Design strategies for carbon nanotube-based biosensors
von Florian Alexander Mann
Datum der mündl. Prüfung:2020-06-04
Erschienen:2020-07-09
Betreuer:Dr. Sebastian Kruss
Gutachter:Prof. Dr. Martin Suhm
Gutachter:Prof. Dr. Claudia Steinem
Gutachter:Prof. Dr. Silvio O. Rizzoli
Gutachter:Prof. Dr. Silvio O. Rizzoli
Dateien
Name:200701_FM-PhD-Thesis_final_upload.pdf
Size:60.8Mb
Format:PDF
Description:PhD Thesis
Zusammenfassung
Englisch
Global health crises such as the COVID-19 pandemic clearly show the need for novel and better diagnostic and therapeutic tools. Additionally, understanding underlying molecular processes is crucial. However, current methods face several problems including e.g. the specificity and spatiotemporal resolution of biomarker detection or in vivo targeting of drug delivery vehicles. Single-walled carbon nanotubes (SWCNTs) are all-carbon nanoparticles with the potential to tackle these challenges. They emit near-infrared (NIR) light (> 900 nm), which leads to three distinct advantages. First, NIR light can be used for enhanced in vivo fluorescence imaging with reduced background and deeper tissue penetration. Second, their NIR fluorescence does not bleach or blink enabling continuous monitoring over long time scales (hours to days). Third, the light emission is responsive to its chemical environment, which in combination with the SWCNT’s large surface area promises high-sensitivity optical sensors for e.g. disease biomarkers. However, these beneficial optical properties can only be utilized when the all-carbon surface is chemically modified to generate selectivity either for biomarker detection or for targeting the SWCNTs to the desired place of action (e.g. cellular receptors). This thesis provides new design strategies for SWCNT-based fluorescent biosensors and is organized according to the nature of conjugation (covalent/non-covalent) and the type of the conjugated biomolecule (DNA, peptide, protein). In the first part, the SWCNTs were non-covalently functionalized with different DNA sequences. It has been known that such SWCNT/DNA hybrids show a fluorescence increase in the presence of the important neurotransmitter dopamine. Here, the correlation between sequence and sensitivity/selectivity was quantified leading to dissociation constants (Kd = 2.3 nM - 9.4 μM) and allowing the detection of dopamine in the presence of structurally similar neurotransmitters such as norepinephrine. In the second part, such SWCNT/DNA-based dopamine sensors were modified with small antibody fragments (nanobodies) lending the required specificity to create targeted dopamine sensors. These targeted sensors were not only fully characterized in vitro, but also applied in vivo in embryos of Drosophila melanogaster for deep-tissue NIR immunofluorescence imaging of the spindle apparatus. Furthermore, this new tool allowed for the first time tracking of a single Kinesin motor protein inside a living organism giving rise to deeper understanding of important intracellular processes as e.g. the velocity a motor protein is moving at in vivo (v = 610 ± 330 nm s-1). To expand the structural possibilities for SWCNT modification, peptidic barrels were introduced as a new molecular entity encapsulating SWCNTs with matching diameter. This new strategy, where de novo designed peptide barrels can be chosen to cover the corresponding SWCNT species, allows not only for chirality enrichment, but also for the subsequent attachment of functional units with applications in targeting or fluorescence microscopy. In the last part, two new so-called quantum defects were introduced into SWCNTs generating an anchor site for subsequent covalent functionalization. In contrast to other covalent functionalization approaches, quantum defects create red-shifted emission features corresponding to exciton traps, but do not quench the SWCNT’s NIR fluorescence. By combining this photophysical advantage with anchor groups for protein attachment and peptide growth, it was possible to generate functional, NIR-fluorescent and covalent SWCNT-Nanobody conjugates, multi-color SWCNTs as well as SWCNT-Peptide hybrids. With the superior stability of covalent chemistry, these anchor-quantum-defects can now be used as a platform technology for the generation of NIR-fluorescent tools for biosens- ing or immunofluorescence microscopy. In summary, these four different parts report fundamental insights into SWCNT surface chemistry and its impact on the photophysical properties. Furthermore, it shows the potential of SWCNTs as building blocks for the generation of new SWCNT-based optical sensors, NIR-tools for fluorescence microscopy or vehicles for targeted delivery under continuous NIR optical monitoring ultimately generating new options for detection and/or treatment of diseases.
Keywords: Nanotube; Carbon; Peptide; Protein; Sensor