Implication of thermal signaling in neuronal differentiation revealed by manipulation and measurement of intracellular temperature.

Neuronal differentiation-the development of neurons from neural stem cells-involves neurite outgrowth and is a key process during the development and regeneration of neural functions. In addition to various chemical signaling mechanisms, it has been suggested that thermal stimuli induce neuronal differentiation. However, the function of physiological subcellular thermogenesis during neuronal differentiation remains unknown. Here we create methods to manipulate and observe local intracellular temperature, and investigate the effects of noninvasive temperature changes on neuronal differentiation using neuron-like PC12 cells. Using quantitative heating with an infrared laser, we find an increase in local temperature (especially in the nucleus) facilitates neurite outgrowth. Intracellular thermometry reveals that neuronal differentiation is accompanied by intracellular thermogenesis associated with transcription and translation. Suppression of intracellular temperature increase during neuronal differentiation inhibits neurite outgrowth. Furthermore, spontaneous intracellular temperature elevation is involved in neurite outgrowth of primary mouse cortical neurons. These results offer a model for understanding neuronal differentiation induced by intracellular thermal signaling.

Highly Dispersed 3C Silicon Carbide Nanoparticles with a Polydopamine/Polyglycerol Shell for Versatile Functionalization.

Silicon carbide (SiC) nanoparticles containing lattice defects are attracting considerable attention as next-generation imaging probes and quantum sensors for visualizing and sensing life activities. However, SiC nanoparticles are not currently used in biomedical applications because of the lack of technology for controlling their physicochemical properties. Therefore, in this study, SiC nanoparticles are deaggregated, surface-coated, functionalized, and selectively labeled to biomolecules of interest. A thermal-oxidation chemical-etching method is developed for deaggregating and producing a high yield of dispersed metal-contaminant-free SiC nanoparticles. We further demonstrated a polydopamine coating with controllable thickness that can be used as a platform for decorating gold nanoparticles on the surface, enabling photothermal application. We also demonstrated a polyglycerol coating, which gives excellent dispersity to SiC nanoparticles. Furthermore, a single-pot method is developed to produce mono/multifunctional polyglycerol-modified SiC nanoparticles. Using this method, CD44 proteins on cell surfaces are selectively labeled through biotin-mediated immunostaining. The methods developed in this study are fundamental for applying SiC nanoparticles to biomedical applications and should considerably accelerate the development of various SiC nanoparticles to exploit their potential applications in bioimaging and biosensing.

Quantum nanodiamonds for sensing of biological quantities: Angle, temperature, and thermal conductivity.

Measuring physical quantities in the nanometric region inside single cells is of great importance for understanding cellular activity. Thus, the development of biocompatible, sensitive, and reliable nanobiosensors is essential for progress in biological research. Diamond nanoparticles containing nitrogen-vacancy centers (NVCs), referred to as fluorescent nanodiamonds (FNDs), have recently emerged as the sensors that show great promise for ultrasensitive nanosensing of physical quantities. FNDs emit stable fluorescence without photobleaching. Additionally, their distinctive magneto-optical properties enable an optical readout of the quantum states of the electron spin in NVC under ambient conditions. These properties enable the quantitative sensing of physical parameters (temperature, magnetic field, electric field, pH, etc.) in the vicinity of an FND; hence, FNDs are often described as "quantum sensors". In this review, recent advancements in biosensing applications of FNDs are summarized. First, the principles of orientation and temperature sensing using FND quantum sensors are explained. Next, we introduce surface coating techniques indispensable for controlling the physicochemical properties of FNDs. The achievements of practical biological sensing using surface-coated FNDs, including orientation, temperature, and thermal conductivity, are then highlighted. Finally, the advantages, challenges, and perspectives of the quantum sensing of FND are discussed. This review article is an extended version of the Japanese article, In Situ Measurement of Intracellular Thermal Conductivity Using Diamond Nanoparticle, published in SEIBUTSU BUTSURI Vol. 62, p. 122-124 (2022).

In situ measurements of intracellular thermal conductivity using heater-thermometer hybrid diamond nanosensors.

Understanding heat dissipation processes at nanoscale during cellular thermogenesis is essential to clarify the relationships between the heat and biological processes in cells and organisms. A key parameter determining the heat flux inside a cell is the local thermal conductivity, a factor poorly investigated both experimentally and theoretically. Here, using a nanoheater/nanothermometer hybrid made of a polydopamine encapsulating a fluorescent nanodiamond, we measured the intracellular thermal conductivities of HeLa and MCF-7 cells with a spatial resolution of about 200 nm. The mean values determined in these two cell lines are both 0.11 ± 0.04 W m-1 K-1, which is significantly smaller than that of water. Bayesian analysis of the data suggests there is a variation of the thermal conductivity within a cell. These results make the biological impact of transient temperature spikes in a cell much more feasible, and suggest that cells may use heat flux for short-distance thermal signaling.

Tracking the 3D Rotational Dynamics in Nanoscopic Biological Systems.

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 Caenorhabditis elegans. Collectively, our findings show that the method can record nanoscale 3-DoF rotation in vitro, in cells, and even in vivo. 3-DoF rotation tracking introduces a new perspective on microscopic biological samples, revealing in greater detail the functional mechanisms due to nanoscale dynamics in molecules and cells.

Polydopamine Coating as a Scaffold for Ring-Opening Chemistry To Functionalize Gold Nanoparticles.

Gold nanoparticles (GNPs) are promising nanomaterials for various biomedical applications owing to their remarkable optical properties and biocompatibility. However, their interfacial properties require modification for practical use in such applications. Herein, a simple method for modifying the surface of GNPs with polydopamine (PDA) to serve as a scaffold for the subsequent polymerization of hyperbranched polyglycerol (HPG) is reported. GNPs were first coated with PDA (GNP-PDA), and then ring-opening chemistry was used at this interface to modify GNP-PDA with HPG (GNP-PDA-HPG). The produced GNP-PDA-HPG shows not only excellent dispersibility in a salt-containing solution but also strong resistance to aggregation in high- and low-pH solutions, even after 10 days. Moreover, we demonstrate a one-pot method for functionalizing GNP-PDA with HPG and either COOH or trimethylammonium. Finally, we conjugated the trimethylammonium-functionalized GNP-PDA-HPG with fluorescent nanodiamonds to investigate the photothermal ability of the functional GNPs.

Bioorthogonal Fluorescent Nanodiamonds for Continuous Long-Term Imaging and Tracking of Membrane Proteins.

Real-time tracking of membrane proteins is essential to gain an in-depth understanding of their dynamics on the cell surface. However, conventional fluorescence imaging with molecular probes like organic dyes and fluorescent proteins often suffers from photobleaching of the fluorophores, thus hindering their use for continuous long-term observations. With the availability of fluorescent nanodiamonds (FNDs), which have superb biocompatibility and excellent photostability, it is now possible to conduct the imaging in both short and long terms with high temporal and spatial resolution. To realize the concept, we have developed a facile method (e.g., one-pot preparation) to produce alkyne-functionalized hyperbranched-polyglycerol-coated FNDs for bioorthogonal labeling of azide-modified membrane proteins and azide-modified antibodies of membrane proteins. The high specificity of this labeling method has allowed us to continuously monitor the movements of the proteins of interest (such as integrin α5) on/in living cells over 2 h. The results open a new horizon for live cell imaging with functional nanoparticles and fluorescence microscopy.

Fluorescent nanodiamonds as a robust temperature sensor inside a single cell.

Thermometers play an important role to study the biological significance of temperature. Fluorescent nanodiamonds (FNDs) with negatively-charged nitrogen-vacancy centers, a novel type of fluorescence-based temperature sensor, have physicochemical inertness, low cytotoxicity, extremely stable fluorescence, and unique magneto-optical properties that allow us to measure the temperature at the nanoscale level inside single cells. Here, we demonstrate that the thermosensing ability of FNDs is hardly influenced by environmental factors, such as pH, ion concentration, viscosity, molecular interaction, and organic solvent. This robustness renders FNDs reliable thermometers even under complex biological cellular environment. Moreover, the simple protocol developed here for measuring the absolute temperature inside a single cell using a single FND enables successful temperature measurement in a cell with an accuracy better than ±1°C.

Single-Step Metal-Free Grafting of Cationic Polymer Brushes on Fluorescent Nanodiamonds.

Cationic polymers are often employed in conjugation with nanomaterials, and the resultant hybrids are useful for various bioapplications. Here, a single-step metal-free method for the synthesis of fluorescent nanodiamonds (FNDs) conjugated with cationic polymer brushes is reported. Distinct from the common methods such as atom transfer radical polymerization and reversible addition fragmentation chain transfer, our ring-opening-polymerization-based method is simple and less time consuming and hazardous. Infrared spectroscopy, thermogravimetric analysis, zeta potential, and dynamic light scattering confirmed the synthesis. The produced FND-polymer brushes showed markedly higher cell labeling and internalization efficiency without noticeable cytotoxicity. Our method is general and applicable to other nanoparticles as well for uses in diverse research areas.

One-Pot Synthesis of Highly Dispersible Fluorescent Nanodiamonds for Bioconjugation.

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.

Manipulating the distribution of electric field intensity to effectively enhance the spatial and spectral fluorescence intensity of fluorescent nanodiamonds.

Fluorescent nanodiamonds (FNDs) having nitrogen-vacancy (NV) centers have drawn much attention for their biocompatibility and stable optical properties. Nevertheless, the NV centers are located in the interior of the FNDs, and it has not been possible to increase the fluorescence intensity of FNDs efficiently using previously developed enhancement methods. In this paper, we present a simple nanocavity structure that enhances the fluorescence intensity of FNDs. The designed Al/SiO2 nanocavities are stable and inexpensive, and provide a large region for efficient enhancement of fluorescence that can cover most 100 nm FNDs. By tuning the thickness of the capping SiO2 layer of the Al/SiO2 nanocavities, the distributions of both the spatial and spectral electric field intensities of the FNDs could be controlled and manipulated. In general, the FNDs were excited using a green-yellow laser; the broadband fluorescence of the FNDs comprised the emissions from neutral (NV0) and negatively charged (NV-) NV centers. To enhance the fluorescence intensity from the NV- centers of the FNDs, we designed an Al/70 nm SiO2 nanocavity to function at excitation and emission wavelengths of 633 and 710 nm, respectively, allowing the NV- centers to be excited efficiently; as a result, we achieved an enhancement in fluorescence intensity of 11.2-fold. Moreover, even when we covered 100 nm FNDs with polyglycerol (forming p-FND), the fluorescence intensities of the p-FND particles placed on the nanocavities remained greatly enhanced.

Enrichment of ODMR-active nitrogen-vacancy centres in five-nanometre-sized detonation-synthesized nanodiamonds: Nanoprobes for temperature, angle and position.

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.

Highly stable lipid-encapsulation of fluorescent nanodiamonds for bioimaging applications.

Highly stable lipid-encapsulated fluorescent nanodiamonds (FNDs) are produced by photo-crosslinking of diacetylene-containing lipids physically attached to the FND surface. Not only is this coating method simple and fast, but also it gives the FND-lipid hybrids favorable properties for bioapplications. The hybrids are useful as fluorescent biolabels as well as fiducial markers for correlative light and electron microscopy.

A Nanodiamond-peptide Bioconjugate for Fluorescence and ODMR Microscopy of a Single Actin Filament.

Recently, the importance of conformational changes in actin filaments induced by mechanical stimulation of a cell has been increasingly recognized, especially in terms of mechanobiology. Despite its fundamental importance, however, long-term observation of a single actin filament by fluorescent microscopy has been difficult because of the low photostability of traditional fluorescent molecules. This paper reports a novel molecular labeling system for actin filaments using fluorescent nanodiamond (ND) particles harboring nitrogen-vacancy centers; ND has flexible chemical modifiability, extremely high photostability and biocompatibility, and provides a variety of physical information quantitatively via optically detected magnetic resonance (ODMR) measurements. We performed the chemical surface modification of an ND with the actin filament-specific binding peptide Lifeact and observed colocalization of pure Lifeact-modified ND and actin filaments by the ODMR selective imaging protocol, suggesting the capability of long-term observation and quantitative analysis of a single molecule by using an ND particle.

Selective Labeling of Proteins on Living Cell Membranes Using Fluorescent Nanodiamond Probes.

The impeccable photostability of fluorescent nanodiamonds (FNDs) is an ideal property for use in fluorescence imaging of proteins in living cells. However, such an application requires highly specific labeling of the target proteins with FNDs. Furthermore, the surface of unmodified FNDs tends to adsorb biomolecules nonspecifically, which hinders the reliable targeting of proteins with FNDs. Here, we combined hyperbranched polyglycerol modification of FNDs with the ß-lactamase-tag system to develop a strategy for selective imaging of the protein of interest in cells. The combination of these techniques enabled site-specific labeling of Interleukin-18 receptor alpha chain, a membrane receptor, with FNDs, which eventually enabled tracking of the diffusion trajectory of FND-labeled proteins on the membrane surface.