Functionalized Red Fluorescent Nanodiamond
Fluorescent nanodiamonds (FNDs) are a chemically and mechanically robust, infinitely photostable, and biocompatible fluorophore. The fluorescence of diamond originates from color centers within the diamond crystal lattice, and typically consist of nitrogen complexes with vacancies (empty carbon sites in the diamond lattice. The nitrogen-vacancy (NV) center, it the most well known color center in diamond, and provides fluorescence emission in the red-NIR region. Negatively charged NV centers (NV-) have unique optoelectronic properties that are utilized in nanoscale quantum sensing applications. Adámas offers a wide range of NV containing nanodiamonds in the size range of 10 nm up to 300 nm. A variety of surface functionalities are available, including: Carboxylic acids, amines, hydrophobic alkyl chains, core-shell polymer coated particles, and ligands for click-chemistry. Application areas for red FNDs include: super-resolution microscopy, correlative microscopy, lifetime gated fluorescence imaging, cellular labeling and tracking, in vivo imaging, lateral flow assay, and nanoscale quantum sensing.
Size Range: 10-13 nm
The level of fluorescence and the population of fluorescent diamond particles in the sub-15 nm range is generally very low. These particles are not suitable for direct cellular imaging, and require users equipped with advanced optical setups to use effectively. These particles best suited for users interested in single photon emitter applications. The quality of NV- centers in these particles is low due to large lattice distortion and damage induced from milling from larger sizes. Single particle fluorescence characterization confirmed the presence of active NV- centers, and determined that the approximate NV-/NV0 ratio was ~0.6, so fluorescing particles contain ~60% NV0 and ~40% NV-. 10 nm particles are available with carboxylic acid enriched surfaces resulting from acid treatment during processing.
A typical fluorescence emission spectra for a solution of 10 nm FNDs is shown in Figure 3 under 532 nm laser excitation (note the strong water Raman scatter). High resolution TEM (HRTEM) images are shown in Figure 4. Figure 5 and Figure 6 show size distributions of 10 nm FNDs from AFM height analysis and dynamic light scattering (DLS), respectively.
Size Range: 50-300 nm
The 50-300 nm size range offers significantly higher brightness as compared to the smaller size ranges. These larger particles are typically easier to work with if additional chemical processing at the customer’s side is desired. We strongly encourage customers that are performing preliminary experimental work or who do not have prior experience working with fluorescent nanodiamonds to start with these sizes if possible as they provide a good balance between fluorescent intensity and usability for bioconjugation or targeting schemes.
Electron paramagnetic resonance EPR measurements determined the NV- concentration was on the order of 3 ppm (this equates to ~300 centers per 100 nm particle and ~800 centers per 140 nm particle.
Typical DLS size distributions for 200 and 300 nm FND particles are shown in Figure 11. These larger particles have been successfully deployed in quantum sensing, in vitro and in vivo imaging. 100 nm FND particles were conjugated with human vascular endothelial growth factor (VEGf) and demonstrated effective conjugation and targeting. 200 nm particles have also been used for whole body in vivo imaging in mice (Figure 12). Intravenous injection into mice with non-targeting FNDs showed spleen and liver accumulation over time with no observed toxicity over a 24 hour period. Ex vivo digestion analysis of the spleen and liver tissue confirmed the presence of diamond. This was the first commercial demonstration of direct fluorescence whole body imaging using FNDs without external field modulation.
Size Range: 20-40 nm
The 20-40 nm size range provides the smallest but brightest commercially available fluorescent diamond particles on the market. These sizes are suitable for intracellular imaging. The brightness of particles depends on the particle size. The larger the particle, the higher the brightness due to the larger number of color centers that can be accommodated by larger particle volumes. If small size is necessary for your work, then the 20-40nm size range offers the best compromise between brightness and size. For first time users, it Is recommended to start with larger particle sizes to determine if fluorescent nanodiamonds will provide the necessary contrast in your application. Typical size distributions measured with DLS for 20-40 nm particles are shown in Figure 7.
Fluorescence emission spectra for FNDs contain characteristic Zero Phonon Lines (ZPLs) for the NV0 and NV- defect centers located at 575 nm and 638 nm denoted by green arrows in Figure 8, respectively. ZPLs become less pronounced in smaller particles.
Nanoscale FND particles are produced by crushing of larger particles, lattice damage induced from this processing can significantly impact NV quality. Generally, the quality of NV centers tends to decrease with particle size. In addition to lattice damage, as NV centers have closer proximity to the surface, their charge state can be come less stable, and a higher amount of neutral NV0 can be expected for smaller particle sizes.
The average number of NV- emitters per particle in 20 – 40 nm products is estimated from a fit to the measured g(2) autocorrelation function using a Hanbury-Brown-Twiss setup. The crushing process also leaves a population of some particles that do not emit fluorescence.
Electron paramagnetic resonance EPR was used to measure the concentration of NV- in 40 nm and larger diamonds. For the 40 nm material, the NV- concentration was determined to be on the order of 1 ppm.
Surface Chemistry
A variety of surface functionalities are available and are summarized in the table on the following page. If you need a specific surface functionalization that is not provided, please contact info@adamasnano.com to discuss your application.
Brightness (NV Content)
For 100 nm and 140 nm particles, Adámas provides NV contents of either 1.5 ppm or 3 ppm (measured from EPR). The 1.5 ppm material provides a cheaper alternative if brightness is not essential.
