We introduced the use of functionalized SWNTs, lying-down on metal surfaces, as efficient linking spacers between redox proteins and metal surfaces, for biodevice application, for two main reasons:

- a direct metal-protein interaction could induce the protein denaturation [1], and this can be overcome by introducing organic molecules as linking spacers [2,3];

- the conduction perpendicular to the main nanotube axis (transverse conduction) should occur over a carrier path shorter than the phonon scattering mean free path (the diameters ranging between 0.5 and 5 nm), and then, an efficient electrical conjugation is expected between proteins and metal surfaces when connected by a lying-down SWNT.

Molecular Topographic Properties

Yeast Cytochrome c (YCC) molecules directly adsorbed on a gold surface, images in TM-AFM in fluid, show a mean height of 2.6 nm, with respect to a crystallographic size of 3.4 nm [4]. We verified that the introduction of an organic linker spacer allows YCC proteins to recover their original size, in three different cases: by using organic chains, such as SATP or SMP, or oxidized SWNTs [2].

To achieve protein electrostatical adsorbtion of SWNT sidewalls, these latter have to be treated with acid solution, in order to create carboxylic groups on their sidewalls, which electrostatically bound the lysine residues of proteins. Such a bond can also become covalent, if properly activated [5], without changing the protein topography.

SWNT Transverse Conduction Properties

A new metode based on Current Sensing AFM (CS-AFM) has been introduced to discriminate metallic from semiconducting SWNTs (when lying on a conductive substrate) [6]. This method is mainly based on the concept of transverse current, i.e. the current across the SWNT as measured in the direction perpendicular to the main nanotube axis.

Indeed, both the contrast in current imaging and the trend of the current response as a function of the applied force depend on the electronic character of the SWNT analyzed.

Within the framework of a tunneling transport model, the data have shown that, at applied bias in the ±1 V range, for semiconductor SWNTs the transport is always tunneling-like, while for the metallic ones, it switches from tunneling to band-like, to tunneling again, with increasing the applied force load, being the second switching probably due to the nanotube deformation induced by the mechanical stress [6].

SWNTs as electrically efficient linkers

Their capability of preserve the original protein crystallographic size and their peculiar transverse conduction properties, allow metallic SWNTs to play a predominant role in increasing the electronic coupling between proteins adsorbed on their sidewalls and the underlying metal electrode. By means of CS-AFM, we measured also the conduction across metallic SWNTs covalently coated with YCC molecules and we compared it with that across single YCC molecules directly chemisorbed on a gold surface. We obtained that the electrical coupling between the redox protein and the metal surface is enhanced when a metallic SWNTs is used as covalent linking spacer, due to the participation of their electronic band to the transverse transport across the hybrid system [5,7].

Such an effect has been observed twice, for two different covalent immobilizations of the YCC proteins on the metallic SWNT sidewalls: (1) oxidation of the nanotubes and subsequent activation of the amide bond formation among the carboxylic groups on the nanotube sidewalls and the Lysine residues of the protein surface [5]; (2) functionalization of the SWNTs with organic chains maleimide-terminated, in order to control the protein-to-nanotube (i.e. the protein-to-surface) orientation by targeting the thiol groups, which are scarcely present on the protein surfaces [7].

[1] D. L. Johnson, C. J. Maxwell, D. Losic, J. G. Shapter, and L. L. Martin, Bioelectrochem. 58, (202) 137. [2] I. Delfino, B. Bonanni, L. Andolfi, C. Baldacchini, A. R. Bizzarri, and S. Cannistraro, J. Phys.: Condens. Matter 19, (2007) 225009.

[3] B. Bonanni, A. R. Bizzarri, and S. Cannistraro, J. Phys. Chem. B 110, (2006) 14574.

[4] Bonanni et al., CHEMPHYSCHEM 4, (2003) 1183.

[5] C. Baldacchini, and S. Cannistraro, J. Nanosci. Nanotech. 10 (2010) 2753.

[6] C. Baldacchini, and S. Cannistraro, Appl. Phys. Lett. 91, (2007) 122103.

[7] C. Baldacchini, M. A. Herrero Chamorro, M. Prato, and S. Cannistraro, Adv. Funct. Mat. 21 (2011) 153.