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Sub-millimeter untethered locomoting robots hold promise to radically change multiple areas of human activity such as microfabrication/assembly or health care. To overcome the associated hurdles of such a degree of robot miniaturization, radically new approaches are being adopted, often relying on soft actuating polymeric materials. Here, we present light-driven, crawling microrobots that locomote by a single degree of freedom actuation of their light-responsive tail section. The direction of locomotion is dictated by the robot body design and independent of the spatial modulation of the light stimuli, allowing simultaneous multidirectional motion of multiple robots. Moreover, we present a method for steering such robots by reversibly deforming their front section, using ultraviolet (UV) light as a trigger. The deformation dictates the robot locomotion, performing right- or left-hand turning when the UV is turned on or off respectively. The robots' motion and navigation are not coupled to the position of the light sources, which enables simultaneous locomotion of multiple robots, steering of robots and brings about flexibility with the methods to deliver the light to the place of robot operation.
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Gene silencing using small interfering RNAs (siRNAs) is a selective and promising approach for treatment of numerous diseases. However, broad applications of siRNAs are compromised by their low stability in a biological environment and limited ability to penetrate cells. Nanodiamonds (NDs) coated with cationic polymers can enable cellular delivery of siRNAs. Recently, we developed a new type of ND coating based on a random copolymer consisting of (2-dimethylaminoethyl) methacrylate (DMAEMA) and N-(2-hydroxypropyl) methacrylamide (HPMA) monomers. These hybrid ND-polymer particles (Cop+-FND) provide near-infrared fluorescence, form stable complexes with siRNA in serum, show low toxicity, and effectively deliver siRNA into cells in vitro and in vivo. Here, we present data on the mechanism of cellular uptake and cell trafficking of Cop+-FND : siRNA complexes and their ability to selectively suppress mRNA levels, as well as their cytotoxicity, viability and colloidal stability. We identified clathrin-mediated endocytosis as the predominant entry mechanism for Cop+-FND : siRNA into U-2 OS human bone osteosarcoma cells, with a substantial fraction of Cop+-FND : siRNA following the lysosome pathway. Cop+-FND : siRNA potently inhibited the target GAPDH gene with negligible toxicity and sufficient colloidal stability. Based on our results, we suggest that Cop+-FND : siRNA can serve as a suitable in vivo delivery system for siRNA.
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Etilaminas , Metacrilatos , Nanodiamantes , Polímeros , Humanos , ARN Interferente Pequeño/metabolismo , Línea Celular Tumoral , CationesRESUMEN
Photoluminescent gold nanoclusters are widely seen as a promising candidate for applications in biosensing and bioimaging. Although they have many of the required properties, such as biocompatibility and photostability, the luminescence of near infrared emitting gold nanoclusters is still relatively weak compared to the best available fluorophores. This study contributes to the ongoing debate on the possibilities and limitations of improving the performance of gold nanoclusters by combining them with plasmonic nanostructures. We focus on a detailed description of the emission enhancement and compare it with the excitation enhancement obtained in recent works. We prepared a well-defined series of gold nanoclusters attached to gold nanorods whose plasmonic band is tuned to the emission band of gold nanoclusters. In the resultant single-element hybrid nanostructure, the gold nanorods control the luminescence of gold nanoclusters in terms of its spectral position, polarization and lifetime. We identified a range of parameters which determine the mutual interaction of both particles including the inter-particle distance, plasmon-emission spectral overlap, dimension of gold nanorods and even the specific position of gold nanoclusters attached on their surface. We critically assess the practical and theoretical photoluminescence enhancements achievable using the above strategy. Although the emission enhancement was generally low, the observations and methodology presented in this study can provide a valuable insight into the plasmonic enhancement in general and into the photophysics of gold nanoclusters. We believe that our approach can be largely generalized for other relevant studies on plasmon enhanced luminescence.
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Diamond magnetometry can provide new insights on the production of free radicals inside live cells due to its high sensitivity and spatial resolution. However, the measurements often lack intracellular context for the recorded signal. In this paper, the possible use of single-particle tracking and trajectory analysis of fluorescent nanodiamonds (FNDs) to bridge that gap is explored. It starts with simulating a set of different possible scenarios of a particle's movement, reflecting different modes of motion, degrees of confinement, as well as shapes and sizes of that confinement. Then, the insights from the analysis of the simulated trajectories are applied to describe the movement of FNDs in glycerol solutions. It is shown that the measurements are in good agreement with the previously reported findings and that trajectory analysis yields meaningful results, when FNDs are tracked in a simple environment. Then the much more complex situation of FNDs moving inside a live cell is focused. The behavior of the particles after different incubation times is analyzed, and the possible intracellular localization of FNDs is deducted from their trajectories. Finally, this approach is combined with long-term magnetometry methods to obtain maps of the spin relaxation dynamics (or T1) in live cells, as FNDs move through the cytosol.
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Nanodiamantes , Diamante , Colorantes Fluorescentes , GlicerolRESUMEN
Temperature is one of the most relevant parameters for the regulation of intracellular processes. Measuring localized subcellular temperature gradients is fundamental for a deeper understanding of cell function, such as the genesis of action potentials, and cell metabolism. Notwithstanding several proposed techniques, at the moment detection of temperature fluctuations at the subcellular level still represents an ongoing challenge. Here, for the first time, temperature variations (1 °C) associated with potentiation and inhibition of neuronal firing is detected, by exploiting a nanoscale thermometer based on optically detected magnetic resonance in nanodiamonds. The results demonstrate that nitrogen-vacancy centers in nanodiamonds provide a tool for assessing various levels of neuronal spiking activity, since they are suitable for monitoring different temperature variations, respectively, associated with the spontaneous firing of hippocampal neurons, the disinhibition of GABAergic transmission and the silencing of the network. Conjugated with the high sensitivity of this technique (in perspective sensitive to < 0.1 °C variations), nanodiamonds pave the way to a systematic study of the generation of localized temperature gradients under physiological and pathological conditions. Furthermore, they prompt further studies explaining in detail the physiological mechanism originating this effect.
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Nanodiamantes , Hipocampo , Neuronas , Nitrógeno , TemperaturaRESUMEN
Photoluminescent (PL) gold nanoclusters (AuNCs) show many advantages over conventional semiconductor quantum dots, however, their application potential is limited by their relatively low absorption cross-section and quantum yield. Plasmonic enhancement is a common strategy for improving the performance of weak fluorophores, yet in the case of AuNCs this method is still poorly explored. Here a robust synthetic approach to a compact plasmonic nanostructure enhancing significantly the PL of AuNCs is presented. Two gold nanostructures, AuNCs and plasmonic gold nanorods (AuNRs), are assembled in a compact core-shell nanostructure with tunable geometry and optical properties. The unprecedented degree of control over the structural parameters of the nanostructure allows to study the effects of several parameters, such as excitation wavelength, AuNC-AuNR distance, and relative loading of AuNCs per single AuNR. Consequently, a more general method to measure and evaluate enhancement independently of the absolute particle concentrations is introduced. The highest PL intensity enhancement is obtained when the excitation wavelength matches the strong longitudinal plasmonic band of the AuNRs and when the separation distance between AuNCs and AuNRs decreases to 5 nm. The results presented are relevant for the application of AuNCs in optoelectronic devices and bioimaging.
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Bromination of high-pressure, high-temperature (HPHT) nanodiamond (ND) surfaces has not been explored and can open new avenues for increased chemical reactivity and diamond lattice covalent bond formation. The large bond dissociation energy of the diamond lattice-oxygen bond is a challenge that prevents new bonds from forming, and most researchers simply use oxygen-terminated NDs (alcohols and acids) as reactive species. In this work, we transformed a tertiary-alcohol-rich ND surface to an amine surface with â¼50% surface coverage and was limited by the initial rate of bromination. We observed that alkyl bromide moieties are highly labile on HPHT NDs and are metastable as previously found using density functional theory. The strong leaving group properties of the alkyl bromide intermediate were found to form diamond-nitrogen bonds at room temperature and without catalysts. This robust pathway to activate a chemically inert ND surface broadens the modalities for surface termination, and the unique surface properties of brominated and aminated NDs are impactful to researchers for chemically tuning diamond for quantum sensing or biolabeling applications.
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Free radicals are crucial indicators for stress and appear in all kinds of pathogenic conditions, including cancer, cardiovascular diseases, and infection. However, they are difficult to detect due to their reactivity and low abundance. We use relaxometry for the detection of radicals with subcellular resolution. This method is based on a fluorescent defect in a diamond, which changes its optical properties on the basis of the magnetic surroundings. This technique allows nanoscale MRI with unprecedented sensitivity and spatial resolution. Recently, this technique was used inside living cells from a cell line. Cell lines differ in terms of endocytic capability and radical production from primary cells derived from patients. Here we provide the first measurements of phagocytic radical production by the NADPH oxidase (NOX2) in primary dendritic cells from healthy donors. The radical production of these cells differs greatly between donors. We investigated the cell response to stimulation or inhibition.
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Nanodiamantes , Células Dendríticas , Diamante , Radicales Libres , Humanos , Magnetismo , Nanodiamantes/químicaRESUMEN
Fluorophores functionalized with heavy elements show enhanced intersystem crossing due to increased spin-orbit coupling, which in turn shortens the fluorescence decay lifetime (τPL). This phenomenon is known as the heavy-atom effect (HAE). Here, we report the observation of increased τPL upon functionalisation of near-infrared photoluminescent gold nanoclusters with iodine. The heavy atom-mediated increase in τPL is in striking contrast with the HAE and referred to as inverse HAE. Femtosecond and nanosecond transient absorption spectroscopy revealed overcompensation of a slight decrease in lifetime of the transition associated with the Au core (ps) by a large increase in the long-lived triplet state lifetime associated with the Au shell, which contributed to the observed inverse HAE. This unique observation of inverse HAE in gold nanoclusters provides the means to enhance the triplet excited state lifetime.
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We present a method for creating ordered 2D structures with material anisotropy from self-assembling micro-sized hydrogel particles (microgels). Microgel platelets of polygonal shapes (hexagon, square, and rhombus), obtained by a continuous scalable lithographic process, are suspended in an aqueous environment and sediment on an inclined plane. As a consequence of gravitational pull, they slide over the plane. Each half of the microgel is composed of a different type of hydrogel [poly(N-isopropylacrylamide) (PNIPAM), and poly(ethylene glycol) diacrylate (PEGDA), respectively] which exhibit different frictional coefficients when sheared over a substrate. Hence the microgels self-orientate as they slide, and the side with the lower frictional coefficient positions in the direction of sliding. The self-oriented microgels concentrate at the bottom of the tilted plane. Here they form densely packed structures with translational as well as orientational order.
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Fricción , Microgeles/química , Resinas Acrílicas/química , Ensayo de Materiales , Estructura Molecular , Polietilenglicoles/químicaRESUMEN
Correction for 'Inverse heavy-atom effect in near infrared photoluminescent gold nanoclusters' by Goutam Pramanik et al., Nanoscale, 2021, DOI: 10.1039/d1nr02440j.
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The long-dreamed-of capability of monitoring the molecular machinery in living systems has not been realized yet, mainly due to the technical limitations of current sensing technologies. However, recently emerging quantum sensors are showing great promise for molecular detection and imaging. One of such sensing qubits is the nitrogen-vacancy (NV) center, a photoluminescent impurity in a diamond lattice with unique room-temperature optical and spin properties. This atomic-sized quantum emitter has the ability to quantitatively measure nanoscale electromagnetic fields via optical means at ambient conditions. Moreover, the unlimited photostability of NV centers, combined with the excellent diamond biocompatibility and the possibility of diamond nanoparticles internalization into the living cells, makes NV-based sensors one of the most promising and versatile platforms for various life-science applications. In this review, we will summarize the latest developments of NV-based quantum sensing with a focus on biomedical applications, including measurements of magnetic biomaterials, intracellular temperature, localized physiological species, action potentials, and electronic and nuclear spins. We will also outline the main unresolved challenges and provide future perspectives of many promising aspects of NV-based bio-sensing.
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Diamante , Nanopartículas , Magnetismo , Nitrógeno , TemperaturaRESUMEN
The optical properties of colloidal near-infrared (NIR) emitting gold nanoclusters (AuNCs) are thoroughly investigated at variable temperatures and excitation powers. Both absorption and photoluminescence (PL) excitation spectra reveal optical transitions expected from literature models of thiolated AuNCs - with the exception of the lowest energy transition which has the form of a featureless absorption tail partially overlapping with the PL band. The absorption cross section is determined via the PL saturation and PL modulation techniques to be in the range of 2-3 × 10-14 cm2 for excitation at 405 nm (relatively large value for such small clusters) and decreases â¼20 times toward 633 nm. Slow transient quenching (perfectly reversible) of PL is observed when the excitation power exceeds the saturation threshold, i.e. when the probability of achieving the second absorption in an excited AuNC before its relaxation is significant. A stable PL quenched level is reached within a fraction of a minute or a few minutes after the start of the excitation. Similar time intervals are needed for AuNCs to relax back to the original state in the dark. By comparing thermally-induced and light-induced PL decreases and PL kinetics speed up, we conclude that the transient quenching is due to heating caused by the dissipated excitation power. The light-induced PL amplitude reduction is much stronger (up to â¼80% under 405 nm, 60 W cm-2 excitation) than changes in PL decay time (â¼20%), which is due to PL blinking and PL switching-off in a fraction of the AuNC ensemble. The potential application of these AuNCs in nanothermometry is discussed.
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Diamond nanoparticles (nanodiamonds) can transport active drugs in cultured cells as well as in vivo. However, in the latter case, methods allowing the determination of their bioavailability accurately are still lacking. A nanodiamond can be made fluorescent with a perfectly stable emission and a lifetime ten times longer than that of tissue autofluorescence. Taking advantage of these properties, we present an automated quantification method of fluorescent nanodiamonds (FND) in histological sections of mouse organs and tumors, after systemic injection. We use a home-made time-delayed fluorescence microscope comprising a custom pulsed laser source synchronized on the master clock of a gated intensified array detector. This setup allows ultra-high-resolution images (120 Mpixels in size) of whole mouse organ sections to be obtained, with subcellular resolution and single-particle sensitivity. As a proof-of-principle experiment, we quantified the biodistribution and aggregation state of new cationic FNDs capable of transporting small interfering RNA inhibiting the oncogene responsible for Ewing sarcoma. Image analysis showed a low yield of nanodiamonds in the tumor after intravenous injection. Thus, for the in vivo efficacy assay, we injected the nanomedicine into the tumor. We achieved a 28-fold inhibition of the oncogene. This method can readily be applied to other nanoemitters with ≈100 ns lifetime.
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Nanodiamantes , Neoplasias , Animales , Fluorescencia , Ratones , ARN Interferente Pequeño , Distribución TisularRESUMEN
Biocompatible nanoscale probes for sensitive detection of paramagnetic species and molecules associated with their (bio)chemical transformations would provide a desirable tool for a better understanding of cellular redox processes. Here, we describe an analytical tool based on quantum sensing techniques. We magnetically coupled negatively charged nitrogen-vacancy (NV) centers in nanodiamonds (NDs) with nitroxide radicals present in a bioinert polymer coating of the NDs. We demonstrated that the T1 spin relaxation time of the NV centers is very sensitive to the number of nitroxide radicals, with a resolution down to â¼10 spins per ND (detection of approximately 10-23 mol in a localized volume). The detection is based on T1 shortening upon the radical attachment, and we propose a theoretical model describing this phenomenon. We further show that this colloidally stable, water-soluble system can be used dynamically for spatiotemporal readout of a redox chemical process (oxidation of ascorbic acid) occurring near the ND surface in an aqueous environment under ambient conditions.
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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.
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Núcleo Celular/ultraestructura , Imagen Molecular/métodos , Nanodiamantes , Línea Celular Tumoral , Células Cultivadas , Técnicas Citológicas , Pulpa Dental/citología , Pulpa Dental/diagnóstico por imagen , Colorantes Fluorescentes , Humanos , Espectrometría RamanRESUMEN
Protein corona formation has been regarded as an obstacle to developing diagnostic and therapeutic nanoparticles for in vivo applications. Serum proteins that assemble around nanoparticles can hinder their targeting efficiency. Virus-based nanoparticles should be naturally predisposed to evade such barriers in host organisms. Here, we demonstrate that virus-like particles derived from mouse polyomavirus do not form a rich protein corona. These particles can be efficiently targeted to cells that overproduce transferrin receptors, e.g., cancer cells, by conjugating transferrin to the particle surface. In this study, we provide evidence that the interaction of virus-like particles with their newly assigned target receptor is not obstructed by serum proteins. The particles enter target cells via a clathrin-dependent endocytic pathway that is not naturally used by the virus. Our results support the notion that the natural properties of virus-like particles make them well-suited for development of nanosized theranostic tools resistant to detargeting by protein coronas.
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Nanopartículas/química , Poliomavirus/química , Corona de Proteínas/química , Corona de Proteínas/metabolismo , Receptores de Transferrina/metabolismo , Animales , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Humanos , RatonesRESUMEN
Biohydrogels, composed of naturally occurring biopolymers are typically preferred over their synthetic analogues in bioapplications thanks to their biocompatibility, bioactivity, mechanical or degradation properties. Shaping biohydrogels on the single-cell length scales (micrometers) is a key ability needed to create bioequivalent artificial cell/tissue constructs and cannot be achieved with current methods. This work introduces a method for photolithographic synthesis of arbitrarily shaped microgels composed purely of a biopolymer of choice. The biopolymer is mixed with a sacrificial photocrosslinkable polymer, and the mixture is photocrosslinked in a lithographic process, yielding anisotropic microgels with the biopolymer entrapped in the network. Subsequent ionic or covalent biopolymer crosslinking followed by template cleavage yields a microgel composed purely of a biopolymer with the 3D shape dictated by the photocrosslinking process. Method feasibility is demonstrated with two model polysaccharide biopolymers (alginate, chitosan) using suitable crosslinking methods. Next, alginate microgels were used as microtaggants on a pharmaceutical oral solid dose formulation to prevent its counterfeiting. Since the alginate is approved as an additive in the food and pharmaceutical industries, the presented tagging system can be implemented in practical use much easier than systems comprising synthetic polymers.
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Alginatos/química , Biopolímeros/química , Hidrogeles/química , Microgeles/química , Dextranos/química , Fluorescencia , Iridoides/química , Metacrilatos/químicaRESUMEN
Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been devoted to their development. In this protocol, a recently developed, facile method for preparation of water soluble, biocompatible, and colloidally stable near-infrared emitting AuNCs have been described in detail. This room-temperature, bottom-up chemical synthesis provides easily functionalizable AuNCs capped with thioctic acid and thiol-modified polyethylene glycol in aqueous solution. The synthetic approach requires neither organic solvents or additional ligand exchange nor extensive knowledge of synthetic chemistry to reproduce. The resulting AuNCs offer free surface carboxylic acids, which can be functionalized with various biological molecules bearing a free amine group without adversely affecting the photoluminescent properties of the AuNCs. A quick, reliable procedure for flow cytometric quantification and confocal microscopic imaging of AuNC uptake by HeLa cells also been described. Due to the large Stokes shift, proper setting of filters in flow cytometry and confocal microscopy is necessary for efficient detection of near-infrared photoluminescence of AuNCs.