A Novel Method for Carbon Nanotube Functionalization Using Immobilized Candida antarctica Lipase

Abstract

Carbon nanotubes (CNTs) have been proposed as nanovehicles for drug or antigen delivery since they can be functionalized with different biomolecules. For this purpose, different types of molecules have been chemically bonded to CNTs; however, this method has low efficiency and generates solvent waste. Candida antarctica lipase is an enzyme that, in an organic solvent, can bind a carboxylic to a hydroxyl group by esterase activity. The objective of this work was to functionalize purified CNTs with insulin as a protein model using an immobilized lipase of Candida antarctica to develop a sustainable functionalization method with high protein attachment. The functionalized CNTs were characterized by scanning electron microscope (SEM), Raman spectroscopy, Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzymatic functionalization of insulin on the surface of the CNTs was found to have an efficiency of 21%, which is higher in conversion and greener than previously reported by the diimide-activated amidation method. These results suggest that enzymatic esterification is a convenient and efficient method for CNT functionalization with proteins. Moreover, this functionalization method can be used to enhance the cellular-specific release of proteins by lysosomal esterases.

Keywords: Candida antarctica lipase; carbon nanotubes; covalent functionalization; enzymatic functionalization; ester bond.

Figure 1

Schematic representation of the functionalization of CNTs using NOVOZYME 435. Acid-treated CNTs display carboxyl groups due to oxidation, and these carboxyl groups are susceptible to reacting with the hydroxyl groups of some amino acids (threonine, serine, or tyrosine) present in proteins—a reaction that is catalyzed by Novozyme 345 (lipase) under anhydrous conditions.

Figure 2

Microphotographs obtained by SEM. Microphotographs show the aspects of (A) pristine CNTs, (B) purified CNT-COOH, (C) CNT-INSp, and (D) CNT-INSs. Arrows indicate spherical bodies detected on the CNT surface.

Figure 3

Raman spectra for the different CNTs. In each figure, the D band is shown at 1338 cm⁻¹, and the G band is shown at 1600 cm⁻¹ (laser excitation at 632.8 nm).

Figure 4

FTIR for the different CNTs. The signals due to the C=O bond (1733 cm⁻¹), C-O (1146 and 1239 cm⁻¹), O-H (3300–3600 cm⁻¹), N-H (700 cm⁻¹) as well as C-H stretching (2875–2950 cm⁻¹) are shown.

Figure 5

Thermogravimetric analysis of functionalized CNTs.

Figure 6

SDS–PAGE of functionalized CNTs. MWM; molecular weight marker, C1; CNTs without enzyme, C2; CNTs without protein, C3; CNTs-INSs, C4; CNTs-INSp. 10% SDS–PAGE. Silver staining.