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

Abstract

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.

Bacteriomimetic invasin-functionalized nanocarriers for intracellular delivery

“…[8] A prerequisite for binding to the integrin α5β1-receptor is that the it to a semi-stable amine-reactive NHS ester, thereby increasing the coupling efficiency. [22][23][24] In the second step, after washing off excess of non-covalently bound EDC, activated carboxylic groups on the liposomes react with the N-terminus of the surface-exposed InvA497 domain leaving the C-terminus of the adhesion molecule free for the interaction with the β1-integrin receptors. This two-step procedure of protein coupling avoided loss of InvA497 activity as well as interprotein conjugation leading to protein supramolecules or even aggregates.…”

“…This two-step procedure of protein coupling avoided loss of InvA497 activity as well as interprotein conjugation leading to protein supramolecules or even aggregates. [24] Liposomes were functionalized with InvA497 or BSA without any observed aggregation. Results of particle characterization are shown in Table 1.…”

Development of effective nanobiocatalytic systems through the immobilization of hydrolases on functionalized carbon-based nanomaterials

“…The physical adsorption of enzymes on carbon-based nanomaterials is mostly governed by hydrophobic interactions (Gao and Kyratzis, 2008). For the covalent immobilization of enzymes, the bifunctional reagent glutaraldehyde was used as a cross-linking agent between the amine functionalized nanomaterials and the enzyme, in a similar manner as described for other hybrid biocatalysts (Wu et al, 2009).…”

“…Taking into consideration that the non-covalent immobilization process relies mostly on hydrophobic interactions between the enzyme and the support (Gao and Kyratzis, 2008), the higher immobilization yield observed in the case of CNTs might result from the higher hydrophobicity of CNTs derivatives compared to the GO ones (data not shown), which results in stronger hydrophobic interactions between CNTs and the enzyme molecules. Moreover, the lower immobilization yield observed with covalent immobilization procedure for both carbonbased nanomaterials used could be due to the limited number of free amine groups on their surface which are available for covalent attachment of enzyme molecules (Cang-Rong and Pastorin, 2009).…”

“…These characteristics facilitated their use in electronic devices , fuel cells (Kauffman and Star, 2010), as carriers for drug delivery (Bianco et al, 2005), or as supports for biomacromolecules immobilization (Cang-Rong and Pastorin, 2009;Gao and Kyratzis, 2008;Pavlidis et al, 2010a,c). Graphene is a single layer of carbon atoms in a honeycomb two-dimensional lattice which has a high specific surface area and can be fabricated from graphite (Park and Ruoff, 2009).…”