Enzymes Immobilized on Carbon Nanotubes

Carbon nanotubes, due to their unique properties, have numerous applications, especially when conjugated with biomacromolecules. As far as the immobilization of proteins concerns, the high specific surface area of these nanomaterials facilitates the immobilization of more protein molecules on the carrier material, which is accompanied by an increased specific enzyme activity. 

CNTs present good electrical communication, which renders feasible the electron transfer from protein to the electrode. For this reason many laboratories have turned their scientific interests in the fabrication of CNT-modified electrodes onto which enzymes or nucleic acids are immobilized. As it can be seen from Table 2.3, most of the works in the field of CNT-protein conjugates are about the development of new biosensors. CNT-biosensors have shown efficient electrical communications and promising sensitivities required for applications as antigen recognition, enzyme-catalyzed reactions and deoxyribonucleic acid (DNA) hybridizations [124]. The presence of CNTs facilitates the transportation of the signal from the enzyme to the electrode. The use of CNT-modified electrodes permits 

The development and application of biosensors has been discussed in several reviews [125, 126]. For instance, fabrication of cholesterol biosensors relies on the immobilization of cholesterol oxidase and cholesterol esterase onto CNTs, while horseradish peroxidase and flavocytochrome P450scc are also used for the same reason [125, 126]. Beyond the biosensing field, carbon nanotubes are also used as carriers for peptide, nucleic acid and drug delivery, due to their intrinsic property to cross cell membranes [102, 124]. The fact that the functionalized CNTs (f-CNTs) are not immunogenic and low-toxic opens the pathway for more research in the field of CNT-abetted drug delivery [102]. 

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The non-specific adsorption of proteins on carbon nanotubes is an interesting phenomenon but represents a relatively less controllable mode of protein-CNT interaction. Moreover, in non-covalent immobilization process, the immobilized protein is in equilibrium between the surface of the carbon nanotubes and the solution and can therefore be gradually detached from the nano-material surface, a phenomenon called protein leakage [127]. To prevent the leaching of enzymes, covalent bonds have been used to attach the enzyme molecules to the nanostructured materials, which lead to more robust and predictable conjugation. Experimental evidences prove that proteins can be immobilized either in their hollow cavity or on the surface of carbon nanotubes [130]. 

Liu X, Bu C, Nan Z, Zheng L, Qiu Y, Lu X (2013) Enzymes immobilized on amine-terminated ionic liquid-functionahzed carbon nanotube for hydrogen peroxide determination. Talanta 105 63-68... 

In view of the conductive and electrocatalytic features of carbon nanotubes (CNTs), AChE and choline oxidases (COx) have been covalently coimmobilized on multiwall carbon nanotubes (MWNTs) for the preparation of an organophosphorus pesticide (OP) biosensor [40, 41], Another OP biosensor has also been constructed by adsorption of AChE on MWNTs modified thick film [8], More recently AChE has been covalently linked with MWNTs doped glutaraldehyde cross-linked chitosan composite film [11], in which biopolymer chitosan provides biocompatible nature to the enzyme and MWNTs improve the conductive nature of chitosan. Even though these enzyme immobilization techniques have been reported in the last three decades, no method can be commonly used for all the enzymes by retaining their complete activity. 

In this chapter we will focus on the most recent developments in the use of nanostructured materials such as nanoclays, carbon nanotubes and magnetic nanoparticles for enzyme immobilization and stabilization, together with their potential applications in various fields, such as development of biosensors and biofuel cells, biocatalytic processes, enzyme purification/separation, intracellular protein transportation etc. 

Another technique to increase the dispersability and the biocompatibility of the carbon nanotubes is the insertion of terminal groups on the surface of CNTs with a procedure called functionalization [104, 105, 118, 123, 136]. The creation of free carboxylic acid and amino acid moieties on the surface are the most commonly used functionalization for enzyme immobilization and stabilization [104, 105, 118,123, 136]. Apart from these two functional groups, other groups are also used, for various purposes. For example, Jeykumari and Narayanan functionalized MWCNTs with toluidine blue, in order to prevent the leakage of the redox mediator from their bienzymic biosensor [114]. 

The interaction of nanoparticles with the proteins is governed from the same type of interactions described for carbon nanotubes. Since NPs carry charges, they can electrostatically adsorb biomolecules with different charges, which depend on the pH that the immobilization takes place and the pi of the protein [3,191]. Moreover, hydrophobic interactions, hydrogen bonds and non-specific absorption can play a role for enzyme non-covalent adsorption onto the surface of nanoparticles. 

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