Functional Interfaces for Biomimetic Energy Harvesting: CNT-DNA Matrix for Enzyme Assembly

The development of 3D structures exploring the properties of nano-materials and biological molecules allows us to mimic natural metabolic processes such as Krebs or Calvin cycle by creating “artificial” metabolic pathways called metabolons. The utilization of metabolons in biomimetic energy-harvesting systems leads to increased fuel oxidation and as a result increased Columbic efficiency. The specific order and minimized distance between the enzymes from those metabolons are the main reasons for subsequent and more effective fuel consumption.

In this study a complex carbon nanotubes (CNTs) – DNA matrix for specific enzyme immobilization was created. It composed of “brush” type aligned CNTs covalently attached on electrochemically modified gold surface. This forest like CNTs structure was used as a substrate for the development of a DNA scaffold. This DNA scaffold had a designed sequence that allows strong attachment of DNA to the CNTs surface by wrapping the nanotubes through pi-pi stacking and at the same time has a unique nine base pair motif that was further hybridized. The hybridized part of the DNA was used to specifically attach zinc finger (Znf268) that was genetically fused to small laccase (SLAC).

Zinc fingers are small protein structural motifs that can recognize dsDNA and specifically bind to it. We used this property to control the SLAC immobilization by fusing the Znf268 to the enzyme and attaching it to the CNTs-DNA scaffold (Fig. 1). Due to the specific interactions of zinc fingers with DNA we will be able to immobilize enzymes in particular order and distance creating metabolons.

Figure 2 shows Atomic Force Microscopy (AFM) images of the gold surface at the different stages of its modification to the creation of the CNTs forest. The modification procedures were also confirmed with x-ray photoelectron spectroscopy (XPS).

To prove the concept we used SLAC, which is an enzyme from the family of well-known multi-copper oxidases that are able to reduce oxygen and form water [1]. The enzyme was immobilized by the described approach and characterized by XPS (Fig. 3).  The presence of copper in the XPS spectrum unambiguously proofs the success of the immobilization procedure. The developed herein approach for specific enzyme immobilization can be applied for all types of enzymes for the development of bio-cathodes as well as bio-anodes in the design of energy-harvesting systems.

The developed herein approach for specific enzyme immobilization can be applied for all types of enzymes for the development of bio-cathodes as well as bio-anodes in the design of energy-harvesting systems.

[1] Szilvay, G., S. Brocato, D. Ivnitski, C. Li, P. De La Iglesia, C. Lau, E. Chi, M. Werner-Washburne, S. Banta P. Atanassov Chem. Commun., 2011, 47, 7464–7466.