Diamond Nanosensors for Age and Stress Related Changes in Cells

Free radicals are produced naturally during cell metabolism. Cells also have a specific mechanism to convert those chemicals to be less reactive and easy to eliminate. However, disturbance of the cell system, which lead to unbalanced level of free radicals inside the cells results in oxidative stress condition and aging process. Developing free radical biosensors is challenging since all existing methods have some issues with sensitivity and specificity. Fluorescent nanodiamonds (FNDs) containing defects called NV centers are promising biosensors. A technique called diamond magnetometry, combines the advantages of fluorescence techniques with magnetic resonance methods. It uses NV centers FNDs that can convert magnetic signals into an optical signal. A specific type of magnetometry measurements that we used in this thesis are called T1 measurements. These are specific for spin noise and thus great candidates for free radicals sensors. This work provides some information about how FNDs can be used as free radicals biosensor

Faktor dalam swamedikasi antibiotika untuk penanganan penyakit periodontal oleh masyarakat di Kecamatan Godean, Sleman, Yogyakarta

Penyakit periodontal merupakan salah satu penyakit ronggal mulut dengan prevalensi yang relatif tinggi di Indonesia yaitu 60%. Salah satu hal yang dilakukan untuk mengatasi penyakit tersebut di masyarakat adalah swamedikasi antibiotika. Swamedikasi didefinisikan sebagai upaya pengobatan menggunakan obat-obatan yang dibeli baik di apotek maupun toko obat tanpa konsultasi dan resep dokter. Tujuan dari penelitian ini adalah mengevaluasi faktor swamedikasi antibiotika pada pengobatan penyakit periodontal oleh masyarakat di Kecamatan Godean, Kabupaten Sleman, Daerah Istimewa Yogyakarta. Penelitian ini merupakan penelitian cross-sectional deskriptif dengan menggunakan purposive sampling. Jumlah responden sebanyak 195 orang yang memiliki pengalaman menderita penyakit periodontal dan melakukan swamedikasi antibiotika. Datadiperoleh melalui kuesioner yang didistribusikan secara online.Hasil menunjukan bahwa perempuan memiliki kecenderungan melakukan swamedikasi lebih tinggi (44,6%) dibandingkan laki-laki. Sementara kelompok usia 17-25 tahun (52,8%) dengan pendidikan terakhir SMA (69,2%) lebih banyak melakukan swamedikasi. Ditinjau dari pekerjaan dan pendapatan, kelompok pelajar (53,8%) dan kelompok dengan pendapatan lebih dari 2 juta per bulan (20,5%) banyak melakukan swamedikasi

Micro Versus Macro:The Effect of Environmental Confinement on Cellular Nanoparticle Uptake

While the microenvironment is known to alter the cellular behavior in terms of metabolism, growth and the degree of endoplasmic reticulum stress, its influence on the nanoparticle uptake is not yet investigated. Specifically, it is not clear if the cells cultured in a microenvironment ingest different amounts of nanoparticles than cells cultured in a macroenvironment (for example a petri dish). To answer this question, here we used J774 murine macrophages and fluorescent nanodiamonds (FND) as a model system to systematically compare the uptake efficiency of cells cultured in a petri dish and in a microfluidic channel. Specifically, equal numbers of cells were cultured in two devices followed by the FND incubation. Then cells were fixed, stained and imaged to quantify the FND uptake. We show that the FND uptake in the cells cultured in petri dishes is significantly higher than the uptake in a microfluidic chip where the alteration in CO(2)environment, the cell culture medium pH and the surface area to volume ratio seem to be the underlying causes leading to this observed difference

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

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

Relaxometry for detecting free radical generation during Bacteria's response to antibiotics

Free radical generation plays a key role in killing bacteria by antibiotics. However, radicals are short-lived and reactive, and thus difficult to detect for the state of the art. Here we use a technique which allows optical nanoscale magnetic resonance imaging (MRI) to detect radical generation on the scale of single bacteria. We demonstrate that the radical generation in Staphylococcus aureus increases in the presence of UV irradiation as well as vancomycin and is dependent on the antibiotic's dose. With a method based on ensembles of nitrogen vacancy (NV) centers in diamond, we were able to follow the radical formation near individual bacteria over the whole duration of the experiment to reveal the dynamics of radical generation. Using this new approach, we observed free radical concentrations within nanoscale voxels around the diamond particles and determined its exact timing depending on the antibiotic dose. Since changes in the response to antibiotics emerge in only a few bacteria of the entire population, such a single-cell approach can prove highly valuable for research into drug resistance

Targeting Nanodiamonds to the Nucleus in Yeast Cells

Nanodiamonds are widely used for drug delivery, labelling or nanoscale sensing. For all these applications it is highly beneficial to have control over the intracellular location of the particles. For the first time, we have achieved targeting the nucleus of yeast cells. In terms of particle uptake, these cells are challenging due to their rigid cell wall. Thus, we used a spheroplasting protocol to remove the cell wall prior to uptake. To achieve nuclear targeting we used nanodiamonds, which were attached to antibodies. When using non-targeted particles, only 20% end up at the nucleus. In comparison, by using diamonds linked to antibodies, 70% of the diamond particles reach the nucleus

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