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

Abstract

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.

Fabrication and Functionalisation of Nanocarbon‐Based Field‐Effect Transistor Biosensors

“…Coming under the same wing as noncovalent functionalisation, this approach again benefits from the undisrupted sp2 network; but is at risk of pushing biomolecules beyond the Debye length. [146,174] Pyrene based linkers represent a significant proportion of these linkers, utilizing their aromatic pyrene group to π-stack onto nanocarbon. [40,175] Commonly employed across graphene and SWCNTs, is 1-pyrenebutanoic acid succinimidyl ester (PBASE), which uses the ester group in a nucleophilic substitution reaction with free amines on a protein, anchoring the protein to the pyrene and thus the nanocarbon.…”

“…Xu et al, made an important development by incorporating these azide mutations at specific residues, allowing the receptor protein to be conjugated to nanocarbon in different, defined orientations, which resulted in differential gating effects being observed. [174] Polymeric linkers are amphiphilic molecules typically consisting of a hydrophobic alkyl backbone with protruding polar groups for biofunctionalisation and water solubilisation. [179] Acting in much the same way as pyrene-based linkers, polymeric linkers are larger in size and can wrap helically around carbon nanotubes, [148] whilst films are formed on graphene.…”

“…Linker molecules represent the final category of functionalisation and can be characterised through their bifunctional ability to covalently or non‐covalently interact with nanocarbon, whilst simultaneously providing a reactive handle to covalently interface with a biomolecule. Coming under the same wing as non‐covalent functionalisation, this approach again benefits from the undisrupted sp2 network; but is at risk of pushing biomolecules beyond the Debye length [146,174] …”

“…This uses the click chemistry reaction of strain‐promoted azide‐alkyne cycloaddition when reacted with proteins containing an azide group. Xu et al ., made an important development by incorporating these azide mutations at specific residues, allowing the receptor protein to be conjugated to nanocarbon in different, defined orientations, which resulted in differential gating effects being observed [174] …”

“…The first, mentioned above, uses bioorthogonal click chemistry to attach a receptor protein to linkers (see section 4.3). The second is a direct link between biomolecule and nanocarbon via UV irradiation; a nitrene radical is formed that can directly insert within the nanocarbon framework [170,171,174,193–195] . This method of conjugation supersedes that of using native biology because the protein can be anchored in a specific orientation, ensuring the analyte‐binding site remains accessible.…”