Diamond magnetometry for sensing in biological environment

This research explored the use of nanodiamonds as magnetic sensors and their interaction with biological samples. The research covers three areas. First, surface functionalisation of the nanoparticles and its effect when interacting with cells. Second, a new approach, based on nanodiamonds, to collect biomarkers from a sample of human serum. Finally, we used Nitrogen-Vacancy centers implanted in nanodiamonds as a sensor to detect magnetic biomolecules. The results from this project enable further investigations of Diamond Magnetometry applied to measure stress response in cells

Quantum Sensing of Free Radical Generation in Mitochondria of Human Keratinocytes during UVB Exposure

Ultraviolet (UV) radiation is known to cause skin issues, such as dryness, aging, and even cancer. Among UV rays, UVB stands out for its ability to trigger problems within cells, including mitochondrial dysfunction, oxidative stress, and DNA damage. Free radicals are implicated in these cellular responses, but they are challenging to measure due to their short lifetime and limited diffusion range. In our study, we used a quantum sensing technique (T1 relaxometry) involving fluorescent nanodiamonds (FNDs) that change their optical properties in response to magnetic noise. This allowed us to monitor the free radical presence in real time. To measure radicals near mitochondria, we coated FNDs with antibodies, targeting mitochondrial protein voltage-dependent anion channel 2 (anti-VDAC2). Our findings revealed a dynamic rise in radical levels on the mitochondrial membrane as cells were exposed to UVB (3 J/cm2), with a significant increase observed after 17 min.</p

Nanodiamond Relaxometry-Based Detection of Free-Radical Species When Produced in Chemical Reactions in Biologically Relevant Conditions

Diamond magnetometry is a quantum sensing method involving detection of magnetic resonances with nanoscale resolution. For instance, T1 relaxation measurements, inspired by equivalent concepts in magnetic resonance imaging (MRI), provide a signal that is equivalent to T1 in conventional MRI but in a nanoscale environment. We use nanodiamonds (between 40 and 120 nm) containing ensembles of specific defects called nitrogen vacancy (NV) centers. To perform a T1 relaxation measurement, we pump the NV center in the ground state (using a laser at 532 nm) and observe how long the NV center can remain in this state. Here, we use this method to provide real-time measurements of free radicals when they are generated in a chemical reaction. Specifically, we focus on the photolysis of H2O2 as well as the so-called Haber-Weiss reaction. Both of these processes are important reactions in biological environments. Unlike other fluorescent probes, diamonds are able to determine spin noise from different species in real time. We also investigate different diamond probes and their ability to sense gadolinium spin labels. Although this study was performed in a clean environment, we take into account the effects of salts and proteins that are present in a biological environment. We conduct our experiments with nanodiamonds, which are compatible with intracellular measurements. We perform measurements between 0 and 10(8) nM, and we are able to reach detection limits down to the nanomolar range and typically find T1 times of a few 100 mu s. This is an important step toward label-free nano-MRI signal quantification in biological environments.</p

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

Evaluation of the Oxidative Stress Response of Aging Yeast Cells in Response to Internalization of Fluorescent Nanodiamond Biosensors

Fluorescent nanodiamonds (FNDs) are proposed to be used as free radical biosensors, as they function as magnetic sensors, changing their optical properties depending on their magnetic surroundings. Free radicals are produced during natural cell metabolism, but when the natural balance is disturbed, they are also associated with diseases and aging. Sensitive methods to detect free radicals are challenging, due to their high reactivity and transiency, providing the need for new biosensors such as FNDs. Here we have studied in detail the stress response of an aging model system, yeast cells, upon FND internalization to assess whether one can safely use this biosensor in the desired model. This was done by measuring metabolic activity, the activity of genes involved in different steps and the locations of the oxidative stress defense systems and general free radical activity. Only minimal, transient FND-related stress effects were observed, highlighting excellent biocompatibility in the long term. This is a crucial milestone towards the applicability of FNDs as biosensors in free radical research

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

The Response of HeLa Cells to Fluorescent NanoDiamond Uptake

Fluorescent nanodiamonds are promising probes for nanoscale magnetic resonance measurements. Their physical properties predict them to have particularly useful applications in intracellular analysis. Before using them in intracellular experiments however, it should be clear whether diamond particles influence cell biology. While cytotoxicity has already been ruled out in previous studies, we consider the non-fatal influence of fluorescent nanodiamonds on the formation of reactive oxygen species (an important stress indicator and potential target for intracellular sensing) for the first time. We investigated the influence of different sizes, shapes and concentrations of nanodiamonds on the genetic and protein level involved in oxidative stress-related pathways of the HeLa cell, an important model cell line in research. The changes in viability of the cells and the difference in intracellular levels of free radicals, after diamond uptake, are surprisingly small. At lower diamond concentrations, the cellular metabolism cannot be distinguished from that of untreated cells. This research supports the claims of non-toxicity and includes less obvious non-fatal responses. Finally, we give a handhold concerning the diamond concentration and size to use for non-toxic, intracellular measurements in favour of (cancer) research in HeLa cells

The fate of lipid-coated and uncoated fluorescent nanodiamonds during cell division in yeast

Fluorescent nanodiamonds are frequently used as biolabels. They have also recently been established for magnetic resonance and temperature sensing at the nanoscale level. To properly use them in cell biology, we first have to understand their intracellular fate. Here, we investigated, for the first time, what happens to diamond particles during and after cell division in yeast (Saccharomyces cerevisiae) cells. More concretely, our goal was to answer the question of whether nanodiamonds remain in the mother cells or end up in the daughter cells. Yeast cells are widely used as a model organism in aging and biotechnology research, and they are particularly interesting because their asymmetric cell division leads to morphologically different mother and daughter cells. Although yeast cells have a mechanism to prevent potentially harmful substances from entering the daughter cells, we found an increased number of diamond particles in daughter cells. Additionally, we found substantial excretion of particles, which has not been reported for mammalian cells. We also investigated what types of movement diamond particles undergo in the cells. Finally, we also compared bare nanodiamonds with lipid-coated diamonds, and there were no significant differences in respect to either movement or intracellular fate

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