Yeast cytochrome c integrated with electronic elements: a nanoscopic and spectroscopic study down to single-molecule level

Abstract

Various aspects of redox protein integration with nano-electronic elements are addressed by a multi-technique investigation of different yeast cytochrome c (YCC)-based hybrid systems. Three different immobilization strategies on gold via organic linkers are explored, involving either covalent bonding or electrostatic interaction. Specifically, Au surfaces are chemically modified by self-assembled monolayers (SAMs) exposing thiol-reactive groups, or by acidoxidized single-wall carbon nanotubes (SWNTs). Atomic force microscopy and scanning tunnelling microscopy are employed to characterize the morphology and the electronic properties of single YCC molecules adsorbed on the modified gold surfaces. In each hybrid system, the protein molecules are stably assembled, in a native configuration. A standing-up arrangement of YCC on SAMs is suggested, together with an enhancement of the molecular conduction, as compared to YCC directly assembled on gold. The electrostatic interaction with functionalized SWNTs allows several YCC adsorption geometries, with a preferential high-spin haem configuration, as outlined by Raman spectroscopy. Moreover, the conduction properties of YCC, explored in different YCC nanojunctions by conductive atomic force microscopy, indicate the effectiveness of electrical conduction through the molecule and its dependence on the electrode material. The joint employment of several techniques confirms the key role of a well-designed immobilization strategy, for optimizing biorecognition capabilities and electrical coupling with conductive substrates at the single-molecule level, as a starting point for advanced applications in nano-biotechnology.

Highly Conductive Redox Protein–Carbon Nanotube Complex for Biosensing Applications

“…After incubation with an YCC solution, the flat gold electrode appears covered by a layer of globular objects, which correspond to proteins chemisorbed via their exposed thiol,14, 22 and the nanotubes are decorated by lumps (Figure 1b) that have the same linear density (34 ± 11 μm −1 ) but a larger mean height (2.8 ± 1.1 nm) than those corresponding to the maleimide‐terminated functional chains (shown in Figure 1a). Such larger lumps have a mean height that is close to that previously measured by TM‐AFM for YCC molecules chemisorbed on bare (2.6 ± 0.7 nm)22 and maleimide‐modified (3.1 ± 0.7 nm)14 gold surfaces. Therefore, they are reasonably attributed to YCC molecules bound to Mal‐SWNT sidewalls with native‐like topological properties, in a 1:1 ratio with respect to the nanotube functional sites.…”

“…In this context, design and development of nano‐biodevices coupling redox proteins with single‐walled carbon nanotubes (SWNTs) have extraordinarily grown. Many strategies have been employed to conjugate redox proteins with SWNTs: non‐covalent adsorption,7, 8 electrostatic interaction,9–14 π‐stacking modification,15 and covalent functionalization10, 16–20 have been exploited. Such a coupling should meet a number of crucial requirements: i) the protein functionality has to be preserved; ii) the protein orientation towards the environment has to be properly controlled, in order to facilitate the exposition of the active site; iii) the proteins should be endowed with sufficient reorientational diffusional freedom, to favor the biorecognition process; iv) an efficient electrical conduction across the protein–nanotube–electrode system has to be established.…”

“…The thiol group belonging to the single exposed cysteine of YCC, which is located in the region opposite to the lysine‐rich protein docking site, should covalently react with malemide, ensuring a strong, oriented nanotube‐protein bond that leaves the docking site free to interact. Topological and functional properties of cytochrome c coupled with SWNTs have been already described, with nano‐biosensor application aims 7–14, 20. Nevertheless, no attention has been paid to achieve simultaneous control of the protein orientation and of the protein–nanotube electrical connection.…”

Polarity‐Controlled Attachment of Cytochrome C for High‐Performance Cytochrome C/Graphene van der Waals Heterojunction Photodetectors

“…Despite exciting progress made in molecular electronics, main challenges remain in fabrication of the molecular electronic devices. They include (1) complicated and tedious electrode fabrication using advanced lithography on individual molecules and (2) difficulties in implementation of “conventional” diode or transistor designs on molecular devices, such as a p–n type of interface for dissociation of the photoexcited excitons and charge transfer from molecule to electrode . Addressing these challenges is paramount to the practical applications of molecular electronics.…”

Lying-Down Metallic Single-Walled Carbon Nanotubes as Efficient Linkers for Metalloprotein-Based Nanodevices

“…In order to overcome these problems, suitable organic molecules can be used as spacers, connecting metalloproteins and electrodes. [14][15][16][17][18] Specifically designed linkers may help in targeting suitable protein residues, so that the self-assembly can be appropriately driven, controlling, to the desired extent, the strength of the bonding, the orientation of the molecule, and its flexibility. Among these spacers, single-walled carbon nanotubes (SWNTs) recently emerged as highly attractive candidates.…”