Hexagonal boron nitride (hBN) has emerged as a unique platform for room temperature quantum photonics, yet translating its optically active defects into a practical single molecule sensing technology requires two stringent conditions: (i) deterministic and spatially controlled generation of emitters and (ii) engineering nanoscale confinement geometries that reliably bring labelled biomolecules in proximity to the hBN emitters while suppressing background fluorescence. This dissertation develops and connects these two capabilities through complementary routes based on optical and strain nano-engineering in layered hBN.

We initially establish a microsphere-assisted femtosecond-laser approach to enhance light–matter interaction during defect formation and readout. By exploiting the combination of photonic nanojets with whispering gallery mode-assisted signal collection, the method enables deterministic emitter generation with improved spatial confinement and higher collection efficiency compared to microsphere-free processing. Specifically, the approach reduces the emission area by a factor of five and increases fluorescence collection efficiency by approximately tenfold.

A second result is the generation and characterization of hBN wrinkle networks in multilayer hexagonal boron nitride,which form from thermal expansion coefficient mismatch with the substrate during annealing. We demonstrate that wrinkles function as planar nanoscale confinements, and can therefore be used as a feature rather than a limitation. Liquid infiltration and retention are validated by time dependent optical imaging, Raman mapping of the water OH stretch band, and capacitance gradient mapping, consistent with liquid retention exceeding 10 h. This self-assembly process provides a lithography-free route to obtain 1D nanochannels and multi-junctions directly on-chip.

For the purposes of biomolecule confinement and imaging, however, such confinements alone do not guarantee clear optical readouts because wide-field imaging remains limited by fluorescence background from surface adsorbed molecules. This shortcoming motivated a background suppression strategy that we implement via vertical hBN/graphene stacks. By using few-layer hBN as a precise physical spacer between biomolecules and graphene, non-radiative energy transfer can be tuned in a predictable manner. This yields a parameter i.e. spacer thickness, that can be exploited to control the degree of quenching and fluorescence recovery. In this way, graphene suppresses unwanted background fluorescence from molecules adsorbed on hBN wrinkles, while preserving the emission from molecules confined deeper inside the wrinkle volumes. As a result, the imaging contrast is starkly improved.

Overall, this dissertation demonstrates how hBN emitter engineering, strain defined confinement, and interface controlled background suppression can be combined into a framework for high-throughput, fluorescence based biosensing using hBN, forming the first steps towards optical protein fingerprinting at 2D material interfaces. ... Read more

The bottom-up manufacturing of DNA origami structures allows for precise control of DNA-based nanostructures’ shape, size, and functionality, making it a powerful tool in nanotechnology. Single-molecule fluorescence measurements, including Forster resonance energy transfer (FRET), are often used to visualise the nanoscale movements of these structures. However, FRET is prone to crosstalk when labelled structures are within a 10 nm range of each other. DNA origami immobilisation within nanoarrays can minimise the crosstalk, since their spatial spacing would be beyond the effective FRET range.

This research aimed to generate nanoarrays with at least 5x5 binding spots and develop a method for filling up this nanoarray with at least 50% DNA origami structures without altering their dynamics. Using nanosphere lithography (NSL), a technique in which the nanospheres self-assemble into a hexagonal closed-packed (HCP) structured monolayer mask, with 300, 600, and 800 nm nanospheres, two types of nanoarrays were fabricated. (1) Oxygen plasma exposure of the nanosphere of the mask, followed by gold sputtering,
resulted in binding spots with a diameter as small as 191.1 nm for biotin-BSA surface functionalization. (2) Heat treatment of the nanosphere mask, reducing the hole size between nanospheres to a minimum of 60 nm. Annealing resulted in regularly spaced small gold islands with a diameter of ∼173.2 nm for thiol-modified DNA immobilization. Both array types were able to create 5x5 binding spot areas confidently.

Fluorescent experiments were performed to analyse the nanoarray occupancy and Holliday Junctions (HJ) dynamics. Within the reduced sphere size via oxygen plasma treatment array, ∼4.3% of 700 analysed traces were HJs, while only ∼0.7% showed their characteristic switching states. Therefore, the proposed method did not reach the occupancy goal of 50%, but the HJ dynamics were preserved. Two traces within the array were compared with baseline traces and gave t-values of 0.59 and 8.73, indicating no significant change in dynamics. 

The novelty of using gold sputtering to create nanoarrays while placing a dynamic DNA origami structure (HJ) led to functional DNA behaviour, as verified by their dynamics, but more research is required to optimise further the results found. ... Read more

This research focuses on the utility of using DNA origami nanoactuator arrays. The goal of this research is to control the formation of DNA origami nanoactuator arrays, and their immobilization on hBN. By controlling the formation of DNA origami arrays, large site-specific templates can be produced and used as single-molecule sensing platforms for preliminary cancer diagnostics. By inserting sticky end staples at the edges of the origamis, arrays were realized. Moreover, two fluorescent docking probes for ATTO647n and 12 immobilization 'docking staples' were implemented in the origami design. The docking staples consist of 30 Adenine bases and were placed on the largest deflection sites of the origamis. The origami with docking staples showed better results on pristine hBN than origamis without. In both cases, an interference layer between origami and hBN was observed, no origamis were immobilized directly on the surface. By creating two different samples, both with different sticky end staples at the edges, large arrays were created. With this method, 7-8 binding sites were utilized per origami interface. When placing them on pristine hBN, no arrays were visible, only a background mesh. When placing single origami structures with docking staples on defective hBN, they appeared stable without an interference layer. Also, when placing the arrays on hBN, they appeared stable and visible, but the length was reduced. By inserting an excess of the full set of edge staples, 17 binding sites were created per origami interface. When placing these arrays on hBN, larger arrays with better visibility were observed, but resulted in a significant reduction on yield per flake. Subsequently, fluorescent measurements were made by using the red channel, the ATTO647N dye attached to the origami could be imaged. By comparing the ATTO647n results to the arrays on hBN, a difference in intensity was detected. This could indicate multiple fluorescent dyes in one spot, and could thus indicate the fluorescent detection of arrays. ... Read more

Single-molecule detection is essential for investigating molecular interactions and dynamics but is often constrained by weak fluorescence signals and the potential for photodamage under high excitation intensities. Conventional enhancement approaches typically involve dried samples or introduce quenching and compatibility issues, making them unsuitable for in-solution measurements. This work presents a hybrid photonic platform that integrates a photonic crystal (PhC) slab with a hexagonal boron nitride (hBN) layer on top as a biocompatible substrate, offering a promising route for fluorescence enhancement under physiological conditions. Additionally, the atomically flat surface of hBN minimizes fluorophore trapping at edges or defects, enabling more uniform and reproducible single-molecule measurements across large areas.

Electromagnetic field simulations based on Rigorous Coupled-Wave Analysis (RCWA) revealed that the PhC structure can concentrate light at the hBN surface, leading to electric field intensity enhancements of up to 259-fold. To experimentally validate this, hBN flakes were successfully stamped onto the center of the PhC patches and characterized by optical microscopy and atomic force microscopy (AFM). The PhC patterns themselves were analyzed with both AFM and scanning electron microscopy (SEM). Single-stranded DNA (ssDNA) labeled with the fluorophore Atto647N was deposited and imaged by confocal fluorescence microscopy.

The experimental results demonstrated consistent fluorescence enhancement, with average increases of up to 13.15-fold across full flakes and localized enhancements up to 18.22-fold for individual fluorophores, likely positioned in high-field regions. Despite these successes, autofluorescence from the PhC introduced background signal and different flake thicknesses, complicating quantitative analysis.

This work confirms that the PhC–hBN platform can significantly enhance fluorescence in solution, providing a viable path forward for high-sensitivity single-molecule biosensing. It also highlights current challenges, such as autofluorescence and structural sensitivity, that can be subject of future research. ... Read more

Understanding the relationship between the sequence, structure, and function of biomolecules, which can serve as biomarkers for early disease detection, is vital for advancing molecular diagnostics. Consequently, highly sensitive optical readout methods to identify biomolecules, such as proteins, are being developed. Single-molecule Förster resonance energy transfer (smFRET) has recently emerged as a promising technique for accurately identifying low-abundance proteins in samples as small as a single cell. However, the effectiveness of this fluorescence-based method depends on the precise design of donor-acceptor probe pairs to sequence labelled proteins. Recent advances have introduced the use of optically active atomic defects in two-dimensional hexagonal boron nitride (2D h-BN), also known as quantum emitters, as promising donor probes. Yet, achieving control over their spatial and spectral properties remains a significant challenge. This thesis investigates thermally induced wrinkles in exfoliated 2D h-BN crystals as potential nanochannels for the localization of both quantum emitters and biomolecules, the latter typically being in solution. The study reveals that the formation and morphology of these wrinkles are strongly influenced by the mismatch in thermal expansion coefficient between h-BN and its substrate. Additionally, the wrinkles exhibit variations in strain and effectively localize quantum emitters in the visible range. Initial experiments with water and ethanol provided a first indication of solution-based effects on wrinkle and emitter properties. The wrinkles remained stable upon exposure and even swelled, suggesting liquid uptake. Additionally, both liquids enhanced the fluorescence intensity of emitters on the wrinkles without necessarily activating those on the flat regions of the h-BN flakes. These findings highlight new research opportunities for using wrinkled 2D h-BN in optofluidic applications, potentially advancing the integration of quantum emitters in smFRET-based sensing devices. ... Read more

In-vitro 3D organoid cultures constitute an essential component of modern-day biological research, with key application areas in drug and therapy testing, and Organ-on-a-Chip models. The increasing demand for reliable models comes with the challenge of boosting the throughput of production of these cultures. The currently researched 3D culture methods pose the challenge of not being contact-free, of limited versatility and throughput in organoid culturing, and often lack a stimulus to promote cell agglomeration. This work presents acoustofluidics as a stimulus-driven solution to promote the clustering of cells in a levitated, suspended environment. In order to establish this objective experimentally, an SBAW resonator was designed, fabricated, and tested as a PoC (Proof-of-Concept). The experiments were carried out with the fabricated PoC which was characterized to assess the trapping performance and determine the controllable experimental variables to tune the acoustofluidic effects aptly, leading to the formation of stable clusters in SBAW nodal planes. On successful fabrication, the device showed repeatable trapping over a wide bandwidth of 300 kHz, and trap stiffnesses of up to 1.7 fN/μm determined experimentally based on the processing of particle agglomerate imaging. It was observed that the trap stiffness was adequate for levitation over several hours, and yet allowed for the formation of a 3D agglomerate of particles. It was also shown that a frequency-sweep actuation was successful in overcoming fabrication limitations and suppressing streaming, and a suitable working range of experimental parameters could be determined to achieve initial clustering in under 3 minutes. A futuristic outlook on the in-plane particle confinement methods to further improve the target performance, and considerations for biological experiments as the immediate next step have been presented as a concluding note in this thesis. This work thus paves the way for the integration of this technique into laboratory organoid culture formats. This positively complements the objective of cutting down the agglomeration time for initial organoid clustering, for an anticipated positive impact on the production throughput and developmental aspects of organoid cultures, with the in-house fabricated and characterized PoC presented in this work as an enabler. ... Read more

Surface acoustic wave (SAW) is widely used in biological research and sensor applications, acomprehensive understanding of SAW propagation and visualization on suspended 2D membranes is crucial for the manipulation of nanoparticles in biological research and sensitivity improvement of SAW sensors based on 2D material. The initial step of this project involved exciting a standing SAW field on the SAW device and attempting to use Digital Holography Microscopy (DHM) for imaging the standing SAW field on the substrate. This attempt was very challenging and unfortunately was not successful. We used an Atomic Force Microscope (AFM) as an alternative solution for SAW field imaging. Initially, AFM was used to image the SAW field on the SAW device substrate, and the impacts of input frequency and power on the amplitude of the SAW field were investigated. Subsequently, microcavities were fabricated on the SAW device to record images of the SAW field within the microcavities and their surrounding areas, aiming to investigate the impact of microcavities on the SAW field. Finally, the prepared graphene membrane was transferred to the microcavities, the SAW propagation was recorded on the suspended membrane and the influence of the suspended membrane on SAW propagation was analyzed. ... Read more

This master thesis explores strain-induced quantum emitters in hexagonal boron nitride (hBN) as novel optical nanoprobes for Förster resonance energy transfer (FRET)-based biosensors. These types of emitters could outperform conventionally used fluorophores due to their high brightness, stability in harsh environments, biocompatibility and ease of integration with solid state devices. Ultimately, the aim is to combine optically-active hBN emitters with protein fingerprinting devices, which could impact the field of molecular diagnostics by detecting clinically relevant protein biomarkers.

To date, however, it is unclear which parameters are crucial for the generation of hBN quantum emitters with strain in both CVD grown and exfoliated hBN crystals. To address this gap in the field, this thesis systematically investigates the generation of strain by mechanically exfoliating pristine hBN crystals onto a variety of rigid micro/nanostructures with different aspect ratios, including 5 µm and 10 µm microbeads, femtosecond laser-ablated cavities, and CD/Blu-ray micro-nanostructures. We characterised the samples with fluorescence microscopy and atomic force microscopy in order to correlate the optical properties of the hBN with the topography of the substrate. Among the tested structures, samples displayed clear fluorescent emission at the location where the hBN was deposited on the femtosecond laser-ablated cavities with sharp edges. The presence of strain in these regions was verified with Raman spectroscopy, and the spectral properties of the fluorescent regions were determined with photoluminescence spectroscopy. We additionally studied the temporal behavior of the identified emitters and observed effects such as blinking with intensities reduced up to a 38 % and photobleaching with quantum emitters’ lifetimes between 6.57 s and 44.17 s.

While there were no clear threshold values of curvature, substrate structure height, and thickness of hBN that led to reproducible localized fluorescence, these findings open up further research opportunities for the use of strain engineering to generate quantum emitters in hBN.
... Read more

Mechanical forces are integral to the functionality and behavior of biological systems, from the cellular to the molecular level. Acoustic force spectroscopy (AFS), an emerging field, seeks to explore these intricate dynamics. Traditional methods in AFS, particularly those using bulk acoustic wave (BAW) devices, still lack sample visibility and have a low throughput. These limitations hinder the amount and type of data that can be gathered in the study of cellular and molecular mechanics. This research project addresses the challenge of enhancing visibility and throughput in AFS by developing a novel BAW device. Here we show the fabrication and implementation of a glass transversal resonator by femtosecond laser ablation and a simple glass-glass bonding technique using polyethylene film and cyanoacrylate glue, which enables two-dimensional particle trapping. Compared to existing BAW setups in AFS, our approach offers improved sample visibility and is capable of positioning particles at arbitrary locations between half- and full-wave mode equilibria by rapid mode-switching. It shows that the position can be predicted using a straightforward method to determine the stiffness of each mode. This advancement of acoustic force spectroscopy could pave the way for new approaches in biomedical research. For example, the ability to alter the force field in a predictable manner allows to form both force- and distance-clamps. More functional BAW devices could thereby not only enhance our understanding of cellular processes but also find broader application in fields like tissue engineering and medical diagnostics. ... Read more

Nanopores are narrow channels in cell membranes that control the passage of small molecules. In recent years, they have been repurposed for diverse applications, including single molecule sensing and drug delivery. Due to the small diameters of conventional protein-based, current research efforts are directed to designing nanopores from other materials to achieve larger diameters. DNA origami has emerged as a promising method for the precise fabrication of nanoscale structures. Using advances in structurally-adaptable DNA origami nanotechnology, here we investigate DNA-based nanoactuators with size-adjustable diameters, which can potentially reach diameters of up to 100 nm, that could be used for macromolecules translocation. The focus of this thesis is the study of the mechanical states of these nanoactuators, which can be triggered to change shape in response to a specific molecular trigger. Given the nanoscopic dimensions of our actuators, we select DNA PAINT for imaging, which is a type of super resolution technique, that can achieve a resolution of 10 nm, beating the optical diffraction limit (~200 nm) of conventional light microscopy. DNA PAINT experiments are performed in combination with total internal reflection fluorescence (TIRF) microscopy to characterize the behavior of DNA origami nanopores in physiological conditions. By testing various parameters sample related and imaging software ones we identify optimal conditions suggesting 5mM Mg^(2+) ions in buffer solution and 1nM DNA nanopores. Laser power (40mW), exposure time (400ms), waiting time between frames (300ms), and image duration (50s) are optimized, resulting in the expected fluorescent blinking behavior which enables us to perform single-molecule localization. The individual corners of the nanopores were, however, not resolved with this technique due to limitations in the resolution of the imaging system. We recommend that future work could exploit the even better resolution of a modified DNA PAINT approach i.e. Exchange PAINT, which has been proven to achieve Angstrom level resolution for imaging of DNA nanostructures. ... Read more

Proteins play a key role in many biological processes and are thus useful indicators of health and disease states. Since the size of proteins is few hundred nanometers, it is necessary to explore new techniques to manipulate and 'read' them. This thesis aims to identify and build a platform that enables sub-micron bead manipulation, which will be used to actively deliver the biomolecules attached to the beads to the single-molecule sensor. During this process, accurate positioning, speed control, and multiplexing of the beads need to be solved to give high-resolution and high-throughput sequencing capabilities. We identify surface acoustics wave (SAW) devices as powerful tools to achieve both beads transport and massively parallel manipulation. Our hypothesis is that we can increase the efficiency and precision of bead capture and manipulation by using SAW devices combined with a microcavity layer. In this master thesis, we fabricate the SAW device with an average 2 $\mu m$ thick film, and there are cavity arrays on the film. In addition, we investigate the performance of trapping sub-micron objects during the actuation. Further more, we discuss few challenges found through the experiments and provide possible solutions for them. At last, this thesis is concluded by discussing the work been done and providing suggestions for future experiments. ... Read more

The microscope is an essential tool for biologists. Since the late 16th century, it has given researchers a better understanding of cell processes and greatly advanced healthcare. In this century, Single molecule localization microscopy (SMLM) has revolutionized optical microscopy by breaking the optical diffraction limit. Sparsely activating emitters in a sample labeled with fluorophores, the object can be reconstructed by estimating their positions using the system point spread function (PSF). These localization algorithms are the state of the art
in optical imaging, using unbiased estimators to reach the theoretical minimum uncertainty, or Cramér-Rao lower bound (CRLB).
While SMLM works well when emitters are sparsely activated, overlap of the emitter images is inevitable for thick or densely labeled samples. When SMLM is used on such images, the estimates become biased and the algorithm cannot find the correct number of emitters. Most techniques also make a deterministic estimate and are incapable of representing the uncertainty of estimates for dense samples.
A three-dimensional, Bayesian multiple emitter fitting algorithm is constructed using reversible jump Markov chain Monte Carlo (RJMCMC). While following the structure of Bayesian multiple-emitter fitting (BAMF), novel RJMCMC moves are designed to sample the parameters. The algorithm also jumps through models, estimating the number of emitters. It asymptotically samples from the posterior, revealing uncertainties in three-dimensional imaging that other techniques are incapable of imaging.
The algorithm was tested with astigmatic and biplane imaging. It has proven capable of consistently finding the correct model when a prior on emitter intensity is used. When separating two emitters, posterior density reconstruction revealed non-Gaussian emitter position uncertainties. Upon further investigation, the posterior density was found to be multimodal, with
both modes representative of the data and indistinguishable in terms of likelihood. This shows the algorithm can quantify three-dimensional PSF degeneracy and can become a vital tool for researchers to analyze their imaging setup. We also expect it to be especially effective when combined with modulation-enhanced localization microscopy (meLM) techniques. ... Read more

This report will investigate the manipulation of biomolecules on 2D hexagonal boron nitride (hBN) crystal surfaces. The goal of this research is to explore the potential for a device that utilizes the interaction between biomolecules and hBN as a new approach to protein sequencing. Our hypothesis is that molecules adsorbed to hBN surfaces can be unfolded and moved at a controlled velocity through the use of shear horizontal surface acoustic waves. These surface acoustic waves utilize acoustoelectric effects that create forces on polarizable molecules. Until this point, the use of surface acoustic waves for manipulation of molecules has not been explored in the framework of single-molecule sequencing. Along with this, the surface interaction between biomolecules and hBN has been investigated primarily with simulations, while experimental confirmations are still lacking. To address this gap in the field, we conducted experiments to fluorescently image lambda DNA, M13mp18 DNA, and α-synuclein proteins adsorbed to hBN surfaces and analysed their free diffusion behavior. We subsequently designed, fabricated and characterized shear horizontal surface acoustic wave devices compatible with measurements in fluid and with an inverted fluorescence microscopy setup. Here, we studied the behavior of the same molecules adsorbed to an hBN surface when subject to acoustic actuation. It was found that limited diffusion effects were visible for α-synuclein proteins and M13mp18 DNA. However, it was possible to observe fragmented lambda DNA freely diffusing on the hBN surface. When acoustically actuated it was found that α-synuclein proteins and M13mp18 DNA located on the hBN surface could not be manipulated. Lambda DNA molecules that were in contact with the hBN surface could be manipulated through acoustic actuation. These findings open up further research opportunities for the use of shear horizontal waves in manipulation of molecules on 2D material surfaces. ... Read more

The discovery of DNA origami nanotechnology has opened new opportunities in this field due to the versatility of the shapes and sizes that can be generated, the ease of modification and functionalization with molecular resolution, and its biocompatibility. Using Watson-Crick base pairing as the main driver in the self-assembly procedure, DNA origami’s simple assembly process has paved the way for the design of static and dynamic nanostructures. The main methods of characterization are AFM and TEM. While AFM allows the characterization of static and dynamic nanostructures, TEM is only limited to static. However, the TEM’s dynamic imaging ability might be enhanced due to a new technique known as Liquid Cell Electron Microscopy. Graphene, the primary material for the fabrication of Liquid Cells, enables reduced electron scattering, reduced radiation damage and allows enhanced contrast compared to conventional carbon supports. If successful, liquid cell microscopy could enable higher lateral resolution and reduced invasiveness related to AFM measurements while imaging the sample in physiological conditions. However, graphene has been a hostile substrate for DNA origami nanostructures. Due to π−π bonding of graphene, DNA bases react with the delocalized π electrons of graphene and are denatured, causing unwanted deformations in the nanostructures. Additionally, other 2D materials with π −π bonds like MoS2, which was also involved in liquid cell microscopy, have shown similar deformations with DNA origami structures. Various functionalizations have enhanced the biocompatibility of graphene and MoS2 surfaces, reducing the degree of deformation of DNA origami nanostructures. A drawback of all these studies is the lack of similarity between the substrates and the methodologies used for the transfer of 2D materials. This impedes the comparison of the interaction of DNA origami with pristine and functionalized 2D materials. This project aims to qualitatively assess the interaction of DNA origami with pristine and functionalized 2D materials. First, measurements on mica were taken as a baseline for comparison. It was found that the location where the measurement was taken affects the surface’s cleanliness due to the morphology of the cleaved mica. A reduced amount of salt was found present in the centre of the mica compared to the side, which allowed an accurate characterization of DNA origami nanostructures. In addition, rinsing 2-3 times reduced the roughness, increased the adhesion of DNA origami nanostructures and diminished the concentration of salt on the mica substrate. Next, the deposition of DNA origami on graphite substrates has shown a shrinking of DNA origami triangles (≈ 10 nm) due to the melting of dsDNA to ssDNA. A similar deformation was observed in hBN substrates that have a similar shape to graphene but have localized π electrons. Functionalization with poly-l-lysine decreases the degree of deformation of DNA origami nanoarchitectures. However, the values still do not match the ones experienced on mica. Conclusionally, pristine graphite and hBN supports cannot serve as an alternative substrate for imaging DNA origami nanostructures. The optimization of the functionalization protocol might enable the use of graphite as TEM grids. ... Read more