Non-contact measurement of internal body temperature using subcutaneously implanted diamond microparticles.

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.

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.

Nanodiamonds for bioapplications-specific targeting strategies.

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.

Self-Assembly of Polymer-Encased Lipid Nanodiscs and Membrane Protein Reconstitution.

The absence of detergent and curvature makes nanodiscs excellent membrane mimetics. The lack of structural and mechanistic model of polymer-encapsulated lipid nanodiscs limits their use in the study of the structure, dynamics, and functions of membrane proteins. In this study, we parameterized and optimized the coarse-graining (CG) bead mapping for two differently charged and functionalized copolymers, containing styrene-maleic acid (SMAEA) and polymethacrylate (PMAQA), for the Martini force-field framework and showed nanodisc formation (<8 nm diameter) on a time scale of tens of microseconds using molecular dynamics (MD) simulations. Structural models of ∼2.0 or 4.8 kDa PMAQA and ∼2.2 kDa SMAEA polymer-based lipid nanodiscs highlight the importance of the polymer chemical structure, size, and polymer-lipid ratio in the optimization of the nanodisc structure. The ideal spatial arrangement of polymers in nanodiscs, nanodisc size, and thermal stability obtained from our MD simulation correlates well with the experimental observations. The polymer-nanodiscs were tested for the reconstitution of single-pass or multipass transmembrane proteins. We expect this study to be useful in the development of novel polymer-based lipid nanodiscs and for the structural studies of membrane proteins.

A cationic polymethacrylate-copolymer acts as an agonist for ß-amyloid and an antagonist for amylin fibrillation.

In humans, ß-amyloid and islet amyloid polypeptide (IAPP, also known as amylin) aggregations are linked to Alzheimer's disease and type-2 diabetes, respectively. There is significant interest in better understanding the aggregation process by using chemical tools. Here, we show the ability of a cationic polymethacrylate-copolymer (PMAQA) to quickly induce a ß-hairpin structure and accelerate the formation of amorphous aggregates of ß-amyloid-1-40, whereas it constrains the conformational plasticity of amylin for several days and slows down its aggregation at substoichiometric polymer concentrations. NMR experiments and microsecond scale atomistic molecular dynamics simulations reveal that PMAQA interacts with ß-amyloid-1-40 residues spanning regions K16-V24 and A30-V40 followed by ß-sheet induction. For amylin, it binds strongly close to the amyloid core domain (NFGAIL) and restrains its structural rearrangement. High-speed atomic force microscopy and transmission electron microscopy experiments show that PMAQA blocks the nucleation and fibrillation of amylin, whereas it induces the formation of amorphous aggregates of ß-amyloid-1-40. Thus, the reported study provides a valuable approach to develop polymer-based amyloid inhibitors to suppress the formation of toxic intermediates of ß-amyloid-1-40 and amylin.

Alzheimer's amyloid-beta intermediates generated using polymer-nanodiscs.

Polymethacrylate-copolymer (PMA) encased lipid-nanodiscs (∼10 nm) and macro-nanodiscs (>15 nm) are used to study Aß1-40 aggregation. We demonstrate that PMA-nanodiscs form a ternary association with Aß and regulate its aggregation kinetics by trapping intermediates. Results demonstrating the reduced neurotoxicity of nanodisc-bound Aß oligomers are also reported.

Nanodisc-Forming Scaffold Protein Promoted Retardation of Amyloid-Beta Aggregation.

Peptidic nanodiscs are useful membrane mimetic tools for structural and functional studies of membrane proteins, and membrane interacting peptides including amyloids. Here, we demonstrate anti-amyloidogenic activities of a nanodisc-forming 18-residue peptide (denoted as 4F), both in lipid-bound and lipid-free states by using Alzheimer's amyloid-beta (Aß40) peptide as an example. Fluorescence-based amyloid fibrillation kinetic assays showed a significant delay in Aß40 amyloid aggregation by the 4F peptide. In addition, 4F-encased lipid nanodiscs, at an optimal concentration of 4F (>20 µM) and nanodisc size (<10 nm), significantly affect amyloid fibrillation. A comparison of experimental results obtained from nanodiscs with that obtained from liposomes revealed a substantial inhibitory efficacy of 4F-lipid nanodiscs against Aß40 aggregation and were also found to be suitable to trap Aß40 intermediates. A combination of atomistic molecular dynamics simulations with NMR and circular dichroism experimental results exhibited a substantial change in Aß40 conformation upon 4F binding through electrostatic and π-π interactions. Specifically, the 4F peptide was found to interfere with the central ß-sheet-forming residues of Aß40 through substantial hydrogen, π-π, and π-alkyl interactions. Fluorescence experiments and coarse-grained molecular dynamics simulations showed the formation of a ternary complex, where Aß40 binds to the proximity of peptidic belt and membrane surface that deaccelerate amyloid fibrillation. Electron microscopy images revealed short and thick amyloid fibers of Aß40 formed in the presence of 4F or 4F-lipid nanodsics. These findings could aid in the development of amyloid inhibitors as well as in stabilizing Aß40 intermediates for high-resolution structural and neurobiological studies.

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.