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.

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.

Toward Using Fluorescent Nanodiamonds To Study Chronological Aging in Saccharomyces cerevisiae.

One of the theories aiming to explain cellular aging is the free radical theory of aging, which describes the possible role of increased production and accumulation of free radicals. Fluorescent nanodiamonds (FNDs) are proposed to provide a tool to detect these radicals, as they function as magnetic sensors that change their optical properties depending on their magnetic surrounding. Therefore, they could enable the study of aging at a molecular level and unravel the exact role of free radicals in this process. In this study, important steps toward this goal are made. FNDs are introduced in chronologically aging yeast cells. Furthermore, the behavior of FNDs in these aging cells is studied to demonstrate the potency of using FNDs in the search for causes of cellular aging.

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.

Generally Applicable Transformation Protocols for Fluorescent Nanodiamond Internalization into Cells.

Fluorescent nanodiamonds (FNDs) are promising nanoprobes, owing to their stable and magnetosensitive fluorescence. Therefore they can probe properties as magnetic resonances, pressure, temperature or strain. The unprecedented sensitivity of diamond defects can detect the faint magnetic resonance of a single electron or even a few nuclear spins. However, these sensitivities are only achieved if the diamond probe is close to the molecules that need to be detected. In order to utilize its full potential for biological applications, the diamond particle has to enter the cell. Some model systems, like HeLa cells, readily ingest particles. However, most cells do not show this behavior. In this article we show for the first time generally applicable methods, which are able to transport fluorescent nanodiamonds into cells with a thick cell wall. Yeast cells, in particular Saccharomyces cerevisiae, are a favored model organism to study intracellular processes including aging on a cellular level. In order to introduce FNDs in these cells, we evaluated electrical transformation and conditions of chemical permeabilization for uptake efficiency and viability. 5% DMSO (dimethyl sulfoxide) in combination with optimized chemical transformation mix leads to high uptake efficiency in combination with low impact on cell biology. We have evaluated all steps in the procedure.