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Optically active spin defects in solids offer promising platforms to investigate nuclear spin clusters with high sensitivity and atomic-site resolution. To leverage near-surface defects for molecular structure analysis in chemical and biological contexts using nuclear magnetic resonance (NMR), further advances in spectroscopic characterization of nuclear environments are essential. Here, we report Fourier spectroscopy techniques to improve localization and mapping of the test bed ^{13}C nuclear spin environment of individual, shallow nitrogen-vacancy centers at room temperature. We use multidimensional spectroscopy, well-known from classical NMR, in combination with weak measurements of single-nuclear-spin precession. We demonstrate two examples of multidimensional NMR: (i) improved nuclear spin localization by separate encoding of the two hyperfine components along spectral dimensions and (ii) spectral editing of nuclear-spin pairs, including measurement of internuclear coupling constants. Our work adds important tools for the spectroscopic analysis of molecular structures by single-spin probes.
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Fragment-based drug design is a well-established strategy for rational drug design, with nuclear magnetic resonance (NMR) on high-field spectrometers as the method of reference for screening and hit validation. However, high-field NMR spectrometers are not only expensive, but require specialized maintenance, dedicated space, and depend on liquid helium cooling which became critical over the recurring global helium shortages. We propose an alternative to high-field NMR screening by applying the recently developed approach of fragment screening by photoinduced hyperpolarized NMR on a cryogen-free 80â MHz benchtop NMR spectrometer yielding signal enhancements of up to three orders in magnitude. It is demonstrated that it is possible to discover new hits and kick-off drug design using a benchtop NMR spectrometer at low micromolar concentrations of both protein and ligand. The approach presented performs at higher speed than state-of-the-art high-field NMR approaches while exhibiting a limit of detection in the nanomolar range. Photoinduced hyperpolarization is known to be inexpensive and simple to be implemented, which aligns greatly with the philosophy of benchtop NMR spectrometers. These findings open the way for the use of benchtop NMR in near-physiological conditions for drug design and further life science applications.
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Nanodiamonds containing fluorescent Nitrogen-Vacancy (NV) centers are the smallest single particles, of which a magnetic resonance spectrum can be recorded at room temperature using optically-detected magnetic resonance (ODMR). By recording spectral shift or changes in relaxation rates, various physical and chemical quantities can be measured such as the magnetic field, orientation, temperature, radical concentration, pH or even NMR. This turns NV-nanodiamonds into nanoscale quantum sensors, which can be read out by a sensitive fluorescence microscope equipped with an additional magnetic resonance upgrade. In this review, we introduce the field of ODMR spectroscopy of NV-nanodiamonds and how it can be used to sense different quantities. Thereby we highlight both, the pioneering contributions and the latest results (covered until 2021) with a focus on biological applications.
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Nanodiamantes , Nanodiamantes/química , Nitrógeno/química , Espectroscopía de Resonancia Magnética/métodos , Microscopía Fluorescente , Imagen por Resonancia MagnéticaRESUMEN
5 nanometer sized detonation nanodiamonds (DNDs) are studied as potential single-particle labels for distance measurements in biomolecules. Nitrogen-vacancy (NV) defects in the crystal lattice can be addressed through their fluorescence and optically-detected magnetic resonance (ODMR) of a single particle can be recorded. To achieve single-particle distance measurements, we propose two complementary approaches based on spin-spin coupling or optical super-resolution imaging. As a first approach, we try to measure the mutual magnetic dipole-dipole coupling between two NV centers in close DNDs using a pulse ODMR sequence (DEER). The electron spin coherence time, a key parameter to reach long distance DEER measurements, was prolonged using dynamical decoupling reaching T 2,DD ≈ 20 µs, extending the Hahn echo decay time T 2 by one order of magnitude. Nevertheless, an inter-particle NV-NV dipole coupling could not be measured. As a second approach, we successfully localize the NV centers in DNDs using STORM super-resolution imaging, achieving a localization precision of down to 15 nm, enabling optical nanometer-scale single-particle distance measurements.
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We demonstrate room-temperature 13C hyperpolarization by dynamic nuclear polarization (DNP) using optically polarized triplet electron spins in two polycrystalline systems: pentacene-doped [carboxyl-13C] benzoic acid and microdiamonds containing nitrogen-vacancy (NV-) centers. For both samples, the integrated solid effect (ISE) is used to polarize the 13C spin system in magnetic fields of 350-400â¯mT. In the benzoic acid sample, the 13C spin polarization is enhanced by up to 0.12â¯% through direct electron-to-13C polarization transfer without performing dynamic 1H polarization followed by 1H-13C cross-polarization. In addition, the ISE has been successfully applied to polarize naturally abundant 13C spins in a microdiamond sample to 0.01â¯%. To characterize the buildup of the 13C polarization, we discuss the efficiencies of direct polarization transfer between the electron and 13C spins as well as that of 13C-13C spin diffusion, examining various parameters which are beneficial or detrimental for successful bulk dynamic 13C polarization.
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Nanodiamonds containing negatively charged triplet (having an electron spin S = 1) nitrogen-vacancy (NV-) centers are an extraordinary room-temperature quantum system, whose electron spins may be polarized and read out optically even in a single nanocrystal. In this Viewpoint we promote a simple but reliable method to identify, attribute, and quantify these triplet defects in a polycrystalline sample using electron paramagnetic resonance (EPR) spectroscopy. The characterization relies on a specific "forbidden" transition ("ΔMS = 2"), which appears at about half the central magnetic field and shows a remarkably small anisotropy. In particular, we emphasize that this method is by far not limited to NV- centers in diamond but could become an important characterization tool for novel triplet defects in various types of nanoparticles.
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BACKGROUND: Nanodiamonds (NDs) provide a unique multitasking system for drug delivery and fluorescent imaging in biological environments. Owing to their quantum properties, NDs are expected to be employed as multifunctional probes in the future for the accurate visualization of biophysical parameters such as temperature and magnetic fields. However, the use of NDs for the selective targeting of the biomolecules of interest within a complicated biological system remains a challenge. One of the most promising solutions is the appropriate surface design of NDs based on organic chemistry and biochemistry. The engineered NDs have high biocompatibility and dispersibility in a biological environment and hence undergo cellular uptake through specific pathways. SCOPE OF REVIEW: This review focuses on the selective targeting of NDs for biomedical and biophysical applications from the viewpoint of ND surface functionalizations and modifications. These pretreatments make possible the specific targeting of biomolecules of interest on or in a cell by NDs via a designed biochemical route. MAJOR CONCLUSIONS: The surface of NDs is covalently or noncovalently modified with silica, polymers, or biomolecules to reshape them, control their size, and enhance the colloidal stability and biomolecular selectivity toward the biomolecules of interest. Electroporation, chemical treatment, injection, or endocytosis are the methods generally adopted to introduce NDs into living cells. The pathway, efficiency, and the cell viability depend on the selected method. GENERAL SIGNIFICANCE: In the biomedical field, the surface modification facilitates specific delivery of a drug, leading to a higher therapeutic efficacy. In biophysical applications, the surface modification paves the way for the accurate measurement of physical parameters to gain a better understanding of various cell functions.
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Portadores de Fármacos , Nanodiamantes/química , Nanotecnología/métodos , Animales , Materiales Biocompatibles/química , Membrana Celular/química , Supervivencia Celular , Endocitosis , Humanos , Lípidos/química , Nanopartículas/química , Polímeros/química , Dióxido de Silicio/química , Electricidad Estática , Propiedades de SuperficieRESUMEN
Nanoscale measurements provide insight into the nano world. For instance, nanometric spatiotemporal distribution of intracellular pH is regulated by and regulates a variety of biological processes. However, there is no general method to fabricate nanoscale pH sensors. Here, we, to endow pH-sensing functions, tailor the surface properties of a fluorescent nanodiamond (FND) containing nitrogen-vacancy centers (NV centers) by coating the FND with an ionic chemical layer. The longitudinal relaxation time T1 of the electron spins in the NV centers inside a nanodiamond modified by carboxyl groups on the particle surface was found to depend on ambient pH between pH 3 and pH 7, but not between pH 7 and pH 11. Therefore, a single particle of the carboxylated nanodiamond works as a nanometer-sized pH meter within a microscopic image and directly measures the nanometric local pH environment. Moreover, the pH dependence of an FND was changed by coating it with a polycysteine layer, which contains a multitude of thiol groups with higher pKa. The polycysteine-coated nanodiamond obtained a pH dependence between pH 7 and pH 11. The pH dependence of the FND was also observed in heavy water (D2O) buffers. This indicates that the pH dependence is not caused by magnetic noise induced by 1H nuclear spin fluctuations, but by electric noise induced by ion exchanges. Via our method, the sensitive pH range of the nanodiamond pH sensor can potentially be controlled by changing the ionic layer appropriately according to the target biological phenomena.
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Técnicas Biosensibles/métodos , Nanopartículas/química , Concentración de Iones de Hidrógeno , Nanodiamantes/química , Péptidos/químicaRESUMEN
Nanodiamonds containing negatively charged nitrogen-vacancy (NV-) centers are versatile nanosensors thanks to their optical and spin properties. While currently most fluorescent nanodiamonds in use have at least a size of a few tens of nanometers, the challenge lies in engineering the smallest nanodiamonds containing a single NV- defect. Such a tiny nanocrystal with a single NV- center is an "optical spin label" for biomolecules, which can be detected in a fluorescence microscope. In this paper, we address two key issues toward this goal using detonation nanodiamonds (DNDs) of 4-5 nm in size. The DND samples are treated first with electron irradiation to create more vacancies. With the aid of electron paramagnetic resonance (EPR) spectroscopy, we confirm a steady increase of negatively charged NV- centers with higher fluence. This leads to a 4 times higher concentration in NV- defects after irradiation with 2 MeV electrons at a fluence of 5 × 1018 e-/cm2. Interestingly, we observe that the annealing of DND does not increase the number of NV- centers, which is in contrast to bulk diamond and larger nanodiamonds. Since DNDs are strongly aggregated after the irradiation process, we apply a boiling acid treatment as a second step to fabricate monodisperse DNDs enriched in NV- centers. These are two important steps toward "optical spin labels" having a single-digit nanometer range size that could be used for bioimaging and nanosensing.
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The development of sensors to estimate physical properties, and their temporal and spatial variation, has been a central driving force in scientific breakthroughs. In recent years, nanosensors based on quantum measurements, such as nitrogen-vacancy centres (NVCs) in nanodiamonds, have been attracting much attention as ultrastable, sensitive, accurate and versatile physical sensors for quantitative cellular measurements. However, the nanodiamonds currently available for use as sensors have diameters of several tens of nanometres, much larger than the usual size of a protein. Therefore, their actual applications remain limited. Here we show that NVCs in an aggregation of 5-nm-sized detonation-synthesized nanodiamond treated by Krüger's surface reduction (termed DND-OH) retains the same characteristics as observed in larger diamonds. We show that the negative charge at the NVC are stabilized, have a relatively long T2 spin relaxation time of up to 4 µs, and are applicable to thermosensing, one-degree orientation determination and nanometric super-resolution imaging. Our results clearly demonstrate the significant potential of DND-OH as a physical sensor. Thus, DND-OH will raise new possibilities for spatiotemporal monitoring of live cells and dynamic biomolecules in individual cells at single-molecule resolution.
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Fourier transform (FT) electron paramagnetic resonance (EPR) correlation spectroscopy usually requires broader excitation bandwidth than can be achieved by monochromatic rectangular pulses. Replacement of such pulses by frequency-swept pulses affords the correlation spectra, which, however, may not look the same as those that would be obtained with sufficiently broad-banded monochromatic rectangular pulses. This was recently observed for correlating nuclear frequencies to FT-EPR spectra by a three-pulse electron spin echo envelope modulation experiment. Here we analyze the origin of the additional cross peaks, whose position depends on the direction of the frequency sweep. We find that such peaks arise if coherence or polarization is transferred to an electron spin transition already before this transition is actually passed during the frequency sweep. This happens by excitation of a chain of transitions that connect levels of the source transition, where coherence resides before mixing, and the target transition, where it resides after mixing. The correlation spectra can be simplified by combining data from frequency up and down sweeps.
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Water accessibility is a key parameter for the understanding of the structure of biomolecules, especially membrane proteins. Several experimental techniques based on the combination of electron paramagnetic resonance (EPR) spectroscopy with site-directed spin labeling are currently available. Among those, we compare relaxation time measurements and electron spin echo envelope modulation (ESEEM) experiments using pulse EPR with Overhauser dynamic nuclear polarization (DNP) at X-band frequency and a magnetic field of 0.33 T. Overhauser DNP transfers the electron spin polarization to nuclear spins via cross-relaxation. The change in the intensity of the (1)H NMR spectrum of H2O at a Larmor frequency of 14 MHz under a continuous-wave microwave irradiation of the nitroxide spin label contains information on the water accessibility of the labeled site. As a model system for a membrane protein, we use the hydrophobic α-helical peptide WALP23 in unilamellar liposomes of DOPC. Water accessibility measurements with all techniques are conducted for eight peptides with different spin label positions and low radical concentrations (10-20 µM). Consistently in all experiments, the water accessibility appears to be very low, even for labels positioned near the end of the helix. The best profile is obtained by Overhauser DNP, which is the only technique that succeeds in discriminating neighboring positions in WALP23. Since the concentration of the spin-labeled peptides varied, we normalized the DNP parameter ϵ, being the relative change of the NMR intensity, by the electron spin concentration, which was determined from a continuous-wave EPR spectrum.
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Membrana Dobles de Lípidos/química , Péptidos/química , Agua/química , Espectroscopía de Resonancia por Spin del Electrón/métodos , Espectroscopía de Resonancia Magnética/métodos , Microondas , Modelos Químicos , Fosfatidilcolinas/química , Pirroles/química , Marcadores de Spin , Compuestos de Sulfhidrilo/químicaRESUMEN
Methanol synthesis by CO2 hydrogenation is attractive in view of avoiding the environmental implications associated with the production of the traditional syngas feedstock and mitigating global warming. However, there still is a lack of efficient catalysts for such alternative processes. Herein, we unveil the high activity, 100 % selectivity, and remarkable stability for 1000â h on stream of In2 O3 supported on ZrO2 under industrially relevant conditions. This strongly contrasts to the benchmark Cu-ZnO-Al2 O3 catalyst, which is unselective and experiences rapid deactivation. In-depth characterization of the In2 O3 -based materials points towards a mechanism rooted in the creation and annihilation of oxygen vacancies as active sites, whose amount can be modulated inâ situ by co-feeding CO and boosted through electronic interactions with the zirconia carrier. These results constitute a promising basis for the design of a prospective technology for sustainable methanol production.
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The main limitation of pulse electron paramagnetic resonance (EPR) spectroscopy is its narrow excitation bandwidth. Ultra-wideband (UWB) excitation with frequency-swept chirp pulses over several hundreds of megahertz overcomes this drawback. This allows to excite electron spin echo envelope modulation (ESEEM) from paramagnetic copper centers in crystals, whereas up to now, only ESEEM of ligand nuclei like protons or nitrogens at lower frequencies could be detected. ESEEM spectra are recorded as two-dimensional correlation experiments, since the full digitization of the electron spin echo provides an additional Fourier transform EPR dimension. Thus, UWB hyperfine-sublevel correlation experiments generate a novel three-dimensional EPR-correlated nuclear modulation spectrum.
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Homogeneous line-widths that arise from transverse relaxation tend to be masked by B0 field inhomogeneity and by multiplets due to homonuclear J-couplings. Besides well-known spin-locking sequences that lead to signals that decay with a rate R1ρ without any modulations, alternative experiments allow one to determine the transverse relaxation rates R2 in systems with scalar-coupled spins. We evaluate three recent strategies by experiment and simulation: (i) moderate-amplitude SITCOM-CPMG sequences (Dittmer and Bodenhausen, 2006), (ii) multiple-quantum filtered (MQF) sequences (Barrère et al., 2011) and (iii) PROJECT sequences (Aguilar et al., 2012). Experiments where the J-evolution is suppressed by spin-locking measure the pure relaxation rate R2(Ix) of an in-phase component. Experiments based on J-refocusing yield a mixture of in-phase rates R2(Ix) and antiphase rates R2(2IySz), where the latter are usually faster than the former. Moderate-amplitude SITCOM-CPMG and PROJECT methods can be applied to systems with many coupled spins, but applications of MQF sequences are limited to two-spin systems since modulations in larger systems can only partly be suppressed.
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In the long bygone days of continuous-wave nuclear magnetic resonance (NMR) spectroscopy, a selected transition within a multiplet of a high-resolution spectrum could be irradiated by a highly selective continuous-wave (CW) radio-frequency (rf) field with a very weak amplitude ω(2)/(2π)≤J. This causes splittings of connected transitions, allowing one to map the connectivities of all transitions within the energy-level diagram of the spin system. Such "tickling" experiments stimulated the invention of two-dimensional spectroscopy, but seem to have been forgotten for nearly 50 years. We show that tickling can readily be achieved in homonuclear systems with Fourier transform spectrometers by applying short pulses in the intervals between the sampling points. Extensions to heteronuclear systems are even more straightforward since they can be carried out using very weak CW rf fields.
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Análisis de Fourier , Espectroscopía de Resonancia Magnética/normas , Estándares de ReferenciaRESUMEN
A half-century quest for improving resolution in Nuclear Magnetic Resonance (NMR) and Magnetic Resonance Imaging (MRI) has enabled the study of molecular structures, biological interactions, and fine details of anatomy. This progress largely relied on the advent of sophisticated superconducting magnets that can provide stable and homogeneous fields with temporal and spatial variations below ΔB(0)/B(0)<0.01 ppm. In many cases however, inherent properties of the objects under investigation, pulsating arteries, breathing lungs, tissue-air interfaces, surgical implants, etc., lead to fluctuations and losses of local homogeneity. A new method dubbed "long-lived-coherence correlation spectroscopy" (LLC-COSY) opens the way to overcome both inhomogeneous and homogeneous broadening, which arise from local variations in static fields and fluctuating dipole-dipole interactions, respectively. LLC-COSY makes it possible to obtain ultrahigh resolution two-dimensional spectra, with linewidths on the order of Δν=0.1 to 1 Hz, even in very inhomogeneous fields (ΔB(0)/B(0)>10 ppm or 5000 Hz at 9.7 T), and can improve resolution by a factor up to 9 when the homogeneous linewidths are determined by dipole-dipole interactions. The resulting LLC-COSY spectra display chemical shift differences and scalar couplings in two orthogonal dimensions, like in "J spectroscopy." LLC-COSY does not require any sophisticated gradient switching or frequency-modulated pulses. Applications to in-cell NMR and to magnetic resonance spectroscopy (MRS) of selected volume elements in MRI appear promising, particularly when susceptibility variations tend to preclude high resolution.
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Window-acquired tetrachromatic irradiation allows one to decouple simultaneously four amide protons in cyclosporineâ A (wavy arrows; see figure) leading to simplified multiplets of the alpha protons. By inserting a manifold of polychromatic pulses in each dwell time, several subsystems can be decoupled simultaneously.
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Sustancias Macromoleculares/química , Espectroscopía de Resonancia Magnética , SolucionesRESUMEN
Among the different fields of research in nuclear magnetic resonance (NMR) which are currently investigated in the Laboratory of Biomolecular Magnetic Resonance (LRMB), two subjects that are closely related to each other are presented in this article. On the one hand, we show how to populate long-lived states (LLS) that have long lifetimes T(LLS) which allow one to go beyond the usual limits imposed by the longitudinal relaxation time T1. This makes it possible to extend NMR experiments to longer time-scales. As an application, we demonstrate the extension of the timescale of diffusion measurements by NMR spectroscopy. On the other hand, we review our work on long-lived coherences (LLC), a particular type of coherence between two spin states that oscillates with the frequency of the scalar coupling constant J(IS) and decays with a time constant T(LLC). Again, this time constant T(LLC) can be much longer than the transverse relaxation time T2. By extending the coherence lifetimes, we can narrow the linewidths to an unprecedented extent. J-couplings and residual dipolar couplings (RDCs) in weakly-oriented phases can be measured with the highest precision.
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Resonancia Magnética Nuclear Biomolecular/métodos , Modelos Teóricos , Factores de Tiempo , Ubiquitina/químicaRESUMEN
Trains of 2π or 4π pulses fail to refocus offsets but can suppress the effects of bilinear interactions such as homonuclear scalar couplings.