Surface Functionalized Fluorescent Nanodiamonds

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Nanodiamonds (NDs) are a family of nanomaterials that shares a core constituted by a common sp3 carbon structure, but differs in size, shape, and surface chemistry. NDs have similar mechanical and optical properties to bulk diamond. In addition, NDs are characterized by high specific surface area, inert carbon core with various impurities and defects, and tunable surface chemistry. NDs also show good tolerability, non-toxicity, and biocompatibility in in vivo studies. Together with the features of the core, NDs are endowed with a versatile surface chemistry, which allows to expand their use in different fields. Both macroscopic (such as hydropathy and colloidal stability) and microscopic (such as reactivity) properties of NDs can be tuned by modifying their surfaces, making the prepared NDs suitable for the desired applications.

Surface Modification of Fluorescent Nanodiamonds

A critical step in therapeutic and diagnostic applications of nanoparticles in biomedicine is to conjugate bioactive agents to the particle surfaces. NDs are clearly advantageous over other non-carbon based nanomaterials in terms of easiness and simplicity of surface modification. The surfaces of NDs provide a versatile platform for conjugation of biomolecules through chemical modifications.

Pristine NDs have various surface moieties, as well as non-diamond carbon structures and impurities generated during synthesis. Non-diamond carbon structures such as carbon dots can affect the fluorescence properties of NDs. Therefore, elimination of impurities and homogenization of surface groups before further reactions on the NDs surface are required to improve purity, control subsequent reactions, and maximize the contribution of nitrogen-vacancy (NV) centers to the optical properties of the fluorescent NDs (FNDs). Carboxylation, hydroxylation, hydrogenation, halogenation, amination, and graphitization are used to establish homogenization procedures for NDs.

Functionalized Red and Green Fluorescent Nanodiamonds

Amerigo Scientific offers FNDs containing NV centers with red to near-infrared fluorescence. The red FNDs with various surface functional groups are available. The negatively charged nitrogen vacancy (NV-) centers have an electron spin with unique magneto-optical properties, and the spin state can be optically detected by optical detection magnetic resonance (ODMR). Since the spin state is highly sensitive to the surrounding environment, environmental changes can be determined by detecting changes in NV fluorescence. Based on these properties, ND-based quantum sensors of magnetic fields, electric currents, temperatures, free radicals, and other phenomena can be developed.

Amerigo Scientific also offers FNDs containing H3 centers (also called NVN centers) with green fluorescence. The H3 center consists of two nitrogen atoms surrounding a vacancy and emits green fluorescence under blue excitation. Similar to the NV- center, the fluorescence emitted by the H3 center is exceptionally stable without photobleaching or flickering.

NV- Center

H3 Center

Fluorescence Characteristics

Fluorescence Color	Peak Excitation	Peak Emission	Multiphoton Excitation	Quantum Yield	Fluorescence Lifetime	Brightness (1 dye molecule vs. single 120 nm particle)
Red (NV centers)	570 nm	680 nm	700 nm, 810 nm	0.7	13 ns	~70x brighter vs A647
Green (H3 centers)	480 nm	520 nm	775 nm	–	20 ns	~90x vs FITC

Carboxylated Red and Green Fluorescent Nanodiamonds

In the process of NDs production, sp2 carbon structures and metal impurities contaminate the surface of NDs. The oxidation treatment is carried out to remove surface impurities, resulting in the formation of carboxyl (COOH) groups on the ND surface. The generated COOH groups on the ND surface provide reliable functionalization strategies through the formation of amide bonds via covalent conjugation of amines. Among various surface functionalized FNDs, carboxylated FNDs can serve as a general starting point for subsequent modifications required for further applications.

Product Name	Size	Particle Size	NV Content
Red Fluorescent Nanodiamonds
Carboxylated 20 nm Red FND in DI water, < 1ppm NV	10 mL (1 mg/mL)	20-25 nm	< 1 ppm NV
Carboxylated 30 nm Red FND in DI water, ~1.5ppm NV	10 mL (1 mg/mL)	30-35 nm	~1.5 ppm NV
Carboxylated 40 nm Red FND in DI water, ~1.5 ppm NV	10 mL (1 mg/mL)	40-45 nm	~1.5 ppm NV
Carboxylated 50 nm Red FND - Fluorescent Nanodiamond in DI water, ~2ppm NV	10 mL (1 mg/mL)	50-55 nm	~2 ppm NV
Carboxylated 60 nm Red FND in DI water, <1 ppm NV	10 mL (1 mg/mL)	60-65 nm	<1 ppm NV
Carboxylated 70 nm Red FND in DI water, ~2ppm NV	10 mL (1 mg/mL)	70-75 nm	~2 ppm NV
Carboxylated 70 nm Red FND in DI water, ~3ppm NV	10 mL (1 mg/mL)	70-75 nm	~3 ppm NV
Carboxylated 90 nm Red FND in DI water, ~3ppm NV	10 mL (1 mg/mL)	90-100 nm	~3 ppm NV
Carboxylated 100 nm Red FND in DI Water, ~3ppm NV	10 mL (1 mg/mL)	100-110 nm	~3 ppm NV
Carboxylated 100 nm Red FND in DI water, ~3ppm NV	10 mL (1 mg/mL)	100 nm	~3 ppm NV
Carboxylated 100 nm Red FND in DI water, ~1.5 ppm NV, Medium Brightness	10 mL (1 mg/mL)	100 nm	~1.5 ppm NV
Carboxylated 120 nm Red FND in DI water, ~3 ppm NV, High Brightness	10 mL (1 mg/mL)	120-130nm	~3 ppm NV
Carboxylated 140 nm Red FND in DI water, ~3ppm NV	10 mL (1 mg/mL)	140-150 nm	~3 ppm NV
Green Fluorescent Nanodiamonds
Carboxylated 120 nm Green FND (NVN centers) from Synthetic Type Ib diamond in DI water	1 mL (1 mg/mL)	120 nm	-
Carboxylated 70 nm Green FND in DI water	10 mL (1 mg/mL)	70-80 nm	-
Carboxylated 90 nm Green FND in DI water	10 mL (1 mg/mL)	90 nm	-
Carboxylated 120 nm Green FND in DI water	10 mL (1 mg/mL)	120 nm	-
Carboxylated 140 nm Green FND in DI water	10 mL (1 mg/mL)	140 nm	-

Hydroxylated Red Fluorescent Nanodiamonds

Because of the wide range of subsequent reactions of hydroxyl (OH) groups, the homogenization of the ND surface with dense OH groups is one of the most common homogenization schemes.

Product Name	Size	Particle Size	NV Content
40 nm Hydroxylated Red FND Suspension, zeta potential +25mV, ~1 ppm NV	2 mL	40-50 nm	~1 ppm NV
60 nm Hydroxylated Red FND Suspension, zeta potential +25mV, ~1.5 ppm NV	2 mL	60-70 nm	~1.5 ppm NV
100 nm Hydroxylated Red FND Suspension, zeta potential +25mV, ~2 ppm NV	2 mL	100 nm	~2 ppm NV
100 nm Hydroxylated Red FND Suspension, zeta potential +18mV, ~3 ppm NV	2 mL	100 nm	~3 ppm NV

Hydrogenated Red Fluorescent Nanodiamonds

The hydrogenated ND surface provides a very convenient starting point for functionalization because it offers a homogeneous surface chemistry for efficient chemistry, specific grafting routes and robust CAC bonds at the interface that are resistant to oxidation or hydrolysis.

Product Name	Size	Particle Size	NV Content
50nm Hydrogenated Red FND Suspension	2 mL (1 mg/mL)	50 nm	-
100 nm Hydrogenated Red FND Suspension, +30mV, 2-3 ppm NV	2 mL	100 nm	2-3 ppm NV
100 nm Hydrogenated Red FND Powder, 2-3ppm NV	10 mg	100 nm	2-3 ppm NV

Other Functionalized Red Fluorescent Nanodiamonds

Amerigo Scientific also offers FND kits and FNDs with Octadecane or Polyglycerol. The Fluorescent Nanodiamond Kit contains 3 vials of 1 mL FND sample with 3 different particle sizes. Please contact us with the specific sizes you are interested in when making the purchase (available sizes of carboxylated red FNDs: 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 100 nm, 140 nm; available sizes of carboxylated green FNDs: 70 nm, 90 nm, 120 nm, 140 nm).

Product Name	Size	Particle Size	NV Content
Fluorescent Nanodiamond Kit	3 bottles, 1 mL (1 mg/mL)	10 nm-140 nm	-
100 nm Red FND Powder with Octadecane	2 mg	100 nm	~3 ppm NV
140 nm Red FND with Polyglycerol	2 mL (1 mg/mL)	140 nm	~3 ppm NV

Applications

The level of fluorescence and the population of fluorescent diamond particles in the sub-15nm range are generally very low. These particles are not suitable for direct cellular imaging, and require users equipped with highly advanced optical setups to use effectively. The brightness of particles depends on the particle size. If small particle size is necessary, FNDs in the size range of 20 to 40nm are the best choice for both brightness and particle size. These FND particles are suitable for intracellular imaging and single molecule tracking. As shown below, the brightness of aggregates of 20nm FNDs is high enough to be detected within cells after internalization in a confocal setup.

Figure. In vitro imaging of 20 nm carboxylated red FNDs 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, Åbo Akademi, Finland.)

Functionalized Multicolor Fluorescent Nanodiamonds

Amerigo Scientific offers novel nanodiamonds with a combination of a NV centers with red to near-infrared fluorescence and H3 centers with green fluorescence within the particle. These FND particles can be used as green fluorescent tags due to the presence of H3 centers, while also being able to sense the surrounding environment due to the presence of NV centers. These dual-color FND particles open new perspectives for multiplexing sensing.

Product Name	Size	Particle Size
Carboxylated 700 nm Green/Red FND (NVN/NV centers) from Synthetic Type Ib diamond in DI water	2 mL (1 mg/mL)	700 nm
Carboxylated 100-120 nm Yellow FND in DI Water (Combination of NV/NVN centers)	1 mL (1 mg/mL)	120 nm

Fluorescence Characteristics

Fluorescence Color	Peak Excitation	Peak Emission	Multiphoton Excitation	Quantum Yield	Fluorescence Lifetime	Brightness (1 dye molecule vs. single 120 nm particle)
Red/Green (NV/NVN) provided by Amerigo	480, 570 nm	520, 575, 680 nm	700-900 nm	–	–	–

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