein immobilization onto SWCNTs, which retain their near-infrared fluorescence following conjugation. The successful demonstration of these bioconjugation strategies serves as a basis for more cost-effective, site-specific immobilization strategies that can help preserve protein folding and functionality.