Tailored near-infrared fluorescent carbon nanotube sensors for pathogen detection
by Robert Nißler
Date of Examination:2021-10-06
Date of issue:2021-10-21
Advisor:Prof. Dr. Sebastian Kruss
Referee:Prof. Dr. Sebastian Kruss
Referee:Prof. Dr. Uwe Groß
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Abstract
English
During the global COVID-19 pandemic, the needs and benefits of fast and specific analytical tools became apparent to everyone. In particular, advances in nanotechnology promise novel healthcare diagnostics, like the identification of bacterial pathogens. However, up to now, optical nanosensors for pathogen detection rarely exist, but could pave the way for fast, label-free in situ detection of infections in the future. One class of nanomaterials with extraordinary photophysical properties are semiconducting single-walled carbon nanotubes (SWCNTs) that can serve as building blocks for such optical biosensors. These tubular carbon allotropes exhibit a diameter-dependent band gap structure, described as ‘chirality’. This leads to fluorescence emission (900-1700 nm) in the near-infrared (NIR), a spectral region most suitable for biosensing applications. To obtain functional and colloidally stable probes, the SWCNT’s hydrophobic surface needs to be non-covalently modified with biomolecules. Such SWCNT-conjugates are able to translate changes in their local chemical environment into fluorescence signals, the basic principle of optical analyte detection by SWCNT-sensors. In this thesis, consecutive steps were undertaken to tailor the functional surface chemistry and optical properties of SWCNTs for detection of pathogens and pathogen-related interactions: 1) Initially, for a better understanding of the SWCNT’s interface, a protocol to quantify adsorbed single-stranded (ss)DNA polymers was established. The calculated amount revealed several hundred DNA molecules on a single SWCNT, depending on oligonucleotide lengths and composition. This displayed the basis for further ratio-specific, bioorthogonal modifications of the nanoconjugates. 2) In addition to tailoring the surface chemistry, SWCNTs with defined emission properties for hyperspectral biosensing were isolated. One strategy made use of chirality specific SWCNT dispersions through polyfluorene polymers and further exchanged the organic interface to those needed for sensing in biological systems. The second approach generated purified samples by aqueous two-phase extraction (ATPE), and demonstrated after subsequent surface exchange a general concept for chemical sensing with chirality-pure SWCNTs. 3) Combination of these approaches facilitated the assembly of nanosensors able to detect bacterial virulence factors like lipopolysaccharides (LPS) and siderophores or secreted enzymes like proteases or nucleases. Integrated into a functional hydrogel-system and combined in an array-structure, multiple of these sensors could be read out simultaneously by a camera-assisted setup. This enabled the remote detection and discrimination of typical infection-associated bacteria (e.g. Escherichia coli, Staphylococcus aureus or Pseudomonas aeruginosa), based on their chemical fingerprint. 4) Specific SWCNT sensors were developed to detect and visualize plant-pathogen interactions. In a rational approach, SWCNTs were designed to sense in vivo (Arabidopsis thaliana) reactive oxygen species (ROS, H2O2), important signaling molecules involved in plant stress response. Lastly, nanosensors for polyphenol detection were identified and used to visualize the spatiotemporal polyphenol secretion from plant roots (Glycine max), a chemical defense response after pathogen stimulus. The presented thesis introduced novel concepts to tailor the functional interface of SWCNTs for molecular recognition of important biomolecules and extends the spectral range to multiplexed approaches. These NIR-fluorescent sensors enabled detection of pathogens and pathogen interactions in vitro and in vivo, paving the way for improved healthcare- and agriculture-diagnostics.
Keywords: carbon nanotubes; Biosensors; imaging; near infrared fluorescence; surface chemistry; pathogen detection