Fabrication and Functionalisation of Nanocarbon-Based Field-Effect Transistor Biosensors.

Nanocarbon-based field-effect transistor (NC-FET) biosensors are at the forefront of future diagnostic technology. By integrating biological molecules with electrically conducting carbon-based platforms, high sensitivity real-time multiplexed sensing is possible. Combined with their small footprint, portability, ease of use, and label-free sensing mechanisms, NC-FETs are prime candidates for the rapidly expanding areas of point-of-care testing, environmental monitoring and biosensing as a whole. In this review we provide an overview of the basic operational mechanisms behind NC-FETs, synthesis and fabrication of FET devices, and developments in functionalisation strategies for biosensing applications.

Tuning Electrostatic Gating of Semiconducting Carbon Nanotubes by Controlling Protein Orientation in Biosensing Devices.

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 ß-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 ß-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.

Site-Specific Protein Photochemical Covalent Attachment to Carbon Nanotube Side Walls and Its Electronic Impact on Single Molecule Function.

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 four different proteins, including the fluorescent protein GFP and a ß-lactamase binding protein (BBP), to carbon nanotube side walls. AFM showed that on attachment BBP could still recognize and bind additional protein components. Single molecule fluorescence revealed that on attachment to SWCNTs function was retained and there was feedback to GFP in terms of fluorescence intensity and improved resistance to photobleaching; 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 chromophore 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 to any protein of choice; the 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.

Better together: building protein oligomers naturally and by design.

Protein oligomers are more common in nature than monomers, with dimers being the most prevalent final structural state observed in known structures. From a biological perspective, this makes sense as it conserves vital molecular resources that may be wasted simply by generating larger single polypeptide units, and allows new features such as cooperativity to emerge. Taking inspiration from nature, protein designers and engineers are now building artificial oligomeric complexes using a variety of approaches to generate new and useful supramolecular protein structures. Oligomerisation is thus offering a new approach to sample structure and function space not accessible through simply tinkering with monomeric proteins.

Optimising CNT-FET biosensor design through modelling of biomolecular electrostatic gating and its application to ß-lactamase detection.

Carbon nanotube field effect transistors (CNT-FET) hold great promise as next generation miniaturised biosensors. One bottleneck is modelling how proteins, with their distinctive electrostatic surfaces, interact with the CNT-FET to modulate conductance. Using advanced sampling molecular dynamics combined with non-canonical amino acid chemistry, we model protein electrostatic potential imparted on single walled CNTs (SWCNTs). We focus on using ß-lactamase binding protein (BLIP2) as the receptor as it binds the antibiotic degrading enzymes, ß-lactamases (BLs). BLIP2 is attached via the single selected residue to SWCNTs using genetically encoded phenyl azide photochemistry. Our devices detect two different BLs, TEM-1 and KPC-2, with each BL generating distinct conductance profiles due to their differing surface electrostatic profiles. Changes in conductance match the model electrostatic profile sampled by the SWCNTs on BL binding. Thus, our modelling approach combined with residue-specific receptor attachment could provide a general approach for systematic CNT-FET biosensor construction.

DNA-Directed Assembly of Carbon Nanotube-Protein Hybrids.

Here, we report the controlled assembly of SWCNT-GFP hybrids employing DNA as a linker. Two distinct, enriched SWCNTs chiralities, (6,5), (7,6), and an unsorted SWCNT solution, were selectively functionalized with DNA and hybridized to a complementary GFPDNA conjugate. Atomic force microscopy images confirmed that GFP attachment occurred predominantly at the terminal ends of the nanotubes, as designed. The electronic coupling of the proteins to the nanotubes was confirmed via in-solution fluorescence spectroscopy, that revealed an increase in the emission intensity of GFP when linked to the CNTs.

Tuning Electrostatic Gating of Semiconducting Carbon Nanotubes by Controlling Protein Orientation in Biosensing Devices.

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 ß-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 ß-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.