We propose a label-free biosensor concept based on the charge state manipulation of nitrogen-vacancy (NV) quantum color centers in diamond, combined with an electrochemical microfluidic flow cell sensor, constructed on boron-doped diamond. This device can be set at a defined electrochemical potential, locking onto the particular chemical reaction, whilst the NV center provides the sensing function. The NV charge state occupation is initially prepared by applying a bias voltage on a gate electrode and then subsequently altered by exposure to detected charged molecules. We demonstrate the functionality of the device by performing label-free optical detection of DNA molecules. In this experiment, a monolayer of strongly cationic charged polymer polyethylenimine is used to shift the charge state of near surface NV centers from negatively charged NV- to neutral NV0 or dark positively charged NV+. Immobilization of negatively charged DNA molecules on the surface of the sensor restores the NV centers charge state back to the negatively charged NV-, which is detected using confocal photoluminescence microscopy. Biochemical reactions in the microfluidic channel are characterized by electrochemical impedance spectroscopy. The use of the developed electrochemical device can also be extended to nuclear magnetic resonance spin sensing.
In recent years, fluorescent nanodiamond (fND) particles containing nitrogen-vacancy (NV) centers gained recognition as an attractive probe for nanoscale cellular imaging and quantum sensing. For these applications, precise localization of fNDs inside of a living cell is essential. Here we propose such a method by simultaneous detection of the signal from the NV centers and the spectroscopic Raman signal from the cells to visualize the nucleus of living cells. However, we show that the commonly used Raman cell signal from the fingerprint region is not suitable for organelle imaging in this case. Therefore, we develop a method for nucleus visualization exploiting the region-specific shape of C-H stretching mode and further use k-means cluster analysis to chemically distinguish the vicinity of fNDs. Our technique enables, within a single scan, to detect fNDs, distinguish by chemical localization whether they have been internalized into cell and simultaneously visualize cell nucleus without any labeling or cell-fixation. We show for the first time spectral colocalization of unmodified high-pressure high-temperature fND probes with the cell nucleus. Our methodology can be, in principle, extended to any red- and near-infrared-luminescent cell-probes and is fully compatible with quantum sensing measurements in living cells.
- MeSH
- buněčné jádro ultrastruktura MeSH
- cytologické techniky MeSH
- fluorescenční barviva MeSH
- kultivované buňky MeSH
- lidé MeSH
- molekulární zobrazování metody MeSH
- nádorové buněčné linie MeSH
- nanodiamanty * MeSH
- Ramanova spektroskopie MeSH
- zubní dřeň cytologie diagnostické zobrazování MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
Nestr.
Špatné hojení ran je závažnou komplikací léčby diabetes zhoršující kvalitu života pacienta. Příčinou této poruchy je nadměrná exprese matrixových metaloproteinas (MMP) při reepitelizaci rány. Inhibitory MMP jsou zatím málo účinné a jejich použití vede k vážným vedlejším efektům. Předkládaný projekt spadá do oblasti translační medicíny a je zaměřený na preklinický vývoj systému pro léčbu špatně se hojících ran. Je založen na vysoce účinné a bezpečné genové terapii - RNA interferenci vybraných MMP. Jednotlivé stavební kameny systému již byly uchazeči funkčně vyzkoušeny a zde jsou synergicky využity. Nosičem jsou nanodiamanty – uhlíkový nanomateriál s nejnižší známou toxicitou – s navázaným polyethyleniminem, umožňujícím stabilizaci terapeutické siRNA a její transfekci do buněk. Celý systém bude inkorporován do nanovláken potřebných pro lokalizované uvolnění terapeutického komplexu a udržení stabilní hladiny siRNA po dobu nutnou k zahojení rány. Vyřešení projektu umožní pokračování in vivo zkoušek na primátech a potenciální translaci terapeutické procedury do klinického použití.; Impaired closure of diabetic ulcers is a serious complication decreasing quality of patient's life. The cause is overexpression of metalloproteinases (MMPs) during reepithelialization. Inhibitors of metalloproteinases are low effective so far and their use leads to serious side effects. The proposed translational research is focused on preclinical development of a system for treatment of impaired closure of ulcers. It’s based on effective and safe gene therapy – RNA interference of selected MMP. The carrier, nanodiamonds – the carbon nanomaterial with the lowest known toxicity – bears polyethyleneimine which stabilizes the therapeutic siRNA and facilitates its transfection. The whole system is incorporated in nanofibers which serve for localized release of the therapeutic complex and maintenance of constant siRNA level for the time necessary for the closure of ulcer. The completion of project will enable to follow with preclinical trials in vivo using primates and possible translation of the therapeutic procedure to clinical outputs.
- MeSH
- hojení ran účinky léků MeSH
- komplikace diabetu farmakoterapie MeSH
- malá interferující RNA terapeutické užití MeSH
- matrixové metaloproteinasy MeSH
- nanodiamanty terapeutické užití MeSH
- nosiče léků MeSH
- terapie založená na RNAi MeSH
- translační biomedicínský výzkum MeSH
- uvolňování léčiv MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- farmacie a farmakologie
- genetika, lékařská genetika
- diabetologie
- NLK Publikační typ
- závěrečné zprávy o řešení grantu AZV MZ ČR
Robust devices for chronic neural stimulation demand electrode materials which exhibit high charge injection (Qinj) capacity and long-term stability. Boron-doped diamond (BDD) electrodes have shown promise for neural stimulation applications, but their practical applications remain limited due to the poor charge transfer capability of diamond. In this work, we present an attractive approach to produce BDD electrodes with exceptionally high surface area using porous titanium nitride (TiN) as interlayer template. The TiN deposition parameters were systematically varied to fabricate a range of porous electrodes, which were subsequently coated by a BDD thin-film. The electrodes were investigated by surface analysis methods and electrochemical techniques before and after BDD deposition. Cyclic voltammetry (CV) measurements showed a wide potential window in saline solution (between -1.3 and 1.2 V vs. Ag/AgCl). Electrodes with the highest thickness and porosity exhibited the lowest impedance magnitude and a charge storage capacity (CSC) of 253 mC/cm2, which largely exceeds the values previously reported for porous BDD electrodes. Electrodes with relatively thinner and less porous coatings displayed the highest pulsing capacitances (Cpulse), which would be more favorable for stimulation applications. Although BDD/TiN electrodes displayed a higher impedance magnitude and a lower Cpulse as compared to the bare TiN electrodes, the wider potential window likely allows for higher Qinj without reaching unsafe potentials. The remarkable reduction in the impedance and improvement in the charge transfer capacity, together with the known properties of BDD films, makes this type of coating as an ideal candidate for development of reliable devices for chronic neural interfacing.
- Publikační typ
- časopisecké články MeSH
Nucleation is a core scientific concept that describes the formation of new phases and materials. While classical nucleation theory is applied across wide-ranging fields, nucleation energy landscapes have never been directly measured at the atomic level, and experiments suggest that nucleation rates often greatly exceed the predictions of classical nucleation theory. Multistep nucleation via metastable states could explain unexpectedly rapid nucleation in many contexts, yet experimental energy landscapes supporting such mechanisms are scarce, particularly at nanoscale dimensions. In this work, we measured the nucleation energy landscape of diamond during chemical vapor deposition, using a series of diamondoid molecules as atomically defined protonuclei. We find that 26-carbon atom clusters, which do not contain a single bulk atom, are postcritical nuclei and measure the nucleation barrier to be more than four orders of magnitude smaller than prior bulk estimations. These data support both classical and nonclassical concepts for multistep nucleation and growth during the gas-phase synthesis of diamond and other semiconductors. More broadly, these measurements provide experimental evidence that agrees with recent conceptual proposals of multistep nucleation pathways with metastable molecular precursors in diverse processes, ranging from cloud formation to protein crystallization, and nanoparticle synthesis.
We show that fluorescent nanodiamonds (FNDs) are among the few types of nanosensors that enable direct optical reading of noncovalent molecular events. The unique sensing mechanism is based on switching between the negatively charged and neutral states of NV centers which is induced by the interaction of the FND surface with charged molecules.
Diamond nanoparticles (DNPs) are very attractive for biomedical applications, particularly for bioimaging. The aim of this study was to evaluate the impact of DNPs on neural cancer cells and thus to assess the possible application of DNPs for these cells imaging. For this purpose, the neuroblastoma SH-SY5Y cell line was chosen. Cells were cultured in medium with different concentrations (15, 50, 100 and 150 μg/ml) of DNPs. After 48 h of incubation, cell metabolic activity was evaluated by the XTT assay. For assessment of cellular metabolic activity, cells were also cultured on differently terminated nanocrystalline diamond (NCD) coatings in medium with 150 μg/ml of DNPs. Cell adhesion and morphology were evaluated by brightfield microscopy. Diamond nanoparticle internalization was determined by confocal microscopy. The obtained results showed that low concentrations (15, 50 and 100 μg/ml) of nanoparticles did not significantly affect the SH-SY5Y cell metabolic activity. However, a higher concentration (150 μg/ml) of DNPs statistically significantly reduced SH-SY5Y cell metabolic activity. After 48 h incubation with 150 μg/ml DNPs, cell metabolic activity was 23% lower than in medium without DNPs on standard tissue culture polystyrene.
- MeSH
- biokompatibilní potahované materiály chemická syntéza farmakologie MeSH
- buněčná adheze účinky léků MeSH
- buněčné kultury MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nanodiamanty chemie MeSH
- neuroblastom patologie MeSH
- proliferace buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
