Unraveling Eumelanin Radical Formation by Nanodiamond Optical Relaxometry in a Living Cell.

Defect centers in a nanodiamond (ND) allow the detection of tiny magnetic fields in their direct surroundings, rendering them as an emerging tool for nanoscale sensing applications. Eumelanin, an abundant pigment, plays an important role in biology and material science. Here, for the first time, we evaluate the comproportionation reaction in eumelanin by detecting and quantifying semiquinone radicals through the nitrogen-vacancy color center. A thin layer of eumelanin is polymerized on the surface of nanodiamonds (NDs), and depending on the environmental conditions, such as the local pH value, near-infrared, and ultraviolet light irradiation, the radicals form and react in situ. By combining experiments and theoretical simulations, we quantify the local number and kinetics of free radicals in the eumelanin layer. Next, the ND sensor enters the cells via endosomal vesicles. We quantify the number of radicals formed within the eumelanin layer in these acidic compartments by applying optical relaxometry measurements. In the future, we believe that the ND quantum sensor could provide valuable insights into the chemistry of eumelanin, which could contribute to the understanding and treatment of eumelanin- and melanin-related diseases.

High-throughput nitrogen-vacancy center imaging for nanodiamond photophysical characterization and pH nanosensing.

The fluorescent nitrogen-vacancy (NV) defect in diamond has remarkable photophysical properties, including high photostability which allows stable fluorescence emission for hours; as a result, there has been much interest in using nanodiamonds (NDs) for applications in quantum optics and biological imaging. Such applications have been limited by the heterogeneity of NDs and our limited understanding of NV photophysics in NDs, which is partially due to the lack of sensitive and high-throughput methods for photophysical analysis of NDs. Here, we report a systematic analysis of NDs using two-color wide-field epifluorescence imaging coupled to high-throughput single-particle detection of single NVs in NDs with sizes down to 5-10 nm. By using fluorescence intensity ratios, we observe directly the charge conversion of single NV center (NV- or NV0) and measure the lifetimes of different NV charge states in NDs. We also show that we can use changes in pH to control the main NV charge states in a direct and reversible fashion, a discovery that paves the way for performing pH nanosensing with a non-photobleachable probe.

Confinement-Free Wide-Field Ratiometric Tracking of Single Fluorescent Molecules.

Single-molecule fluorescence has been highly instrumental in elucidating interactions and dynamics of biological molecules in the past two decades. Single-molecule fluorescence experiments usually rely on one of two detection geometries, either confocal point-detection or wide-field area detection, typically in a total internal reflection fluorescence (TIRF) format. However, each of these techniques suffers from fundamental drawbacks that limit their application. In this work, we present a new technique, solution wide-field imaging (SWiFi) of diffusing molecules, as an alternative to the existing methods. SWiFi is a simple extension to existing objective-type TIRF microscopes that allows wide-field observations of fast-diffusing molecules down to single fluorophores without the need of tethering the molecules to the surface. We demonstrate that SWiFi enables high-throughput ratiometric measurements with several thousands of individual data points per minute on double-stranded DNA standard (dsDNA) samples containing Förster resonance energy transfer pairs. We further display the capabilities of SWiFi by reporting on mobility and ratiometric characterization of fluorescent nanodiamonds, DNA Holliday junctions, and protein-DNA interactions. The ability of SWiFi for high-throughput, ratiometric measurements of fast-diffusing species renders it a valuable tool for the single-molecule research community by bridging between confocal and TIRF detection geometries in a simple and efficient way.

Positive zeta potential of nanodiamonds.

In this paper, the origin of positive zeta potential exhibited by nanodiamond particles is explained. Positive zeta potentials in nano-structured carbons can be explained by the presence of graphitic planes at the surface, which leave oxygen-free Lewis sites and so promotes the suppression of acidic functional groups. Electron Microscopy and Raman Spectroscopy have been used to show that positive zeta potential of nanodiamond is only exhibited in the presence of sp2 carbon at the surface.