Data processing and applications of fluorescent nanodiamonds for bio-technology

This thesis focusses on different aspects related to sensing with ensembles of NV-centers in nanodiamonds (~70nm). First off, there is a chapter about how to analyze the resulting T1 curve. Here different commonly used models are compared with one another and more advanced data analysis methods are introduced. Afterwards the focus is more shifted towards applications of quantum sensing with the NV-center. We performed studies in chemical and biological environments. We show how the T1 changes during a fenton reaction with copper based on the ionic charge of the copper. Here the main advantage of using the NV-center was that the measurement was reversible. Additionally we show that it is possible to track the degradation of a thin polymer film. Here the main advantage of the FNDs was that they can be embedded within the material without affecting it too much. Lastly we introduce the nanodiamonds to cells. Here we used a commonly used virus model to show it is possible to track the change in free radical production over the course of the viral infection. Additionally we showed that at the site of the infection during the fusion of the virus with the cell membrane there is a burst of free radicals

Optimising data processing for nanodiamond based relaxometry

The nitrogen-vacancy (NV) center in diamond is a powerful and versatile quantum sensor for diverse quantities. In particular, relaxometry (or T1), allows to detect magnetic noise at the nanoscale. While increasing the number of NV centers in a nanodiamond allows to collect more signal, a standardized method to extract information from relaxometry experiments of such NV ensembles is still missing. In this article, we use T1 relaxation curves acquired at different concentrations of gadolinium ions to calibrate and optimize the entire data processing flow, from the acquired raw data to the extracted T1. In particular, we use a bootstrap to derive a signal to noise ratio (SNR) that can be quantitatively compared from one method to another. At first, T1 curves are extracted from photoluminescence pulses. We compare integrating their signal through an optimized window as performed conventionally, to fitting a known function on it. Fitting the decaying T1 curves allows to obtain the relevant T1 value. We compared here the three most commonly used fit models that are, single, bi, and stretched-exponential. We finally investigated the effect of the bootstrap itself on the precision of the result as well as the use of a rolling window to allows time-resolution.Comment: With Advanced Quantum Technologies (26/04/2023) / Supplementary information included / 15 pages + 1SI, 6 figures + 1SI, 58 reference

Insight into a Fenton-like Reaction Using Nanodiamond Based Relaxometry

Copper has several biological functions, but also some toxicity, as it can act as a catalyst for oxidative damage to tissues. This is especially relevant in the presence of H2O2, a by-product of oxygen metabolism. In this study, the reactions of copper with H2O2 have been investigated with spectroscopic techniques. These results were complemented by a new quantum sensing technique (relaxometry), which allows nanoscale magnetic resonance measurements at room temperature, and at nanomolar concentrations. For this purpose, we used fluorescent nanodiamonds (FNDs) containing ensembles of specific defects called nitrogen-vacancy (NV) centers. More specifically, we performed so-called T1 measurements. We use this method to provide real-time measurements of copper during a Fenton-like reaction. Unlike with other chemical fluorescent probes, we can determine both the increase and decrease in copper formed in real time

Melt electrowritten scaffolds containing fluorescent nanodiamonds for improved mechanical properties and degradation monitoring

Biocompatible fluorescent nanodiamonds (FNDs) were introduced into polycaprolactone (PCL) – the golden standard material in melt electrowriting (MEW). MEW is an advanced additive manufacturing technique capable of depositing high-resolution micrometric fibres. Due to the high printing precision, MEW finds growing interest in tissue engineering applications. Here, we introduced fluorescent nanodiamonds (FNDs) into polycaprolactone prior to printing to fabricate scaffolds for biomedical applications with improved mechanical properties. Further FNDs offer the possibility of their real-time degradation tracking. Compared to pure PCL scaffolds, the functionalized ones containing 0.001 wt% of 70 nm-diameter nanodiamonds (PCL-FNDs) showed increased tensile moduli (1.25 fold) and improved cell proliferation during 7-day cell cultures (2.00 fold increase). Furthermore, the addition of FNDs slowed down the hydrolytic degradation process of the scaffolds, accelerated for the purpose of the study by addition of the enzyme lipase to deionized water. Pure PCL scaffolds showed obvious signs of degradation after 3 h, not observed for PCL-FNDs scaffolds during this time. Additionally, due to the nitrogen-vacancy (NV) centers present on the FNDs, we were able to track their amount and location in real-time in printed fibres using confocal microscopy. This research shows the possibility for high-resolution life-tracking of MEW PCL scaffolds’ degradation.</p

Fluorescent Nanodiamonds for Detecting Free-Radical Generation in Real Time during Shear Stress in Human Umbilical Vein Endothelial Cells

Free-radical generation is suspected to play a key role in cardiovascular diseases. Another crucial factor is shear stress. Human umbilical vein endothelial cells (HUVECS), which form the lining of blood vessels, require a physiological shear stress to activate many vasoactive factors. These are needed for maintaining vascular cell functions such as nonthrombogenicity, regulation of blood flow, and vascular tone. Additionally, blood clots form at regions of high shear stress within a blood vessel. Here, we use a new method called diamond magnetometry which allows us to measure the dynamics of free-radical generation in real time under shear stress. This quantum sensing technique allows free-radical detection with nanoscale resolution at the single-cell level. We investigate radical formation in HUVECs in a microfluidic environment under different flow conditions typically found in veins and arteries. Here, we looked into free-radical formation before, during, and after flow. We found that the free-radical production varied depending on the flow conditions. To confirm the magnetometry results and to differentiate between radicals, we performed conventional fluorescent reactive oxygen species (ROS) assays specific for superoxide, nitric oxide, and overall ROS

Fluorescent Nanodiamonds for Tracking Single Polymer Particles in Cells and Tissues

Polymer nanoparticles are widely used in drug delivery and are also a potential concern due to the increased burden of nano- or microplastics in the environment. In order to use polymer nanoparticles safely and understand their mechanism of action, it is useful to know where within cells and tissues they end up. To this end, we labeled polymer nanoparticles with nanodiamond particles. More specifically, we have embedded nanodiamond particles in the polymer particles and characterized the composites. Compared to conventional fluorescent dyes, these labels have the advantage that nanodiamonds do not bleach or blink, thus allowing long-term imaging and tracking of polymer particles. We have demonstrated this principle both in cells and entire liver tissues.</p

Quantum monitoring the metabolism of individual yeast mutant strain cells when aged, stressed or treated with antioxidant

Free radicals play a key role in the ageing process. The strongly debated free radical theory of ageing even states that damage caused by free radicals is the main cause of aging on a cellular level. However, free radicals are small, reactive and short lived and thus challenging to measure. We utilize a new technique called diamond magnetometry for this purpose. We make use of nitrogen vacancy centers in nanodiamonds. Via a quantum effect these defects convert a magnetic resonance signal into an optical signal. While this method is increasingly popular for its unprecedented sensitivity in physics, we use this technique here for the first time to measure free radicals in living cells. Our signals are equivalent to T1 signals in conventional MRI but from nanoscale voxels from single cells with sub-cellular resolution. With this powerful tool we are able to follow free radical generation after chemically inducing stress. In addition, we can observe free radical reduction in presence of an antioxidant. We were able to clearly differentiate between mutant strains with altered metabolism. Finally, the excellent stability of our diamond particles allowed us to follow the ageing process and differentiate between young and old cells. We could confirm the expected increase of free radical load in old wild type and sod1{\Delta} mutants. We further applied this new technique to investigate tor1{\Delta} and pex19{\Delta} cells. For these mutants an increased lifespan has been reported but the exact mechanism is unclear. We find a decreased free radical load in, which might offer an explanation for the increased lifespan in these cells.Comment: Main Text: 21 pages, 4 figures / Supplementary information: 13 pages, 13 figure