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.

Site-Specific Protein Conjugation onto Fluorescent Single-Walled Carbon Nanotubes

Ardemis Anoush Boghossian, Shang-Jung Wu, Vitalijs Zubkovs

Semiconducting single-walled carbon nanotubes (SWCNTs) are among the few photostable optical emitters that are ideal for sensing, imaging, drug delivery, and monitoring of protein activity. These applications often require strategies for immobilizing proteins onto the nanotube while preserving the optical properties of the SWCNTs. Site-specific and oriented immobilization strategies, in particular, offer advantages for improving sensor and optical signaling responses. In this study, we demonstrate site-specific protein immobilization of a model of enhanced yellow fluorescent protein with a single engineered cysteine residue, using either single-stranded DNA or a pyrene-containing linker to interact with the SWCNT surface. Protein expression and bioconjugation were characterized using a combination of gel electrophoresis, absorbance, fluorescence, mass spectrometry, and circular dichroism measurements. The results confirm successful protein immobilization onto SWCNTs, which retain their near-infrared fluorescence following conjugation. The successful demonstration of these bioconjugation strategies serves as a basis for more cost-effective, site-specific immobilization strategies that can help preserve protein folding and functionality.

2020

Illuminant estimation and detection using near infrared

Sabine Süsstrunk

Digital camera sensors are sensitive to wavelengths ranging from the ultraviolet (200-400nm) to the near-infrared (700-100nm) bands. This range is, however, reduced because the aim of photographic cameras is to capture and reproduce the visible spectrum (400-700nm) only. Ultraviolet radiation is filtered out by the optical elements of the camera, while a specifically designed "hot-mirror" is placed in front of the sensor to prevent near-infrared contamination of the visible image. We propose that near-infrared data can actually prove remarkably useful in colour constancy, to estimate the incident illumination as well as providing to detect the location of different illuminants in a multiply lit scene. Looking at common illuminants spectral power distribution show that very strong differences exist between the near-infrared and visible bands, e.g., incandescent illumination peaks in the near-infrared while fluorescent sources are mostly confined to the visible band. We show that illuminants can be estimated by simply looking at the ratios of two images: a standard RGB image and a near-infrared only image. As the differences between illuminants are amplified in the near-infrared, this estimation proves to be more reliable than using only the visible band. Furthermore, in most multiple illumination situations, one of the light will be predominantly near-infrared emitting (e.g., flash, incandescent) while the other will be mostly visible emitting (e.g., fluorescent, skylight). Using near-infrared and RGB image ratios allow us to accurately pinpoint the location of diverse illuminant and recover a lighting map.

2009

Mid-infrared surface plasmon resonance sensing applied to refractive index measurements of liquids

Sylvain Herminjard

There has been a very strong development of the sensors based on surface plasmon resonance during the last thirty years, mostly for biological and biomedical applications. If the first experiments in this field were carried out at the beginning of the 20t century, it would have been necessary to wait for the end of the sixties to see the first devices performing accurate measurements of the refractive indices of gas and liquid samples. Indeed, the surface plasmon resonance phenomenon allows to measure refractive index variations with a high sensitivity that is hardly obtained with other optical measurement techniques. The basic principle consists of exciting the free electrons of a metallic layer with light, which induces the formation of a surface wave called surface plasmon. This resonant phenomenon depends on the wavelength of the excitation light and the refractive indices of the metallic layer and the material located close to the metallic layer. A variation of the refractive index of the latter induces a variation of the resonance condition, which allows to realize optical sensors designed to measure determined samples. An appropriate calibration allows to set a relationship between the variation of the concentration of the sample and the measured refractive index value. Among the various parameters that describe the performances of the sensor, one of the most important is the sensitivity. It is possible to demonstrate that the excitation wavelength has an impact on the sensitivity of the system : the latter increases if the measured sample has an absorption peak at a wavelength close to the excitation wavelength. The main goal of this thesis is to explore this sensitivity enhancement technique based on the absorption properties of the probed medium. A proof-of-principle is experimentally demonstrated with an optical setup constituted of laser sources working in the visible range of the electromagnetic spectrum. However, many materials have absorption peaks located in the mid-infrared range due to their fundamental molecular vibrations. Therefore, the realization of a plasmonic sensor working in the mid-infrared could allow to measure these materials with an increased sensitivity. To the best of our knowledge, surface plasmon resonance based sensors have never been developed above the near-infrared. This thesis presents for the first time the design and realization of a plasmonic sensor working in the mid-infrared range able to measure refractive index variations of gas and liquids samples. Even if the optical sources, components and detectors designed for the mid-infrared are clearly different from the ones designed for the visible range, it has been possible to successfully realize two systems working in the mid-infrared range during this project. The first is able to perform gas measurements. As the excitation wavelength depends on the measured sample, it was necessary to select a laser source that had a wavelength located close to the absorption peak of the gas under measurement. For this purpose, we designed a plasmonic sensor working at a 4.4 µm wavelength able to measure carbon dioxide (CO2) absorbing at 4.3 µm. It has been demonstrated that the experimental sensitivity of this system is five times higher than the theoretical sensitivity obtained with a system working in the visible range at a 633 nm wavelength. The second has been conceived to measure liquid samples in order to measure variations of concentration of CO2 dissolved in water. If measurements of refractive index variations (without using the sensitivity enhancement technique) could be performed successfully with variations of concentration of alcohol in water, further work is necessary to get decisive results for CO2 dissolved in water. This project was carried out in collaboration with an industrial partner in order to take into account some industrial constraints during the development of the sensor. Therefore, particular care has been taken in order to design a system that is compact, mechanically robust, without any mechanical moving parts, cheap and easily reproducible. The technological development presented in this thesis finds many applications mainly in the chemical, biological and biomedical fields. Indeed, many proteins show absorption peaks in the mid-infrared. It could be possible to measure their concentration or the modification of their properties – like their composition or structure – with an increased sensitivity. The interest of surface plasmons is that the measurement is not a transmission-based technique, but rather a "reflection-based technique", as the light does not go through the gas or liquid sample. This measurement technique is also adapted for high volume samples that are present in industrial production processes like water or soft drink quality control.

EPFL2010

Mid-infrared surface plasmon resonance sensing applied to refractive index measurements of liquids

Sylvain Herminjard

EPFL2010