Dynamics for High-Sensitivity Detection of Free Radicals in Primary Bronchial Epithelial Cells upon Stimulation with Cigarette Smoke Extract.

Chronic obstructive pulmonary disease (COPD), the third leading cause of death worldwide, is caused by chronic exposure to toxic particles and gases, such as cigarette smoke. Free radicals, which are produced during a stress response to toxic particles, play a crucial role in disease progression. Measuring these radicals is difficult since the complex mixture of chemicals within cigarette smoke interferes with radical detection. We used a new quantum sensing technique called relaxometry to measure free radicals with nanoscale resolution on cells from COPD patients and healthy controls exposed to cigarette smoke extract (CSE) or control medium. Epithelial cells from COPD patients display a higher free radical load than those from healthy donors and are more vulnerable to CSE. We show that epithelial cells of COPD patients are more susceptible to the damaging effects of cigarette smoke, leading to increased release of free radicals.

Diamond Quantum Sensing Revealing the Relation between Free Radicals and Huntington's Disease.

Huntington's disease (HD) is a well-studied yet rare disease caused by a specific mutation that results in the expression of polyglutamine (PolyQ). The formation of aggregates of PolyQ leads to disease and increases the level of free radicals. However, it is unclear where free radicals are generated and how they impact cells. To address this, a new method called relaxometry was used to perform nanoscale MRI measurements with a subcellular resolution. The method uses a defect in fluorescent nanodiamond (FND) that changes its optical properties based on its magnetic surroundings, allowing for sensitive detection of free radicals. To investigate if radical generation occurs near PolyQ aggregates, stable tetracycline (tet)-inducible HDQ119-EGFP-expressing human embryonic kidney cells (HEK PQ) were used to induce the PolyQ formation and Huntington aggregation. The study found that NDs are highly colocalized with PolyQ aggregates at autolysosomes, and as the amount of PolyQ aggregation increased, so did the production of free radicals, indicating a relationship between PolyQ aggregation and autolysosome dysfunction.

Lipid peroxidation in diamond supported bilayers.

Lipid peroxidation is a process that occurs in cells when they are exposed to oxidative stress. During the process reactive oxygen species attack lipids within the lipid bilayers of cells. Since the products of lipid peroxidation are toxic and carcinogenic, it is important to understand where and how it occurs with nanoscale resolution. The radical intermediates of this process are particularly interesting since they are causing chain reactions damaging large parts of the lipid membranes in cells. However, they are also difficult to measure for the state of the art because they are short lived and reactive. Here, we study the lipid peroxidation of three artificial lipid bilayers on a diamonds substrate that can be used to study lipid peroxidation. In particular, we present a diamond quantum sensing method called T1-relaxometry that allows for in situ measurements and imaging of radical intermediates of lipid peroxidation in these membranes.

Detecting free radicals post viral infections.

Free radical generation plays a key role in viral infections. While free radicals have an antimicrobial effect on bacteria or fungi, their interplay with viruses is complicated and varies greatly for different types of viruses as well as different radical species. In some cases, radical generation contributes to the defense against the viruses and thus reduces the viral load. In other cases, radical generation induces mutations or damages the host tissue and can increase the viral load. This has led to antioxidants being used to treat viral infections. Here we discuss the roles that radicals play in virus pathology. Furthermore, we critically review methods that facilitate the detection of free radicals in vivo or in vitro in viral infections.

Quantum monitoring of cellular metabolic activities in single mitochondria.

Free radicals play a vital role in all kinds of biological processes including immune responses. However, free radicals have short lifetimes and are highly reactive, making them difficult to measure using current methods. Here, we demonstrate that relaxometry measurement, or T1, inherited from the field of diamond magnetometry can be used to detect free radicals in living cells with subcellular resolution. This quantum sensing technique is based on defects in diamond, which convert a magnetic signal into an optical signal, allowing nanoscale magnetic resonance measurements. We functionalized fluorescent nanodiamonds (FNDs) to target single mitochondria within macrophage cells to detect the metabolic activity. In addition, we performed measurements on single isolated mitochondria. We were able to detect free radicals generated by individual mitochondria in either living cells or isolated mitochondria after stimulation or inhibition.

Nanodiamond uptake in colon cancer cells: the influence of direction and trypsin-EDTA treatment.

Nanoparticles are routinely used in cell biology. They deliver drugs or function as labels or sensors. For many of these applications it is essential that the nanoparticles enter the cells. While some cell types readily ingest all kinds of particles, others just don't. We report that uptake can be enhanced for some cells if the particles are administered from the basolateral side of the cells (in this case from below). Compared to apical uptake (from above), we report an 8-fold increase in the number of fluorescent nanodiamonds internalized by the colon cancer cell line HT29. Up to 96% of the cells treated by a modified protocol contain at least one nanodiamond, whereas in the control group we could observe nanodiamonds in less than half of the cells. We were also able to show that simple treatment of cell clusters with trypsin-EDTA leads to the same enhancement of the nanodiamond uptake as seeding the cells on top of the nanoparticles. Although our study is focused on nanodiamonds in HT29 cells, we believe that this method could also be applicable for other nanoparticles and cells with a specific directionality.

Interaction of nanodiamonds with bacteria.

Nanocarbons come in many forms and among their applications is the engineering of biocompatible and antibacterial materials. Studies have shown that diamond nanoparticles might have the interesting combination of both properties: they are highly biocompatible, while surprisingly reducing bacterial viability or growth at the same time. In this article, we consider for the first time the interaction of milled HPHT nanodiamonds with bacteria. These nanoparticles are capable of hosting nitrogen-vacancy (NV) centers, which provide stable fluorescence with potential use in sensing applications. An initial study was performed to assess the interaction of partially oxidized monocrystalline nanodiamonds with Gram positive S. aureus ATCC 12600 and Gram negative E. coli ATCC 8739. It was shown that for S. aureus ATCC 12600, the presence of these nanodiamonds leads to a sharp reduction of colony forming ability under optimal conditions. A different effect was observed on Gram negative E. coli ATCC 8739, where no significant adverse effects of ND presence was observed. The mode of interaction was further studied by electron microscopy and confocal microscopy. The effects of NDs on S. aureus viability were found to depend on many factors, including the concentration and size of nanoparticles, the suspension medium and incubation time.

Nanodiamonds as multi-purpose labels for microscopy.

Nanodiamonds containing fluorescent nitrogen-vacancy centers are increasingly attracting interest for use as a probe in biological microscopy. This interest stems from (i) strong resistance to photobleaching allowing prolonged fluorescence observation times; (ii) the possibility to excite fluorescence using a focused electron beam (cathodoluminescence; CL) for high-resolution localization; and (iii) the potential use for nanoscale sensing. For all these schemes, the development of versatile molecular labeling using relatively small diamonds is essential. Here, we show the direct targeting of a biological molecule with nanodiamonds as small as 70 nm using a streptavidin conjugation and standard antibody labelling approach. We also show internalization of 40 nm sized nanodiamonds. The fluorescence from the nanodiamonds survives osmium-fixation and plastic embedding making them suited for correlative light and electron microscopy. We show that CL can be observed from epon-embedded nanodiamonds, while surface-exposed nanoparticles also stand out in secondary electron (SE) signal due to the exceptionally high diamond SE yield. Finally, we demonstrate the magnetic read-out using fluorescence from diamonds prior to embedding. Thus, our results firmly establish nanodiamonds containing nitrogen-vacancy centers as unique, versatile probes for combining and correlating different types of microscopy, from fluorescence imaging and magnetometry to ultrastructural investigation using electron microscopy.

Shape and crystallographic orientation of nanodiamonds for quantum sensing.

Nanodiamonds with dimensions down to a few tens of nanometers containing nitrogen-vacancy (NV) color centers have revealed their potential as powerful and versatile quantum sensors with a unique combination of spatial resolution and sensitivity. The NV centers allow transducing physical properties, such as strain, temperature, and electric or magnetic field, to an optical transition that can be detected in the single photon range. For example, this makes it possible to sense a single electron spin or a few nuclear spins by detecting their magnetic resonance. The location and orientation of these defects with respect to the diamond surface play a crucial role in interpreting the data and predicting their sensitivities. Despite its relevance, the geometry of these nanodiamonds has never been thoroughly investigated. Without accurate data, spherical models have been applied to interpret or predict results in the past. With the use of High Resolution Transmission Electron Microscopy (HR-TEM), Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM), we investigated nanodiamonds with an average hydrodynamic diameter of 25 nm (the most common type for quantum sensing) and found a flake-like geometry, with 23.2 nm and 4.5 nm being the average lateral and vertical dimensions. We have also found evidence for a preferred crystallographic orientation of the main facet in the (110) direction. Furthermore, we discuss the consequences of this difference in geometry on diamond-based applications. Shape not only influences the creation efficiency of nitrogen-vacancy centers and their quantum coherence properties (and thus sensing performance), but also the optical properties of the nanodiamonds, their interaction with living cells, and their surface chemistry.

Investigation of surface magnetic noise by shallow spins in diamond.

We present measurements of spin relaxation times (T1, T1ρ, T2) on very shallow (≲5 nm) nitrogen-vacancy centers in high-purity diamond single crystals. We find a reduction of spin relaxation times up to 30 times compared to bulk values, indicating the presence of ubiquitous magnetic impurities associated with the surface. Our measurements yield a density of 0.01-0.1µB/nm2 and a characteristic correlation time of 0.28(3) ns of surface states, with little variation between samples and chemical surface terminations. A low temperature measurement further confirms that fluctuations are thermally activated. The data support the atomistic picture where impurities are associated with the top carbon layers, and not with terminating surface atoms or adsorbate molecules. The low spin density implies that the presence of a single surface impurity is sufficient to cause spin relaxation of a shallow nitrogen-vacancy center.