Bioconjugated Fluorescent Nanodiamonds

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Nanodiamonds (NDs) have considerable potential as bioimaging probes due to their fluorescence properties, generally associated to ND intrinsic impurities or defects. Nitrogen-based defects produce the broadest array of photoluminescent centers, producing emissions extending through the entire visible spectrum into the near-infrared (NIR). The nitrogen-vacancy (NV) center is a point defect in diamond consisting of a carbon vacancy site to one substitutional nitrogen atom at its adjacent site. There are two charge states of NV: one is neutral NV and the other is NV-. Other major nitrogen-related centers include two nitrogen atoms adjacent to a vacancy (H3 center or NVN center) that produce green fluorescence, and three nitrogen atoms surrounding a vacancy (N3 center) that produce blue fluorescence. Fluorescent nanodiamonds (FNDs) are considered as the best alternative probes to meet the ideal fluorescent labeling criteria due to their unique optical and chemical properties. Covalent and non-covalent attachment of emitting fluorophores improves imaging quality.

Figure 1. Schematic structures of the negatively charged NV center (NV-) on the left, and H3 (NVN) center on the right.

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

Bioconjugated Fluorescent Nanodiamonds

Amerigo scientific offers nanodiamonds with NV centers with red-to-near-infrared fluorescence, NVN centers with green fluorescence, and novel particles with a combination of NV and NVN centers within a single particle with visual yellow fluorescence. The novel particles provide the capability for ratiometric calibration of the fluorescence (monitoring ratio between intensities of emission in the green and red spectral ranges), which helps in tracking particles even in the presence of high background fluorescence.

Biofunctionalized FNDs are photostable labels for fluorescent and multiphoton microscopy, which can be used for cellular tracking and long-term observation of biological processes in the same fields of view in live cell-imaging and fixed cells. 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 fluorescence intensity of FND depends on the NV centers electronic states, which in turn depends on external electromagnetic fields. Fluorescence from NV centers can be magnetically modulated, providing a solution to eliminate the fluorescent background by image subtraction and significantly improve the signal-to-noise ratio. This property could be very useful in future biosensing applications, such as lateral flow assays (LFA) in clinical diagnostics. Relevant applications also include super-resolution microscopy, multiphoton microscopy, time-gated microscopy, and correlative microscopy.

Product Range

Categories	Product Name	NV Content	Conjugate	Particle Size	Size
Red Functionalized Fluorescent Nanodiamonds	100 nm Fluorescent Nanodiamond - Goat AntiRabbit	~3 ppm NV	Goat Anti-rabbit Antibody	100 nm	500 ug
	100 nm Fluorescent Nanodiamond - Goat AntiMouse	~3 ppm NV	Goat Anti-mouse Antibody	100 nm	500 ug
	40 nm Red Fluorescent Nanodiamond with Biotin	~2 ppm NV	Biotin	40-45 nm	2 mg
	40 nm Red Fluorescent Nanodiamond with Streptavidin	~2 ppm NV	Streptavidin	40-45 nm	2 mg
	100 nm Red Fluorescent Nanodiamond with Biotin	~3 ppm NV	Biotin	100 nm	2 mg; 10 mg
	100 nm Red Fluorescent Nanodiamond with Streptavidin	~3 ppm NV	Streptavidin	100 nm	2 mg
	100 nm Red Fluorescent Nanodiamond aminated with ethylenediamine	~3ppm NV	Ethylenediamine	100 nm	2 mL
	140 nm Red Fluorescent Nanodiamond with Polyglycerol	~3ppm NV	Polyglycerol	140 nm	2 mL
Green Functionalized Fluorescent Nanodiamonds	120 nm Green Fluorescent Nanodiamond with Biotin	Trace amounts of NV centers	Biotin	120 nm	2 mg
Green Functionalized Fluorescent Nanodiamonds	120 nm Green Fluorescent Nanodiamond with Biotin	Spectrally pure, depleted from NV centers	Biotin	120 nm	2 mg

The brightness of ND particle depends on its size. The larger the particle and the brighter it is, because a larger particle volume can accommodate more color centers. If small particles are necessary for research, 40nm particles are the best choice for both brightness and size. For first-time users, it is recommended to start with larger particle sizes (about 100 nm and above) to determine if FNDs provide the necessary contrast in the application.

Fluorescence Characteristics

Fluorescence Color	Peak excitation (nm)	Peak Emission (nm)	Multiphoton Excitation (nm)	Quantum Yield (φ）	FL lifetime (τ)	Brightness (1 dye molecule vs. single 120 nm particle)
Red (NV)	570	680	700, 810	0.7	13 ns	~70x brighter vs AF647
Green (NVN)	480	520	75	-	20 ns	~90x vs FITC
Red/Green (NV/NVN) supplied by Amerigo	480, 570	520, 575, 680	700-900	-	-	-

Applications

• Fluorescence Microscopy with Bioconjugated Fluorescent Nanodiamonds

FNDs are emerging as efficient and safe candidates for cellular imaging, due to their bright fluorescence, high photostability with sufficiently long lifetime, and excellent biocompatibility.

Figure 4. Confocal imaging of cells labeled separately with green FNDs and red FNDs under 488 nm excitation and 543 nm excitation. (Small (Weinheim an der Bergstrasse, Germany) vol. 15,48 (2019): e1902151.)

Products available for intracellular labelling:

Product Name	Particle Size	Size
100 nm Fluorescent Nanodiamond - Goat AntiRabbit	100 nm	500 ug
100 nm Fluorescent Nanodiamond - Goat AntiMouse	100 nm	500 ug
40 nm Red Fluorescent Nanodiamond with Biotin	40-45 nm	2 mg
40 nm Red Fluorescent Nanodiamond with Streptavidin	40-45 nm	2 mg
100 nm Red Fluorescent Nanodiamond with Biotin	100 nm	2 mg; 10 mg
100 nm Red Fluorescent Nanodiamond with Streptavidin	100 nm	2 mg

• In Vivo Imaging with Bioconjugated Fluorescent Nanodiamonds

For in vivo work, FNDs have been recognized as a safe and biocompatible material in many studies. 170 nm FND particles were used for whole body in vivo imaging in mice. 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. Longer wavelengths produced slightly better results despite the absence of excitation maxima, probably due to less autofluorescence background and increased tissue penetration. Particles were recovered by tissue digestion in piranha acid solution (Figure 5c) and confirmed by quantitative fluorescence spectroscopy (Figure 5d). The emission is not affected by solvents or strong acids.

Figure 5. Whole-body IVIS imaging of administered 170 nm FND particles with acid digestion recovery. Images of three mice, administered with nanodiamond in 5% dextrose buffer at a dose either of 0, 2, or 4 mg per mouse, imaged with a) 605 nm excitation or b) 640 nm excitation. c) Spleens after piranha acid digestion, showing contrast of accumulated particles. d) Bulk spectra of samples in three indicating dose-dependent recovery and validation of FND presence. (Small (Weinheim an der Bergstrasse, Germany) vol. 15,48 (2019): e1902151.)

Recommended products for in vivo imaging:

Product Name	Conjugate	Particle Size	Size
140 nm Red Fluorescent Nanodiamond with Polyglycerol	Polyglycerol	140 nm	2 mL

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