Carbon Nanotube Biohybrids: From Protein Site-Specific Coupling to the Fabrication of Reconfigurable (multi)Sensing Systems and Devices
Authors: Palma Matteo 
Published: February 2020

We controlled the assembly of different biomolecules, from proteins to DNA and aptamer sequences, on carbon nanotubes (CNTs), so as to couple CNTs electronic output to biomolecular function. First we will present a facile strategy for the site-specific coupling of proteins to single walled carbon nanotubes (SWCNTs): i) in solution and with single-molecule control, with a focus on light harvesting and charge transfer investigations, as well as ii) in device configurations to investigate biomolecular (protein-protein) recognition in the context of antimicrobial resistance enzyme detection.
Using an orthogonal Click reaction, Green Fluorescent Protein (GFP) was engineered to contain a genetically encoded azide group and then bound selectively to SWCNT ends in 1:1 nanohybrids with different protein orientations: in close proximity or at longer distances from the GFP’s functional center. Atomic force microscopy and fluorescence analysis in solution and on surfaces at the single-protein level confirmed the importance of bioengineering optimal protein attachment sites to achieve direct protein−nanotube communication and bridging.[1]
We will then discuss the extension of this rationale into bioelectornic devices for the development of real-time biosensors with engineered protein interfacing. We assembled different variants of a b-lactamase (BL) inhibitory protein (BLIP) onto SWCNT sidewalls in electronic device configurations. We recorded the current responses in real-time for the detection of different concentrations of a class BL enzymes,[2] that degrade antibiotics, in the context of investigating antimicrobial resistance. (Resistance against the most useful antibiotics, the b-lactams, including the penicillins, is arguably the most pressing issue due to the prevalence of many different BL enzymes that degrade the antibiotic; BLIP acts as the ideal detection element in terms of its specificity to BLs and ability to bind a wide variety of different BLs).
Employing a similar strategy, we will report on the fabrication of reconfigurable and solution processable nanoscale biosensing devices with multisensing capability based on SWCNTs functionalized with specific, and different, aptamer sequences employed as selective recognition elements. Distinct, hence water-soluble, SWCNT-aptamer hybrids were immobilized from solution onto different pre-patterned electrodes on the same chip, via a low-cost dielectrophoresis methodology. Multiplexed detection of three different biomarkers indicative of stress and neuro-trauma conditions was successfully performed, and real-time detection was achieved in serum down to physiologically relevant concentrations.[3]
Finally, we present a strategy for the controlled assembly of stimuli-responsive end-to-end SWCNT junctions in aqueous solution, employing DNA as a molecular linker. The assembly/disassembly of the nanotubes was controlled via the intrinsic responsiveness to different stimuli of sequence-specific DNA linkers forming the junctions.[4]
By and large, we demonstrate novel approaches for the designed assembly of solution-processable bioelectronic systems where single biomolecules modulate the properties of carbon nanotubes.This can yield new insights into biomolecular events down to single-molecule resolution and facilitate biomolecules’ use as integrated nanocomponents in molecular electronics and biosensing devices.
[1] Journal of the American Chemical Society, 2017, 139, 17834-17840
[2] Submitted

[3] Nano Letters, 2018, 18, 4130-4135
[4] Under 2nd review in Chemistry of Materials