Biofunctionalization of carbon nanotubes for reversible site-specific protein immobilization

Carbon nanotubes (CNTs) emerge as biosensors due to their extraordinary electrical transport properties. Such applications depend on interfacing proteins with CNTs in an oriented manner while maintaining their structural and functional integrity, opening versatile opportunities for one-dimensional alignment and high-sensitivity sensing of protein interactions and conformational dynamics. Here, we devised a novel surface architecture for reversible protein immobilization on CNTs via a short peptide tag for fabrication of reusable biosensors for multiple protein analysis. To this end, we conjugated pyrene-poly(ethylene glycol) with tris-nitrilotriacetic acid (Py-PEG-tris-NTA) for site-specific immobilization of oligohistidine(His)-tagged proteins. We demonstrate spontaneous self-assembly of Py-PEG-tris-NTA on CNTs and graphene, as well as specific capturing of His-tagged green fluorescent protein after loading the NTA chelators with Ni(II) ions. The protein binding capacity of tris-NTA-functionalized CNTs could be restored by an imidazole wash, allowing for repeated immobilization cycles. Reflectance interference spectroscopy and fluorescence lifetime analysis confirmed a specific and reversible binding of proteins on CNT-coated silica substrates as well as close interfacing to the CNT surface.

Automated summary

By designing a surface architecture for reversible protein immobilization on CNTs, we have successfully fabricated reusable biosensors for multiple protein analysis. The specific capturing of His-tagged proteins was achieved by combining Py-PEG-tris-NTA and Ni(II) ions, with the protein binding capacity being restored by imidazole wash.