Bioconjugated FND Technical Info

 

 

Biofunctionalized Fluorescent Nanodiamond Technical Info

 

Bio-functionalized fluorescent nanodiamonds (FNDs) offer an infinitely photostable label for fluorescent and multiphoton microscopy for cellular and in vivo tracking and long-term observation of biological processes.  We provide nanodiamonds with nitrogen-vacancy (NV) centers and H3 ( NVN) centers (Figure 1). Protein, antibody, biologically active organic molecules, and custom functionalizations for different particle sizes are possible. 

Novel particles are also available (only at Adamas) with a combination of NV and NVN centers within a single particle, providing the capability for ratiometric calibration of the fluorescence (monitoring ratio between intensities of emission in the green and red spectral ranges). This capability helps in tracking particles even with high background fluorescence.

Biofunctionalized FNDs containing negatively charged NV centers are increasingly used as powerful sensors for surrounding spins due to uniquely coupled magneto-optical properties of the NV center. The intensity of FND fluorescence depends on the NV centers’ electronic states, which in turn depends on external electromagnetic fields.

Figure 1. Schematic structures of the negatively charged NV center (NV-) on the left, and H3 (NVN) center on the right. The NV center consists of a neighboring Nitrogen atom and a Vacancy, (absent carbon atom) and emits red/NIR light under green excitation. Its brightness is sensitive to an external magnetic field due to its electron spin (green arrow). An H3 center consists of two Nitrogen atoms surrounding a Vacancy and emits green light under blue excitation.


Spectral Characteristics of NV- and NVN Centers

Figure 2: General emission and excitation spectra for NV- and NVN centers in diamond.

NV Emission Spectra as A Function of Particle Size

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 40nm particles offer the best compromise between brightness and size. For first time users, it is recommended to start with larger particle sizes (~ 100 nm and above) to determine if fluorescent nanodiamonds (FNDs) will provide the necessary contrast in your application.

Emission spectra for FNDs contain characteristic Zero Phonon Lines ZPLs for the NV center with neutral charge (NV0) and negatively charged NV- centers located at 575 nm and 638 nm denoted by green arrows in Figure 3, respectively. As the particle size decreases, the ZPL signature tends to decrease due to the reduced number of emitters per particle, which decreases approximately volumetrically with particle volume. Because particles are produced by milling/crushing larger particles, induced lattice damage can significantly impact the quality of NV centers and the resultant spectroscopic quality.

Figure 3  : Emission spectra of 20, 30, and 40 nm FND (left) and 100 nm and 140 nm (right) particle suspensions in deionized water at approximately 1 mg/mL  0.1% w/v  concentration.  Excitation by 45 mW (15 mW for larger particles) 532 nm CW laser  Coherent – Sapphire.  Spectra collected with Ocean Optics HR2000 USB spectrometer with 500msec integration time.  Black arrows denote water Raman peak at ~650 nm.  ZPL indicated by green arrows.

Electron paramagnetic resonance EPR studies were performed to evaluate the concentration of NV- centers in the particles. For the 40 nm material, the NV- concentration was determined to be on the order of 1 ppm and ~ 3 ppm  for 100-140 nm particles, which equates to approximately ~300 centers per 100 nm particle and ~800 centers per 140 nm particle. In general, a particle volume ratio related dependence on the number of centers per particle is observed with respect to size. 40 nm and 100 nm size products are available, and custom orders for other sizes are possible if you contact us directly.

Fluorescence Microscopy with FND

The brightness of aggregates of 20nm particles is high enough to be detected within cells after internalization in a confocal setup (Figure 4).

Figure 4 : In vitro imaging of 20nm-Hi in MDA-MB-231 Breast Cancer Cells with 488 nm laser excitation following 48 h incubation at 50 μg/mL loading  650-720 nm detection window . N. Prabhakar, Ã…bo Akademi, Finland.

Products available for intracellular labelling: NDNV100nmOPGantiRabbit, NDNV100nmOPGantiMouse, NDNV40nmHibiotin, NDNV40nmHiSA, NDNV100nmHibiotin, NDNV100nmHiSA. Custom functionalization is also available.

In Vivo Imaging with FND

The large size range high brightness particles have been used extensively in in vivo and in vitro studies. The particles have been successfully conjugated with human vascular endothelial growth factor (VEGf) and tumor targeting demonstrated.[1]

170 nm particles have also been used for whole body in vivo imaging in mice.[2] Intravenous injection into mice with non-targeting FNDs showed spleen and liver accumulation over time Figure 5). No observed toxicity was detected over a 24h period.  There is more information on the website . Ex vivo digestion analysis of the spleen and liver tissue confirmed the presence of diamond (Figure 6).

Figure 5:  Top  Whole body imaging on IVIS system of live nude mice with two administered, intravenous doses of 170 nm FND particles. Spleen and kidney accumulation observed over 5 hours. Courtesy of G. Palmer, Duke University School of Medicine. 
Figure 6. (a) Whole-body images of Balb/c mice taken with an IVIS Kinetic after administration of 0, 2, or 4 mg of fluorescent nanodiamond (540 nm excitation and Cy5 emission). Digestion of the spleen in Piranha show recover diamond in a dose-related manner (b), as confirmed by fluorescence (c). Emission is not harmed by solvents or strong acids. Reference [3].

Recommended products for in vivo imaging: NDNV140nmHiPG, with Polyglycerol coating (PG), providing colloidal stability in biological media. Larger sizes with higher brightness are also available as custom orders.

References for future reading:

[1] Torelli MD, Rickard AG, Backer MV, Filonov DS, Nunn NA, Kinev AV, Backer JM, Palmer GM, Shenderova OA. “Targeting Fluorescent Nanodiamonds to Vascular Endothelial Growth Factor Receptors in Tumor”, Bioconjug Chem. 2019;30(3):604-13. Epub 2019/01/12. doi:10.1021/acs.bioconjchem.8b00803. PubMed PMID: 30633508.

[2] M.D. Torelli, N.A. Nunn, and O.A. Shenderova. ” A Perspective on Fluorescent Nanodiamond Bioimaging ” Small 2019, 1902151, DOI: 10.1002/smll.201902151

[3] M.Torelli, N.Nunn, O.Shenderova, et al. High Temperature Treatment of Diamond Particles Toward Enhancement of Their Quantum Properties, Frontiers in Physics 2020, 8:205.

[4] Nicholas Nunn, Neeraj Prabhakar, Philipp Reineck et al. Brilliant blue, green, yellow, and red fluorescent diamond particles: synthesis, characterization, and multiplex imaging demonstrations, Nanoscale Issue 24, 2019. https://doi.org/10.1039/C9NR02593F

ACKNOWLEDGEMENT: Characterization work performed for these products was supported in whole or in part by the National Institutes of Health NIH National Heart, Lung, and Blood Institute NHLBI under contract No. HHSN268201500010C.