Covalent Immobilization of Proteins on Carbon Nanotubes Using the Cross-Linker 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide—a Critical Assessment

Functionalization of carbon nanotubes (CNTs) with proteins is often a key step in their biological applications, particularly in biosensing. One popular method has used the cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to covalently conjugate proteins onto carboxylated CNTs. In this article, we critically assess the evidence presented in these conjugation studies in the literature. As CNTs have a natural affinity for diverse proteins through hydrophobic and electrostatic interactions, it is therefore important to differentiate protein covalent attachment from adsorption in the immobilization mechanism. Unfortunately, many studies of conjugating proteins onto CNTs using EDC lacked essential controls to eliminate the possibility of protein adsorption. In studies where the attachment was claimed to be covalent, discrepancies existed and the observed immobilization appeared to be due to adsorption. So far, bond analysis has been lacking to ascertain the nature of the attachment using EDC. We recommend that this approach of covalent immobilization of proteins on CNTs be re-evaluated and treated with caution.