Fluorescent nanodiamonds for nanosensing and single-molecule imaging

Thesis

Unlike ensemble measurements where individual behaviours are averaged and therefore inaccessible, single-molecule microscopy allows researchers to obtain real-time information about a biomolecule's intracellular localisation, interactions or conformational changes. The observation time of individual biomolecules, typically a few seconds to a few minutes, depends on the photostability of the fluorescent molecules used as imaging probes. The limitation of the observation time prevents us to gain precious insight on long-term individual biomolecular behaviours as biological processes like bacterial cell division take at least 20 minutes to occur.

The fluorescent nitrogen-vacancy defect in nanodiamond (ND) can emit light for hours and is an established nanoscale probe for quantum sensing (e.g., magnetometry); as a result, there has been much interest in using NDs in biological imaging. Nevertheless, the implementation of fluorescent NDs (FNDs) in single-molecule imaging has been limited by the lack of single-molecule characterisation methods and the low brightness of 10-nm FNDs.

In this thesis, we first report the development of a photophysical characterisation method for FNDs down to 5-10 nm in size and present the discovery of the 10-nm ND's potential as direct pH nanosensors. We then pursue the establishment of a new characterisation technique for diffusing NDs and describe an innovative routine to estimate the proportion of FNDs among all the NDs. An optimised biofunctionalisation approach is detailed, thus allowing us to perform in vitro single-molecule work with the smallest conjugated FNDs ever manufactured (10-15 nm). Finally, we explore the application of these small functionalised FNDs for quantum sensing and present in vivo experiments using conjugated FNDs in bacteria.

Authors: Sow, M

• DOI: 10.5287/ora-dpajj5mpz
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: microscopy; biophysics; nitrogen vacancy centre; nanoparticles; photophysics; diamond; surface chemistry; nanosensing; aggregation; ph sensing; nanodiamond; fluorescence; single particle tracking; single molecule imaging; colloidal stability; NV centre