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Nanodiamonds containing negatively charged nitrogen-vacancy (NV−) centers are versatile room-temperature quantum sensors in a growing field of research. Yet, knowledge regarding the NV− formation mechanism in very small particles is still limited. This study focuses on the formation of the smallest NV−-containing diamonds, 5 nm detonation nanodiamonds (DNDs). As a reliable method to quantify NV− centers in nanodiamonds, half-field signals in electron paramagnetic resonance (EPR) spectroscopy are recorded. By comparing the NV− concentration with a series of nanodiamonds from high-pressure high-temperature (HPHT) synthesis (10−100 nm), it is shown that the formation process in 5 nm DNDs is unique in several aspects. NV− centers in DNDs are already formed at the stage of electron irradiation, without the need for high-temperature annealing, an effect related to the very small particle size. Also, the NV− concentration (in atomic ratio) in 5 nm DNDs surpasses that of 20 nm-sized nanodiamonds, which contradicts the observation that the NV− concentration generally increases with particle size. This can be explained by the 10 times higher concentration of substitutional nitrogen atoms in the studied DNDs ([NS ≈ 1000 ppm]) compared to the HPHT nanodiamonds ([NS ≈ 100 ppm]). Upon electron irradiation at a fluence of 1.5 × 10^19 e−/cm2, DNDs show a 12.5-fold increment in the NV− concentration with no sign of saturation reaching 1 out of about 80 DNDs containing an NV− center. These findings can be of interest for the creation of defects in other very small semiconductor nanoparticles beyond NV-nanodiamonds as quantum sensors., The Journal of Physical Chemistry C, 126 (11), ISSN:1932-7455, ISSN:1932-7447

Detonation nanodiamonds (DNDs) are a class of very small and spherical diamond nanocrystals. They are used in polymer reinforcement materials or as drug delivery systems in the field of nanomedicine. Synthesized by detonation, only the final deaggregation step down to the single-digit nanometer size (< 10 nm) unfolds their full potential. Existing deaggregation methods mainly rely on mechanical forces, such as high-power sonication or beads milling. These techniques entail drawbacks such as contamination of the sample and the need for a specialized apparatus. In this paper, we report a purely chemical deaggregation method by simply combining oxidation in air followed by a boiling acid treatment, to produce highly stable single-digit DNDs in a suspension. The resulting DNDs are surface functionalized with carboxyl groups, the final boiling acid treatment removes primary metal contaminants such as magnesium, iron or copper and the nanoparticles remain dispersed over a wide pH range. Our method can be easily carried out in a standard chemistry laboratory with commonly available laboratory apparatus. This is a key step for many DND-based applications, ranging from material science to biological or medical applications.

Nanodiamonds containing negatively-charged Nitrogen-Vacancy (NV-) centers are versatile room-temperature quantum sensors in a growing field of research. Yet, knowledge regarding the formation mechanism in very small particles is still limited. Here, the study was focused on the formation of the smallest NV--containing diamonds, 5 nm detonation nanodiamonds (DNDs). As a reliable method to quantify NV- centers in nanodiamonds, half-field signals in electron paramagnetic resonance (EPR) spectroscopy are recorded. By comparing the NV- concentration in a series of nanodiamonds (5 - 100 nm), it was shown that the formation process in DNDs is unique in several aspects. NV- centers in DNDs are already formed during electron irradiation, without the need for high-temperature annealing. The effect was interpreted in terms of “self-annealing”, where size and type dependent effects enable vacancy migration at lower temperature. Although NV- concentration increases with particle size, the NV- concentration in NDs surpasses that of 20 nm-sized nanodiamonds. Using Monte-Carlo simulations, we show that the higher substitutional Nitrogen concentration compensates the vacancy loss induced by the large particle surface. Upon 1.5 × 1019 e-/cm2 electron irradiation, DNDs show a 12.5-fold NV- increment with no sign of saturation. These findings can be of interest for the creation of defects in other very small semiconductor nanoparticles.

Graphene oxide (GO) can form ultrapermeable and ultraselective membranes that are promising for various gas separation applications, including hydrogen purification. However, GO films lose their attractive separation properties in humid conditions. Here we show that incorporating positively charged nanodiamonds (ND+s) into GO nanolaminates leads to humidity-resistant, yet high-performing, membranes. While native GO membranes fail at a single run, the GO/ND+ composite retains up to ~90% of GO’s H2 selectivity against CO2 after several cycles under an aggressive humidity test. The addition of negatively charged ND to GO brought no such stabilization, suggesting that charge compensation acts as the main mechanism conferring humidity resistance, where ND+s neutralize the negative charge GO sheets. We observed a similar but inferior stabilization effect when positively charged polyhedral oligomeric silsesquioxane replaces ND+. The demonstrated material platform offers a solution for separating H2 gas from its usually humid mixtures generated from fossil fuel sources or water splitting.

With in-cell NMR, the structural and dynamic features of proteins can be analyzed directly in living cells at atomic resolution. However, in mammalian cells, such as human cells, the structural/dynamic features and physiological functions are not fully understood at atomic resolution. In this study, we developed a method for the highly viable and efficient introduction of stable isotope-labeled proteins into human cells by electrotransfection. Thus, it was possible to perform in-cell NMR to study the structural/dynamic features of intracellular proteins with a time resolution of a minute. The transfected proteins were localized in intracellular vesicles and gradually degraded as the vesicles acidified, which could be controlled by adding EDTA, a membrane-impermeable low molecular weight compound, during electrotransfection. With this method, we could then determine the precise structure and dynamics of proteins in endosomal vesicles. Our approach demonstrates that the combination of electrotransfection and in-cell NMR provides an opportunity to study the structural/dynamic features and physiological functions of molecules taken into intact cells.

von Willebrand factor (vWF) is an adhesive plasma protein that is important for platelet adhesion in normal hemostasis in response to vascular injury. Although large vWF multimers are released from storage granules of platelets and (sub-)endothelial cells in response to hemostatic stimuli, for normal physiological function, vWF multimers are required to be cleaved into smaller multimeric forms. The plasma metalloproteinase ADAMTS13 specifically cleaves the peptide bond located in the middle of the A2 domain of vWF (vWF-A2), but the cleavage site is buried inside the structure of vWF and is difficult to access in the absence of elevated flow shear stress. On the other hand, in the presence of high vascular shear stress, the structure of vWF-A2 is supposed to be unfolded, thereby becoming accessible for proteolysis by ADAMTS13. However, the atomic-level mechanism underlying shear-induced structural changes of vWF-A2 remains unclear and to date no solution NMR information is available. In this study, we present the backbone 1H, 13C, and 15N resonance assignments of mouse vWF-A2; side chain assignments of 13Cβ are also provided. Secondary structure propensity analysis based on the assigned chemical shifts showed that mouse vWF-A2 forms similar secondary structures in solution to the previously determined crystal structure of human vWF-A2. The obtained NMR assignment data will contribute to an atomic-level characterization of shear-induced unfolding of vWF-A2 in solution.

Determination of optimal measurement parameters is essential for measurement experiments. They can be manually optimized if the linear correlation between them and the corresponding signal quality is known or easily determinable. However, in practice, this correlation is often nonlinear and not known a priori; hence, complicated trial and error procedures are employed for finding optimal parameters while avoiding local optima. In this work, we propose a novel approach based on machine learning for optimizing multiple measurement parameters, which nonlinearly influence the signal quality. Optically detected magnetic resonance measurements of nitrogen-vacancy centers in fluorescent nanodiamonds were used as a proof-of-concept system. We constructed a suitable dataset of optically detected magnetic resonance spectra for predicting the optimal laser and microwave powers that deliver the highest contrast and signal-to-noise ratio values by means of linear regression, neural networks, and random forests. The model developed by the considered neural network turned out to have a coefficient of determination significantly higher than that of the other methods. The proposed method thus provided a novel approach for the rapid setting of measurement parameters that influence the signal quality in a nonlinear way, opening a gate for fields like nuclear magnetic resonance, electron paramagnetic resonance, and fluorescence microscopy to benefit from it.

Aggregate formation of superoxide dismutase 1 (SOD1) inside motor neurons is known as a major factor in onset of amyotrophic lateral sclerosis. The thermodynamic stability of the SOD1 β-barrel has been shown to decrease in crowded environments such as inside a cell, but it remains unclear how the thermodynamics of crowding-induced protein destabilization relate to SOD1 aggregation. Here we have examined the effects of a protein crowder, lysozyme, on fibril aggregate formation of the SOD1 β-barrel. We found that aggregate formation of SOD1 is decelerated even in mildly crowded solutions. Intriguingly, transient diffusive interactions with lysozyme do not significantly affect the static structure of the SOD1 β-barrel but stabilize an alternative excited "invisible" state. The net effect of crowding is to favor species off the aggregation pathway, thereby explaining the decelerated aggregation in the crowded environment. Our observations suggest that the intracellular environment may have a similar negative (inhibitory) effect on fibril formation of other amyloidogenic proteins in living cells. Deciphering how crowded intracellular environments affect aggregation and fibril formation of such disease-associated proteins will probably become central in understanding the exact role of aggregation in the etiology of these enigmatic diseases.

Polyubiquitin is a multifunctional protein tag formed by the covalent conjugation of ubiquitin molecules. Due to the high rigidity of the ubiquitin fold, the ubiquitin moieties in a polyubiquitin chain appear to be structurally equivalent to each other. It is therefore unclear how a specific ubiquitin moiety in a chain may be preferentially recognized by some proteins, such as the kinase PINK1. Here we show that there is structural dynamic heterogeneity in the two ubiquitin moieties of K48-linked diubiquitin by NMR spectroscopic analyses. Our analyses capture subunit-asymmetric structural fluctuations that are not directly related to the closed-to-open transition of the two ubiquitin moieties in diubiquitin. Strikingly, these newly identified heterogeneous structural fluctuations may be linked to an increase in susceptibility to phosphorylation by PINK1. Coupled with the fact that there are almost no differences in static tertiary structure among ubiquitin moieties in a chain, the observed subunit-specific structural fluctuations may be an important factor that distinguishes individual ubiquitin moieties in a chain, thereby aiding both efficiency and specificity in post-translational modifications.

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 microdia-monds containing NV−centers. For both samples, the integrated solid effect (ISE) is used to polarize the13C spin system in magnetic fields of 350-400 mT. In the benzoic acid sample, the13C spin polarization is enhanced up to 0.12% through directelectron-to-13C polarization transfer without performing dynamic1H polarization followed by 1H-13C cross polarization. In5addition, ISE has been successfully applied for the first time to polarize naturally abundant 13C spins in a microdiamond sample to 0.01%. To characterize the buildup of the13C polarization, we discuss the efficiencies of direct polarization transfer be-tween the electron and13C spins as well as that of13C–13C spin diffusion, examining various parameters which are beneficial or detrimental for successful bulk dynamic13C polarization.

We constructed a highly sensitive fluorescence wide-field imaging system with a microwave source, implanted fluorescent diamond microparticles ("microdiamonds") subcutaneously into the dorsal skin of a mouse after sacrifice, and demonstrated the feasibility of using optically detected magnetic resonance (ODMR) to measure internal body temperature in a mammal.

Ubiquitination is one of the major post-translational modifications and entails conjugation of ubiquitin molecules to target proteins. To make free ubiquitin molecules available for conjugation, in cells ubiquitin is not only synthesized de novo, but is also provided by cleaving off existing conjugated ubiquitin molecules, so-called deubiquitination reaction. Therefore, intracellular ubiquitin molecules are thought to be recycled, but the recycling frequency remains elusive. The main reason for the lack of such mechanistic details is that the original and recycled ubiquitin molecules are indistinguishable in their chemical and physical properties. To tackle this issue, here we applied 18O-labeling to trace how ubiquitin is recycled in a simultaneous ubiquitination/deubiquitination reaction (ubiquitin cycle reaction). Because deubiquitination is a hydrolysis reaction, the two 16O atoms of the C-terminal carboxy group of a ubiquitin molecule can be exchanged with 18O atoms by deubiquitination in 18O-labeled aqueous solution. By using quantitative mass spectrometry, we detected 18O atom incorporation into the C-terminal carboxy group of ubiquitin in the course of a deubiquitination reaction, in addition, we were able to quantify the 18O-incorporation in a ubiquitin cycle reaction. Unexpectedly, kinetic analysis suggested that ubiquitination reactivity was accelerated in the presence of a deubiquitinating enzyme. Collectively, we have established a quantitative method to trace ubiquitin cycle reactions by analyzing deubiquitination-associated 18O-incorporation into ubiquitin.

添付関連ファイル参照, The 4th International Forum on Quantum Metrology and Sensing

Dynamic nuclear polarization (DNP), a technique to transfer spin polarization from electrons to nuclei, has been studied since its early discovery [1] and has opened the way for high sensitive nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging. In "conventional" DNP, which uses thermally-polarized unpaired electrons as the source of polarization, the DNP enhancement factor is limited to γe/γn, where γe(n) are the gyromagnetic ratios of the electron (nuclear) spins. In DNP using the paramagnetic electrons in thermal equilibrium, experiments need to be performed at cryogenic temperature to increase the electron-spin polarization as much as possible. Conversely, the spins of optically created electrons in the triplet state can have much higher polarization than their thermal equilibrium value, DNP using the triplet state, referred to as triplet DNP, can lead to nuclear hyperpolarization beyond the limit of the conventional DNP using thermal electron polarization. Furthermore, experiments can be carried out at room temperature, by irradiating the sample with laser light. Recently, DNP of an ensemble of 13C nuclear spins using negatively charged nitrogen-vacancy (NV−) color centers in a bulk diamond single crystal has been demonstrated at room temperature and the 13C polarization of 6 % has been achieved via the combination of the thermal mixing and the solid effect [2]. DNP using NV− in powdered microdiamonds has been reported by Ajoy et al., who took advantage of the reduced width of the anisotropic electron spin resonance powder pattern of the NV− centers at the magnetic field of ca. 30 mT [3]. However, triplet DNP is much older and achieved a 1H polarization of 34 % at room temperature and a magnetic field of 0.4 T using pentacene in p-terphenyl crystal [4]. Even though triplet DNP in both systems, NV- centers and pentacene, relies on the transfer of spin polarization from optically hyperpolarized triplet electrons to nuclei , there are important differences. While the NV- center has an electronic triplet ground state and is therefore paramagnetic, pentacene has an electronic singlet ground state, is diamagnetic, and only becomes paramagnetic through optical excitation into a triplet state. On the other hand, the zero-field splitting parameter D for pentacene is only half as large as in NV- centers, which is an advantage when disordered powder is used as a sample. In this work, we compare triplet-DNP of NV− centers in diamond and pentacene doped in [carboxyl-13C] benzoic acid (PBA) in polycrystalline samples at room temperature [5]. In the DNP experiments, the integrated solid effect (ISE) was used to transfer the polarization from electrons to nuclei. The ISE employs microwave irradiation and external magnetic-field sweep, so that the Hartmann–Hahn matching is implemented between the electron spins in the rotating frame and the nuclear spins in the laboratory frame. We study the behavior of the 13C polarization buildup in terms of the polarization efficiency of the transfer from the electron to nuclei, exchange rate, and the 13C spin diffusion. As a result, we obtained the 13C polarization of 0.01 % in the microdiamonds, and of 0.12 % in PBA at room temperature in a magnetic field of 0.4 T by using the integrated solid effect and the obtained exchange rate was 0.87 % for microdiamonds and 3.5 % for PBA. The 13C polarization enhancements for the diamond and the PBA were 300 and 3600 compared to the thermal NMR polarization. Besides the initial polarization transfer from the triplet electron to the nuclei, we also shed light on the process of nuclear spin-spin diffusion, which distributes the hyperpolarization within the sample., 2021 MRS Fall Meeting & Exhibit

Cytosine methylation is an epigenetic modification essential for formation of mature heterochromatin, gene silencing, and genomic stability. In plants, methylation occurs not only at cytosine bases in CpG but also in CpHpG and CpHpH contexts, where H denotes A, T, or C. Methyl-CpG binding domain (MBD) proteins, which recognize symmetrical methyl-CpG dinucleotides and act as gene repressors in mammalian cells, are also present in plant cells, although their structural and functional properties still remain poorly understood. To fill this gap, in this study, we determined the solution structure of the MBD domain of the MBD6 protein from Arabidopsis thaliana and investigated its binding properties to methylated DNA by binding assays and an in-depth NMR spectroscopic analysis. The AtMBD6 MBD domain folds into a canonical MBD structure in line with its binding specificity toward methyl-CpG and possesses a DNA binding interface similar to mammalian MBD domains. Intriguingly, however, the binding affinity of the AtMBD6 MBD domain toward methyl-CpG-containing DNA was found to be much lower than that of known mammalian MBD domains. The main difference arises from the absence of positively charged residues in AtMBD6 that supposedly interact with the DNA backbone as seen in mammalian MBD/methyl-CpG-containing DNA complexes. Taken together, we have established a structural basis for methyl-CpG recognition by AtMBD6 to develop a deeper understanding how MBD proteins work as mediators of epigenetic signals in plant cells.

Adenosine triphosphate (ATP) is an immensely well-studied metabolite serving multiple key biochemical roles as the major chemical energy currency in living systems, a building block of ribonucleic acids, and a phosphoryl group donor in kinase-mediated signaling. Intriguingly, ATP has been recently proposed to act as a hydrotrope that inhibits aggregation of amyloidogenic proteins; however, the underlying mechanism and the general physicochemical effect that coexistence with ATP exerts on proteins remain unclear. By combining NMR spectroscopy and MD simulations, here we observed weak but unambiguously measurable and concentration-dependent noncovalent interactions between ATP and various proteins. The interactions were most pronounced for an intrinsically disordered protein (α-synuclein) and for residues in flexible regions (e.g., loops or termini) of two representative folded proteins (ubiquitin and the dimeric ubiquitin-binding domain of p62). As shown by solution NMR, a consequence of the ATP-protein interaction was altered hydration of solvent-exposed residues in the protein. The observation that ATP interacted with all three proteins suggests that ATP is a general nonspecific binder of proteins. Several complementary biophysical methods further confirmed that, at physiological concentrations of ∼5-10 mM, ATP starts to form oligomeric states via magnesium-chelating and chelation-independent mechanisms, in agreement with previous studies. Although the observed ATP-protein interaction was relatively weak overall, the high ratio of ATP (monomeric free ATP, mono- and divalent ion-bound ATP, oligomeric and chelated ATP) to proteins in cells suggests that most proteins are likely to encounter transient interactions with ATP (and chemically similar metabolites) that confer metabolite-mediated protein surface protection.

Formation of protein aggregates or fibrils entails the conversion of soluble native protein monomers via multiple molecular states. No spectroscopic techniques have succeeded in capturing the transient molecular-scale events of fibrillation in situ. Here we report residue- and state-specific real-time monitoring of the fibrillation of amyotrophic lateral sclerosis-related SOD1 by rheology NMR (Rheo-NMR) spectroscopy. Under moderately denaturing conditions, where NMR signals of folded and unfolded monomeric SOD1 are simultaneously observable, the cross-peak intensities of folded monomeric SOD1 decreased faster than those of the unfolded species, and a 310-helix in folded SOD1 was deformed prior to global unfolding. Furthermore, real-time protein dynamics analysis identified residues involved in the core structure formation of SOD1 oligomers. Our findings provide insight into local and global unfolding events in SOD1 and fibril formation. This Rheo-NMR analysis will be applicable not only to atomic-level monitoring of other amyloidogenic proteins but also to quantification of shear-induced structural changes of non-amyloidogenic proteins and elucidation of shear-enhanced chemical phenomena such as viscosity increase and crystallization of various solution-state compounds.

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.

ナノダイヤモンド中のNVセンタの形成の中でも粒形依存性を調べたので報告する。, 第68回 応用物理学会春季学術講演会

第5節 小型超高感度ダイヤモンド量子センサの 開発と応用 〈白川 昌宏，五十嵐 龍治〉 1 はじめに 2 生命計測技術としての量子センサ 3 多機能センサとしての量子センサ 4 超解像イメージングプローブとしての 量子センサ 5 ナノ回転センサとしての量子センサ 6 おわりに

量子技術の応用でもっとも産業化が早いと目されている量子計測・センシング技術の最新研究を概観

Detonation nanodiamonds (DNDs) with sizes below 10 nm have attracted attention as single photon emitters with potential in many research fields from life sciences to quantum technologies. However, while nitrogen-vacancy (NV) color centers are found in nanodiamonds directly after the detonation synthesis without the need for irradiation or annealing, silicon-vacancy (SiV) color centers are not present in these pristine samples. Herein, SiV centers are created in DNDs by an annealing treatment up to 1100 °C in high vacuum. As silicon is not added, the precursor of the SiV centers must be pristine silicon impurities inside the nanodiamond lattice. A sharp emission line at the wavelength of 737 nm with a linewidth of 7.7 nm is observed in DNDs that are electron irradiated before annealing. This wavelength is consistent with the characteristic emission line of SiV centers and its linewidth is comparable with that in larger nanodiamonds created by chemical vapor deposition and subsequent milling. The average lifetime (0.4 ± 0.04 ns) of the fluorescence, which is in the range of reported lifetimes in nanodiamonds, also support that the observed emission peak are due to SiV centers in DNDs., Physica Status Solidi A, 218 (19), ISSN:1862-6300, ISSN:1862-6319, ISSN:0031-8965, ISSN:1521-396X

Nanodiamonds carrying negatively-charged nitrogen-vacancy (NV-) defects are promising biocompatible nano-scale probes for quantum sensing. To maximize the potential of this versatile tool, the smallest type of nanodiamonds that carries NV- defects are highly desirable and are available in the form of 5-nanometer detonation nanodiamonds (DNDs). Previously, we revealed that the common NVformation mechanism may not be applicable to DNDs. To investigate the reasons behind this phenomenon, the NV- formation process has been studied experimentally in a series of nanodiamonds with different sizes, and additional simulations complete the mechanistic understanding., 量子生命科学会第2回大会

我々が超高感度溶液NMRを実現したペンタセンをドープした[calboxyl-13C]安息香酸の系と、近年世界中で盛んに研究されているNV中心を含むダイヤモンドの二つの系でpulsed-DNPによる13Cスピンの直接室温超偏極に成功した。Pulsed-DNPシーケンス一回あたりの13Cスピンへの偏極移動確率が、ダイヤモンドの系は安息香酸の系に比べて約9倍高いが、核スピンT1は5倍短く、またESRスペクトルが2倍ブロードであることなどに起因して、到達偏極率が低くなる。最終的に、ダイヤモンド中に天然存在比で存在する13Cスピンでは0.01%、[calboxyl-13C]安息香酸中では0.13%の偏極率を得ることができた。, 3rd IFQMS

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 NV- centers. For both samples, the integrated solid effect (ISE) is used to polarize the 13C spin system in magnetic fields of 350–400 mT. In the benzoic acid sample, the 13C spin polarization is enhanced up to 0.12 % through direct electron-to-13C polarization transfer without performing dynamic 1H polarization followed by 1H-13C cross polarization. In addition, ISE has been successfully applied for the first time to polarize naturally abundant 13C spins in a microdiamond sample to 0.01 %. 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.

Detonation nanodiamonds (DNDs) are a class of very small and spherical diamond nanocrystals. Used in polymer reinforcement materials, as drug delivery systems or as fluorescent biomarkers, only the final deaggregation step down to the single-digit nanometer size unfolds their full potential. All existing deaggregation methods rely on mechanical forces, such as high-power sonication or beads milling. We report a purely chemical deaggregation method by combining oxidation in air followed by a boiling acid treatment. Our DNDs are surface functionalized with carboxyl groups, the final boiling acid treatment removes primary metal contaminants and the nanoparticles remain dispersed over a wide pH range. Experiments can be easily carried out in a standard chemistry laboratory. This is a key step for many DND-based applications, ranging from material science to biological or medical applications and opens a way for inexpensive mass production on industrial scale.

Ubiquitination is one of the major post-translational modifications and entails conjugation of ubiquitin molecules to target proteins. To make free ubiquitin molecules available for conjugation, in cells ubiquitin is not only synthesized de novo, but is also provided by cleaving off existing conjugated ubiquitin molecules, so-called deubiquitination reaction. Therefore, intracellular ubiquitin molecules are thought to be recycled, but the recycling frequency remains elusive. The main reason for the lack of such mechanistic details is that the original and recycled ubiquitin molecules are indistinguishable in their chemical and physical properties. To tackle this issue, here we applied

The rotation of an object cannot be fully tracked without understanding a set of three angles, namely, roll, pitch, and yaw. Tracking these angles as a three-degrees-of-freedom (3-DoF) rotation is a fundamental measurement, facilitating, for example, attitude control of a ship, image stabilization to reduce camera shake, and self-driving cars. Until now, however, there has been no method to track 3-DoF rotation to measure nanometer-scale dynamics in biomolecules and live cells. Here we show that 3-DoF rotation of biomolecules can be visualized via nitrogen-vacancy centers in a fluorescent nanodiamond using a tomographic vector magnetometry technique. We demonstrate application of the method to three different types of biological systems. First, we tracked the rotation of a single molecule of the motor protein F1-ATPase by attaching a nanodiamond to the γ-subunit. We visualized the 3-step rotation of the motor in 3D space and, moreover, a delay of ATP binding or ADP release step in the catalytic reaction. Second, we attached a nanodiamond to a membrane protein in live cells to report on cellular membrane dynamics, showing that 3D rotational motion of the membrane protein correlates with intracellular cytoskeletal density. Last, we used the method to track nonrandom motions in the intestine of

Fluorescent nanodiamonds (FNDs) have been attracting much attention as promising therapeutic agents and probes for bioimaging and nanosensing. For their biological applications, several hydrophilizing methods to enhance FND colloidal stability have been developed to suppress their aggregation and the nonspecific adsorption to biomolecules in complex biomedical environments. However, these methods involve several complicated synthetic and purification steps, which prohibit the use of FNDs for bioapplications by biologists. In this study, we describe a simple one-pot FND hydrophilization method that comprises coating of the surface of the nanoparticles with COOH-terminated hyperbranched polyglycerol (HPG-COOH). HPG-COOH-coated FNDs (FND-HPG-COOHs) were found to exhibit excellent dispersibility under physiological conditions despite the thinness of the 5 nm HPG-COOH layer. Biotinylated FND-HPG-COOHs specifically captured avidin molecules in the absence of nonspecific protein adsorption. Moreover, we demonstrated that FND-HPG-COOHs conjugated with antibodies can be used to selectively target integrins in fixed HeLa cells. In addition, intracellular temperature changes were measured via optically detected magnetic resonance using FND-HPG-COOHs conjugated with mitochondrial localization signal peptides. Our one-pot synthetic method will encourage the broad use of FNDs among molecular and cellular biologists and pave the way for extensive biological and biomedical applications of FNDs.