These are physical, chemical and combination methods for de-aggregation of NDs. Physical approaches are selected when NDs aggregation is arbitrate through graphitic layering, while chemical methods are base upon the task of the surface. Chemical approaches use the conjugation of different organic or inorganic molecules on the surface of NDs to control aggregation and to transmit them specific characteristics. De-aggregation of ND in suspensions is produced by milling the ceramic microbeads (ZrO2, SiO2) or ultrasonic disintegration with microbeads, dry milling with sodium chloride or coarse sucrose, high-temperature hydrogen treatment, ultrasonic treatment in borane presence . There was a data that allow the purification and oxidation in air to isolate a stable hydrosol of particles 4-5 nm in diameter by centrifugation. The first method yield the colloidal solutions of individual NDs of size 4-5 nm in diameter, but during the bead milling graphitic layer is formed around the primary particles15,70–74. Liquid oxidants are used to remove it, following of the formation of new aggregates. The second method is cheap that allows the particles and small aggregates of size 5- 20 nm to be achieved without any additional contamination. 2-4 nm size NDs are produced by high-temperature hydrogen treatment. Ultrasonic treatment in borane presence, decrease the aggregates size to ~20-nm. The possible re-aggregation of ND particles is obstructed by ultrasound-assisted treatment in the presence of sodium chloride. It is assumed that Na+ repels each other when they are attached to the surface of the individual particles. Functionalization of the surface can also oblige the reduction of the size of the aggregates. The above mentioned method with borane, lice combined method showed the greatest reduction of aggregates after functionalization. Functionalization can be used for biomedical and pharmaceutical utilization, thus allows loading of drug substances. NDs surface improvement can be attained through physical adsorption or chemical interaction. Physical adsorption of NDs has been widely accomplished using proteins, drugs, and nucleic acid. The unification of the surface groups of the NDs is an important step before the chemical functionalization of NDs, in order to conform the similar behavior conjugating from the entire surface. Oxidation or reduction of NDs is selected which is based on the terminal functional groups required on the surface34,71,72. After oxidation, different oxidative agents give variable functional group distribution on the surface. For example, nitric acid and sulphuric acid result in carboxylate (COO-) rich surface, potassium permanganate and sulphuric acid result in SO3- or O- derivatives of phenol. Reduction transform most of the functional groups into hydrogen or hydroxyl groups. Therefore, reduction of NDs through hydrogenation can create either positive surface or negative surface through hydroxylation of ND surfaces. There are three different types of surface chemistries: wet chemistry, gas phase methods or atmospheric plasma treatments. In Wet chemistry treatment, there is use of appropiate solvent systems to introduce the functional groups. Depending on attached functional on the surface, oxidized carboxylated NDs or reduced hydroxylated NDs can be used. Carboxylate functionalized NDs can form acyl chloride functionalities on reacting with thionyl chloride which can be further attached to amine-containing chemical moieties7,34,75. The treatment of NDs in gas or a vapor reactive medium is other different approach. The gas phases can include the use of hydrogen, ammonia, carbon tetrachloride or argon. On treating the NDs with ammonia yield carbonyl, amine or cyano groups on the surface of ND, while on treatment with chlorine results in the production of chloro-NDs or acylchloride functionalized NDs. Functionalized with amino acids and alkyl chains via covalent bonding or alkyl-, amino-, and amino acid-functionalized diamonds have been created by chemical modification of fluorinated NDs with alkyl lithium, ethylenediamine, or glycine ethyl ester hydrochloride, respectively. Another approach for improvig the surface is atom transfers radical polymerization, when radical initiators (benzoyl peroxides, hydroxyethyl-2-bromoisobutyrate or 2, 2, 2-trichloroethanol) are attached covalently to oxidized NDs through esterification. Chemical groups are then introduced in the system which polymerize and arrange as brush arrays on the surface15,70. This process can produce hydrophilic or hydrophobic surface depending of the nature of polymer. Radical generation mechanism is used for successful grafting of carboxylic groups onto NDs. Different functional groups on the surface of NDs give possibilities for their conjugation with different moieties without compromising the useful properties of the diamond core. Since the 1960s all traditional deaggregation techniques known in colloidal science/materials have consistently failed to yield single-digit NDs from DND aggregates. The problem was summarized by E Osawa: ‘The aqueous slurry of micron nanodiamond aggregates can be disintegrated into 60 nm aggregates but never beyond by means of powerful 400 W ultrasound’5 (Table2). Intense aggregation is mainly explained by richsurface chemistry and small size of DNDs. The existence of various functional groups on the ND surface, such as carboxyl, hydroxyl, lactone, etc, may result in the production of multiple hydrogen bonds and even covalent bonds between the adjacent DND particles, making it difficult to separate them. DND primary particles are?5 nm in diameter, thus many biological studies lead with DND aggregates of 100–200 nm can hardly reflect the performance of single-digit ND particles. The shape of nanomaterials also has a great impact on their application as a therapeutic platform . For example, the use of spherical shape graphene oxide is more advantageous for photothermal ablation of tumors as compared to needle-like shape graphene oxide. And in this respect too, nearly spherical shaped primary DND particles are beneficial as compared to the elongated, thin and sharp shapes of other nanocarbons. The problem of ND deaggregation into single-digit particles was solved only in 2005 via bead-assisted ball milling (table2) and its successor bead-assisted sonic disintegration (BASD), ( table 2). A few years later, salt-assisted dry attrition milling (, table 2) became available. Most recently, the salt-assisted ultrasonic deaggregation has been added to the arsenal, opening avenues to easily produced, inexpensive, ultra-pure single-digit ND colloids for a multitude of applications (table 2)76,77.