A Perspective on Fluorescent Nanodiamond Bioimaging.

The field of fluorescent nanodiamonds (FNDs) has advanced greatly over the past few years. Though historically limited primarily to red fluorescence, the wavelengths available for nanodiamonds have increased due to continuous technical advancement. This Review summarizes the strides made in the synthesis, functionalization, and application of FNDs to bioimaging. Highlights range from super-resolution microscopy, through cellular and whole animal imaging, up to constantly emerging fields including sensing and hyperpolarized magnetic resonance imaging.

Targeting Fluorescent Nanodiamonds to Vascular Endothelial Growth Factor Receptors in Tumor.

The increased expression of vascular endothelial growth factor (VEGF) and its receptors is associated with angiogenesis in a growing tumor, presenting potential targets for tumor-selective imaging by way of targeted tracers. Though fluorescent tracers are used for targeted in vivo imaging, the lack of photostability and biocompatibility of many current fluorophores hinder their use in several applications involving long-term, continuous imaging. To address these problems, fluorescent nanodiamonds (FNDs), which exhibit infinite photostability and excellent biocompatibility, were explored as fluorophores in tracers for targeting VEGF receptors in growing tumors. To explore FND utility for imaging tumor VEGF receptors, we used click-chemistry to conjugate multiple copies of an engineered single-chain version of VEGF site-specifically derivatized with trans-cyclooctene (scVEGF-TCO) to 140 nm FND. The resulting targeting conjugates, FND-scVEGF, were then tested for functional activity of the scVEGF moieties through biochemical and tissue culture experiments and for selective tumor uptake in Balb/c mice with induced 4T1 carcinoma. We found that FND-scVEGF conjugates retain high affinity to VEGF receptors in cell culture experiments and observed preferential accumulation of FND-scVEGF in tumors relative to untargeted FND. Microspectroscopy provided unambiguous determination of FND within tissue by way of the unique spectral shape of nitrogen-vacancy induced fluorescence. These results validate and invite the use of targeted FND for diagnostic imaging and encourage further optimization of FND for fluorescence brightness.

Optically Detected Magnetic Resonance for Selective Imaging of Diamond Nanoparticles.

While there is great interest in understanding the fate and transport of nanomaterials in the environment and in biological systems, the detection of nanomaterials in complex matrices by fluorescence methods is complicated by photodegradation, blinking, and the presence of natural organic material and other fluorescent background signals that hamper detection of fluorescent nanomaterials of interest. Optically detected magnetic resonance (ODMR) of nitrogen-vacancy (NV) centers in diamond nanoparticles provides a pathway toward background-free fluorescence measurements, as the application of a resonant microwave field can selectively modulate the intensity from NV centers in nanodiamonds of various diameters in complex materials systems using on-resonance and off-resonance microwave fields. This work represents the first investigation showing how nanoparticle diameter impacts the NV center lifetime and thereby directly impacts the accessible contrast and signal-to-noise ratio when using ODMR to achieve background-free imaging of NV-nanodiamonds in the presence of interfering fluorophores. These results provide new insights that will guide the choice of optimum nanoparticle size and methodology for background-free imaging and sensing applications, while also providing a model system to explore the fate and transport of nanomaterials in the environment.

A method for optical imaging and monitoring of the excretion of fluorescent nanocomposites from the body using artificial neural networks.

In this study, a new approach to the implementation of optical imaging of fluorescent nanoparticles in a biological medium using artificial neural networks is proposed. The studies were carried out using new synthesized nanocomposites - nanometer graphene oxides, covered by the poly(ethylene imine)-poly(ethylene glycol) copolymer and by the folic acid. We present an example of a successful solution of the problem of monitoring the removal of nanocomposites based on nGO and their components with urine using fluorescent spectroscopy and artificial neural networks. However, the proposed method is applicable for optical imaging of any fluorescent nanoparticles used as theranostic agents in biological tissue.

Theory and modelling of diamond fracture from an atomic perspective.

Discussed in this paper are several theoretical and computational approaches that have been used to better understand the fracture of both single-crystal and polycrystalline diamond at the atomic level. The studies, which include first principles calculations, analytic models and molecular simulations, have been chosen to illustrate the different ways in which this problem has been approached, the conclusions and their reliability that have been reached by these methods, and how these theory and modelling methods can be effectively used together.

Nanodiamond-Based Composite Structures for Biomedical Imaging and Drug Delivery.

Nanodiamond particles are widely recognized candidates for biomedical applications due to their excellent biocompatibility, bright photoluminescence based on color centers and outstanding photostability. Recently, more complex architectures with a nanodiamond core and an external shell or nanostructure which provides synergistic benefits have been developed, and their feasibility for biomedical applications has been demonstrated. This review is aimed at summarizing recent achievements in the fabrication and functional demonstrations of nanodiamond-based composite structures, along with critical considerations that should be taken into account in the design of such structures from a biomedical point of view. A particular focus of the review is core/shell structures of nanodiamond surrounded by porous silica shells, which demonstrate a remarkable increase in drug loading efficiency; as well as nanodiamonds decorated with carbon dots, which have excellent potential as bioimaging probes. Other combinations are also considered, relying on the discussed inherent properties of the inorganic materials being integrated in a way to advance inorganic nanomedicine in the quest for better health-related nanotechnology.

Clot Imaging Using Photostable Nanodiamond.

While thrombosis is the leading cause of morbidity and mortality in the United States, an understanding of its triggers, progression, and response to anticoagulant therapy is lacking. Intravital fluorescence microscopy has advanced the study of thrombus formation by providing targeted, multi-color contrast. However, photodegradation of fluorophores limits the application in longitudinal studies (e.g., clot progression and/or dissolution). Fluorescent nanodiamond (FND) is a fluorophore which utilizes intrinsic fluorescence of chromogenic centers within and protected by the diamond crystalline lattice. Recent developments in diamond processing have allowed for the controlled production of nanodiamonds emitting in green or red. Here, the use of FND to label blood clots and/or clot lysis is demonstrated and compared to commonly used organic fluorophores. Model ex vivo clots were formed with incorporated labeled fibrinogen to allow imaging. FND was shown to match the morphology of organic fluorophore labels absent of photobleaching over time. The addition of tissue plasminogen activator (tPa) allowed visualization of the clot lysis stage, which is vital to studies of both DVT and pulmonary embolism resolution.

Optical and electronic spin properties of fluorescent micro- and nanodiamonds upon prolonged ultrahigh-temperature annealing.

High-temperature annealing is a promising but still mainly unexplored method for enhancing spin properties of negatively charged nitrogen-vacancy (NV) centers in diamond particles. After high-energy irradiation, the formation of NV centers in diamond particles is typically accomplished via annealing at temperatures in the range of 800-900 °C for 1-2 h to promote vacancy diffusion. Here, we investigate the effects of conventional annealing (900 °C for 2 h) against annealing at a much higher temperature of 1600 °C for the same annealing duration for particles ranging in size from 100 nm to 15 µm using electron paramagnetic resonance and optical characterization. At this high temperature, the vacancy-assisted diffusion of nitrogen can occur. Previously, the annealing of diamond particles at this temperature was performed over short time scales because of concerns of particle graphitization. Our results demonstrate that particles that survive this prolonged 1600 °C annealing show increased NV T1 and T2 electron spin relaxation times in 1 and 15 µm particles, due to the removal of fast relaxing spins. Additionally, this high-temperature annealing also boosts magnetically induced fluorescence contrast of NV centers for particle sizes ranging from 100 nm to 15 µm. At the same time, the content of NV centers is decreased fewfold and reaches a level of <0.5 ppm. The results provide guidance for future studies and the optimization of high-temperature annealing of fluorescent diamond particles for applications relying on the spin properties of NV centers in the host crystals.

High-performance size exclusion chromatography with online fluorescence and multi-wavelength absorbance detection for isolation of high-purity carbon dots fractions, free of non-fluorescent material.

The carbon dots (CDs) from natural nanographite oxide mixture (NGO-MIX) and from its fraction NGO (3.5-10K) recovered after ultrafiltration and dialysis were analyzed by 3D-excitation/emission matrix and high-performance size exclusion chromatography (HPSEC) combined with online fluorescence and absorbance detections. HPSEC chromatograms obtained simultaneously with absorption within the wavelength range 200-500 nm and fluorescence detection at λexc/λem = 270/450 nm/nm showed that NGO-MIX sample is not homogeneous and consist of well resolved CDs fractions with different sizes, absorption spectra and distinct fluorescence and non-fluorescence properties. Despite the twice higher fluorescence intensity of fraction NGO (3.5-10K) compared to the NGO-MIX, some impurity of non-fluorescent components was detected by HPSEC. The absorbance spectra of chromatographic peaks, extracted from the data of multi-wavelength absorbance detector, demonstrated different combinations of absorbance maxima. It means that different chromatographic peaks correspond to sized and chemically different CDs fractions. This study demonstrated for the first time the possibility of separating oxidized nanographite into homogeneous free from non-fluorescent material CDs fractions with their simultaneous spectroscopic characterization.

Absolute luminescence quantum yield for nanosized carbon particles in water as a function of excitation wavelength.

The absolute luminescence quantum yield Q as a function of excitation wavelength λex in a wide spectral range 270-470 nm was measured for the first time for the group of carbon nanoparticles dispersed in water: carbon dots (CD), detonation nanodiamonds (DND), as well as detonation nanodiamonds decorated with carbon dots (CD-DND). The luminescence quantum yield for DND increased after functionalization; the CD-decorated DND demonstrated significantly higher Q values in the UV region of excitation. We found that the quantum yield for CD luminescence is 4-8 times higher than that for CD-DND luminescence, and 20 times higher than that for DND luminescence. Roughly three spectral regions can be distinguished within the Q(λex): below 330 nm, 330-390 nm and 390-470 nm. Conclusions are drawn about the number of chromophores of the studied nanoparticles and transfer of photoexcitation energy in the systems under consideration.

Review Article: Synthesis, properties, and applications of fluorescent diamond particles.

Diamond particles containing color centers-fluorescent crystallographic defects embedded within the diamond lattice-outperform other classes of fluorophores by providing a combination of unmatched photostability, intriguing coupled magneto-optical properties, intrinsic biocompatibility, and outstanding mechanical and chemical robustness. This exceptional combination of properties positions fluorescent diamond particles as unique fluorophores with emerging applications in a variety of fields, including bioimaging, ultrasensitive metrology at the nanoscale, fluorescent tags in industrial applications, and even potentially as magnetic resonance imaging contrast agents. However, production of fluorescent nanodiamond (FND) is nontrivial, since it requires irradiation with high-energy particles to displace carbon atoms and create vacancies-a primary constituent in the majority color centers. In this review, centrally focused on material developments, major steps of FND production are discussed with emphasis on current challenges in the field and possible solutions. The authors demonstrate how the combination of fluorescent spectroscopy and electron paramagnetic resonance provides valuable insight into the types of radiation-induced defects formed and their evolution upon thermal annealing, thereby guiding FND performance optimization. A recent breakthrough process allowing for production of fluorescent diamond particles with vibrant blue, green, and red fluorescence is also discussed. Finally, the authors conclude with demonstrations of a few FND applications in the life science arena and in industry.

Nanodiamonds and surfactants in water: Hydrophilic and hydrophobic interactions.

HYPOTHESIS: Nanodiamonds, one of the most promising nanomaterials for the use in biomedicine, placed in the organisms are bound to interact with various amphiphilic lipids and their micelles. However, while the influence of surfactants, the close relative of lipids, on the properties of colloidal nanodiamonds is well studied, the influence of nanodiamonds on the properties of surfactants, lipids, and, therefore, on the structure of surrounding tissues, is poorly understood. EXPERIMENT: In this work, the influence of interactions of hydrophobic and hydrophilic nanodiamonds with ionic surfactant sodium octanoate in water on hydrogen bonds, the properties of the surfactant and micelle formation were studied using Raman spectroscopy and dynamic light scattering technique. FINDINGS: Nanodiamonds are found to actively influence the bulk properties only of the premicellar surfactant solutions: the strength of hydrogen bonds, ordering and conformation of hydrocarbon tails, the critical micelle concentration. This influence is deduced to be dependent on two mechanisms not unique to nanodiamonds: (1) the induction of micro-flows around nanoparticles undergoing Brownian motions, and (2) the creation of the chaotic state in the surfactant solutions if two or more incompatible types of interactions between nanoparticles' surfaces and surfactants are similarly favorable, e.g. hydrophobic interaction and Coulomb attraction.

Visible to near-IR fluorescence from single-digit detonation nanodiamonds: excitation wavelength and pH dependence.

Detonation nanodiamonds are of vital significance to many areas of science and technology. However, their fluorescence properties have rarely been explored for applications and remain poorly understood. We demonstrate significant fluorescence from the visible to near-infrared spectral regions from deaggregated, single-digit detonation nanodiamonds dispersed in water produced via post-synthesis oxidation. The excitation wavelength dependence of this fluorescence is analyzed in the spectral region from 400 nm to 700 nm as well as the particles' absorption characteristics. We report a strong pH dependence of the fluorescence and compare our results to the pH dependent fluorescence of aromatic hydrocarbons. Our results significantly contribute to the current understanding of the fluorescence of carbon-based nanomaterials in general and detonation nanodiamonds in particular.

Science and engineering of nanodiamond particle surfaces for biological applications (Review).

Diamond has outstanding bulk properties such as super hardness, chemical inertness, biocompatibility, luminescence, to name just a few. In the nanoworld, in order to exploit these outstanding bulk properties, the surfaces of nanodiamond (ND) particles must be accordingly engineered for specific applications. Modification of functional groups on the ND's surface and the corresponding electrostatic properties determine their colloidal stability in solvents, formation of photonic crystals, controlled adsorption and release of cargo molecules, conjugation with biomolecules and polymers, and cellular uptake. The optical activity of the luminescent color centers in NDs depends on their proximity to the ND's surface and surface termination. In order to engineer the ND surface, a fundamental understanding of the specific structural features and sp(3)-sp(2) phase transformations on the surface of ND particles is required. In the case of ND particles produced by detonation of carbon containing explosives (detonation ND), it should also be taken into account that its structure depends on the synthesis parameters and subsequent processing. Thus, for development of a strategy of surface modification of detonation ND, it is imperative to know details of its production. In this review, the authors discuss ND particles structure, strategies for surface modification, electrokinetic properties of NDs in suspensions, and conclude with a brief overview of the relevant bioapplications.

Functionalization of graphene oxide nanostructures improves photoluminescence and facilitates their use as optical probes in preclinical imaging.

Recently reported photoluminescent nanographene oxides (nGOs), i.e. nanographene oxidised with a sulfuric/nitric acid mixture (SNOx method), have tuneable photoluminescence and are scalable, simple and fast to produce optical probes. This material belongs to the vast class of photoluminescent carbon nanostructures, including carbon dots, nanodiamonds (NDs), graphene quantum dots (GQDs), all of which demonstrate a variety of properties that are attractive for biomedical imaging such as low toxicity and stable photoluminescence. In this study, the nGOs were organically surface-modified with poly(ethylene glycol)-poly(ethylene imine) (PEG-PEI) copolymers tagged with folic acid as the affinity ligand for cancer cells expressing folate receptors. The functionalization enhanced both the cellular uptake and quantum efficiency of the photoluminescence as compared to non-modified nGOs. The nGOs exhibited an excitation dependent photoluminescence that facilitated their detection with a wide range of microscope configurations. The functionalized nGOs were non-toxic, they were retained in the stained cell population over a period of 8 days and they were distributed equally between daughter cells. We have evaluated their applicability in in vitro and in vivo (chicken embryo CAM) models to visualize and track migratory cancer cells. The good biocompatibility and easy detection of the functionalized nGOs suggest that they could address the limitations faced with quantum dots and organic fluorophores in long-term in vivo biomedical imaging.

Optical imaging of fluorescent carbon biomarkers using artificial neural networks.

The principle possibility of extraction of fluorescence of nanoparticles in the presence of background autofluorescence of a biological environment using neural network algorithms is demonstrated. It is shown that the methods used allow detection of carbon nanoparticles fluorescence against the background of the autofluorescence of egg white with a sufficiently low concentration detection threshold (not more than 2 µg/ml for carbon dots 3 µg/ml and for nanodiamonds). It was also shown that the use of the input data compression can further improve the accuracy of solving the inverse problem by 1.5 times.

Core-shell designs of photoluminescent nanodiamonds with porous silica coatings for bioimaging and drug delivery I: fabrication.

A multifunctional core-shell nanocomposite platform consisting of a photoluminescent nanodiamond (ND) core with uniform porous silica coatings is presented. This design intended for drug delivery applications allows simultaneous stable fluorescent imaging with high loading capacity of bioactive molecules. Despite irregularly shaped starting cores, well-dispersed and uniformly shaped nanocomposite particles can be produced. Moreover, after optimization of the silica source-to-diamond ratio, the thickness of the porous layer can be tuned by adjusting the ethanol amount, allowing rational nanoparticle size control. The ND key property, photoluminescence, is not quenched regardless of coating with thick silica layers. The high loading capacity for incorporation of active agents, provided by the introduced porous layer, is demonstrated by adsorption of a hydrophobic model drug to the composite particles. The loading degree, as compared to a pure ND, increased by two orders of magnitude from 1 wt% for the ND to >100 wt% for the composite particles. Combining these two material classes, which both have well-documented excellent performance especially in biomedical applications, for the NDs with emphasis, but not exclusively, on imaging and mesoporous silica (MSN) on drug delivery, the advantages of both are shown here to be synergistically integrated into one multifunctional nanocomposite platform.