Protein Bioconjugation to Carbon Nanotubes for Near-Infrared Sensing

Single-walled carbon nanotubes (SWCNTs) are a promising material for electrochemical and optical biosensing applications. Their small dimensions, large surface area, indefinite photostability, and remarkable electronic properties make SWCNTs an ideal transducer in optical sensors. SWCNTs fluoresce in the near-infrared (NIR)-II region of the light spectrum, between 1000 and 1350 nm. The absorbance of living tissue and cells reaches a minimum within this spectral range, making SWCNTs an excellent material for in vivo sensor implants. Although inherently non-selective, a selective optical response can be imparted to SWCNTs for a specific analyte through appropriate surface functionalization, commonly achieved using oligonucleotides or proteins. The type of biofunctionalization dictates the specificity, biocompatibility, and overall stability of the composite. Therefore, targeted engineering of the SWCNT surface coating is critical for the development of improved optical sensors.

This PhD thesis is focused on producing, and further researching, protein-SWCNT composites, with a specific aim towards their application for glucose sensing. The proposed sensors consist of SWCNTs, acting as the optical transducer element, and a glucose-selective protein, glucose oxidase (GOx), which was used to activate the nanotube surface and impart selectivity. This study presents the design and fabrication of a reversible, mediatorless sensor that undergoes a selective fluorescence increase in the presence of glucose.

The first sensor comprises of non-specifically adsorbed GOx onto the sidewall of SWCNTs. Incorporating this GOx-SWCNT composite into a custom-built sensor device enabled the selective and reversible detection of glucose. Further, the focus was directed towards designing a method to immobilize the protein onto the SWCNT through a non-covalently bound linker molecule, which would impart greater long-term stability.

In order to achieve this goal, a site-specific protein conjugation method was developed and optimized using enhanced yellow fluorescence protein (EYFP) with synthetically-engineered surface cysteine. The protein was conjugated to a N-(1-pyrenyl)-maleimide (PM) linker and immobilized onto SWCNTs via pi-pi stacking. Protein conjugation and immobilization were confirmed using absorbance and fluorescence spectroscopy, and time-resolved NIR spectroscopy was employed to study the protein-/protein-linker-SWCNT interactions. This work demonstrated that the secondary structure of the protein was preserved to a higher degree by employing the PM-linker compared to relying on non-specific protein adsorption. These findings imply that PM-based immobilization is the most promising method for engineering protein-based SWCNT optical sensors.

Based on these findings, the GOx was conjugated to the PM linker in order to immobilize the protein onto the SWCNTs. That was achieved through bioengineering of GOx variants with accessible surface cysteine. The resultant GOx-PM-SWCNT sensor demonstrated a reversible response towards glucose in liquid. Given the requirement of sensors capable of continuous, long-term monitoring in diabetes management, future devices can undoubtedly benefit from the advantages offered by this technology.