Functional integration of proteins with carbon-based nanomaterials such as nanotubes holds great promise in emerging electronic and optoelectronic applications. Control over protein attachment poses a major challenge for consistent and useful device fabrication, especially when utilizing single/few molecule properties. Here, we exploit genetically encoded phenyl azide photochemistry to define the direct covalent attachment of three different proteins, including the fluorescent protein GFP, to carbon nanotube side walls. Single molecule fluorescence revealed that on attachment to SWCNTs GFP’s fluorescence changed in terms of intensity and improved resistance to photobleaching; essentially GFP is fluorescent for much longer on attachment. The site of attachment proved important in terms of electronic impact on GFP function, with the attachment site furthest from the functional center having the larger effect on fluorescence. Our approach provides a versatile and general method for generating intimate protein-CNT hybrid bioconjugates. It can be potentially applied easily to any protein of choice; attachment position and thus interface characteristics with the CNT can easily be changed by simply placing the phenyl azide chemistry at different residues by gene mutagenesis. Thus, our approach will allow consistent construction and modulate functional coupling through changing the protein attachment position.

The ability to detect proteins through gating conductance by their unique surface electrostatic signature holds great potential for improving biosensing sensitivity and precision. Two challenges are: (1) defining the electrostatic surface of the incoming ligand protein presented to the conductive surface; (2) bridging the Debye gap to generate a measurable response. Herein, we report the construction of nanoscale protein-based sensing devices designed to present proteins in defined orientations; this allowed us to control the local electrostatic surface presented within the Debye length, and thus modulate the conductance gating effect upon binding incoming protein targets. Using a β-lactamase binding protein (BLIP2) as the capture protein attached to carbon nanotube field effect transistors in different defined orientations. Device conductance had influence on binding TEM-1, an important β-lactamase involved in antimicrobial resistance (AMR). Conductance increased or decreased depending on TEM-1 presenting either negative or positive local charge patches, demonstrating that local electrostatic properties, as opposed to protein net charge, act as the key driving force for electrostatic gating. This, in turn can, improve our ability to tune the gating of electrical biosensors toward optimized detection, including for AMR as outlined herein.

A central challenge in nanobiotechnology is the bottom-up assembly of platforms capable of monitoring and exploiting biomolecular interactions with nanoscale control; this in turn can allow the development of novel bioelectronics interfaces. In this regard, electrical detection methodologies, using nanomaterials, are one of the most promising candidates for biosensing investigations: they can be effectively merged with miniaturised hardware, offer simplicity, low-cost, portability, ultrahigh sensitivity, selectivity, and (label-free) real-time electrical detection We will present the fabrication of bioelectronic devices for the development of real-time biosensors with engineered protein interfacing. In particular, we assembled b-lactamase (BL) inhibitory proteins (BLIPs) onto SWCNT sidewalls in electronic device configurations, with controlled protein orientation. This allowed us to control the local electrostatic surface presented within the Debye length (see Figure), and thus modulate the conductance gating effect upon sensing protein targets. We recorded the current responses in real-time for the detection of a range of concentrations of a class BL enzymes, that degrade antibiotics, in the context of investigating antimicrobial resistance (AMR). The strategy presented here is of general applicability for the control and detection of protein-protein interactions in nanoscale device configurations, through electrostatic surface profiling; moreover, it may open up new opportunities for the development of AMR-related diagnostic devices. Figure 1

Metalloporphyrins play important roles in areas ranging from biology to nanoscience. Using computational design, we converted metalloporphyrin specificity of cytochrome b562 from iron to fluorogenic zinc. The new variant had a near total preference for zinc representing a switch in specificity, which greatly enhanced the negligible aqueous fluorescence of free ZnPP in vitro and in vivo.

Functionalintegration of proteins with carbon-based nanomaterials such as nanotubes holdsgreat promise in emerging electronic and optoelectronic applications. Control over protein attachmentposes a major challenge for consistent and useful device fabrication,especially when utilizing single/few molecule properties. Here, we exploitgenetically encoded phenyl azide photochemistry to define the direct covalentattachment of three different proteins, including the fluorescent protein GFP, tocarbon nanotube side walls. Single molecule fluorescence revealed that onattachment to SWCNTs GFP’s fluorescence changed in terms of intensity andimproved resistance to photobleaching; essentially GFP is fluorescent for muchlonger on attachment. The site of attachment proved important in terms ofelectronic impact on GFP function, with the attachment site furthest from thefunctional center having the larger effect on fluorescence. Our approachprovides a versatile and general method for generating intimate protein-CNThybrid bioconjugates. It can be potentially applied easily to any protein ofchoice; attachment position and thus interface characteristics with the CNT caneasily be changed by simply placing the phenyl azide chemistry at differentresidues by gene mutagenesis. Thus, our approach will allow consistentconstruction and modulate functional coupling through changing the protein attachmentposition.