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Fluorescent Nanodiamonds for In Vitro and In Vivo Biological Imaging


Summary There is a need to develop a biocompatible fluorescent label that never photobleaches or blinks, and that is brighter than commonly used dyes. Fluorescent nanodiamonds (FNDs) are 10 to 100 nm sized biocompatible particles with indefinite photo-stability that make them superior imaging probes for a wide range of applications. Whereas organic dyes and quantum dots are neither biocompatible nor photo-stable as they photobleach and blink, and gold nanoparticles exhibit weak, size and shape dependent fluorescence, FNDs do not photo-bleach or blink and can provide bright fluorescence. In particular, their near-infrared fluorescence and biocompatibility make them ideally suited for in vivo diagnostic applications. The commercial potential for fluorescent nanodiamonds is enormous. Because of their superior fluorescence characteristics and inherent biocompatibility, FNDs could replace the most commonly used optical probes; quantum dots (QDs) and organic fluorophores. The nanodiamond fluorescence comes from nitrogen-vacancy (N-V) centers, point defects in the diamond structure. By adjusting the number of N-V centers created in a particle, its brightness can be tuned for a desired application. FND near-infrared emissions are not only optimal for in vivo imaging but also can be used as an optical readout of magnetic resonance. Furthermore, the relatively long fluorescence lifetime (~17 ns) of FNDs compared to ~1-2 ns lifetime of in vivo autofluorescence makes FNDs ideal background-free agents for time-gated imaging of, for instance various, cardiac myopathies or blood malignancies where typically blood hemoglobin interference in fluorescence spectrum has limited the uses of optical imaging for these pathologies. At the single-molecule level, they can be used to track labeled biomolecules over extended periods of time, and due to their wide excitation spectra, can be used as stable multispectral fiducial markers for ultra high resolution microscopy across multiple wavelengths to study sub cellular structures with nm precision. While these are just a few of the biomedical applications of FNDs, the energy level structure and electron spin coherence of N-V centers have potential novel applications in ultra-low magnetic field detection, ultra-sensitive NMR, ultra-low power consuming spin-based spintronics, and quantum computing. The commercial routes to develop this product for are numerous and highly profitable. Project Goals Currently there is no commercial source of fluorescent nanodiamonds appropriate for biomedical imaging applications. This is a rapidly emerging field that would be well served by a source of well characterized FNDs that could be further processed by the end user for a wide range of applications in biomedical imaging and nanotechnology. Phase I Activities and Expected Deliverables In Phase I, we expect 100 grams of fluorescent nanodiamonds. The mean diameter of the nanodiamonds should be in the range of 10 to 80 nm, with a coefficient of variation not to exceed 60%. The peak fluorescence emission of the nanodiamonds will be in the range of 650-750 nm and they will be photostable, i.e., not photobleach, under continuous laser excitation of 20 mW or less in the range of 500-600 nm. A minimum of 50% of the fluorescent nanodiamonds will be at least 10 times brighter (i.e., 10-fold higher fluorescence emission at the peak emission wavelength with an optical bandwidth of 30 nm) than Alexa680, which is a commonly used near infrared dye. These specifications can be confirmed with total internal reflection fluorescence microscopy (TIRFM) measurements in which the brightness of fluorescent nanodiamonds and Alexa680 can be compared side-by-side under identical conditions. We are prepared to assist with these measurements if requested. We have successfully made fluorescent nanodiamonds (~30 nm diameter), but their brightness must be improved with optimization of the N-V center creation and annealing process. The contracting company will be expected to optimize the process, deliver well-characterized fluorescent nanodiamonds, and provide a description of the irradiation, annealing, and any additional processing such that an expert in the field could reproduce the process. We can assist the company with the characterization of the nanodiamonds. Phase II Activities and Expected Deliverables In Phase II, the deliverables will be a range of FNDs with different levels of brightness and different sizes. Furthermore, the more challenging deliverable in Phase II will be a high-yield product with narrow size and brightness distributions. These well-defined distributions can either be achieved by determining a method that generates the desired distributions directly, or by separating the fluorescent nanodiamonds based on size and brightness after the fact, a technique that would solve a problem that the fields of nanotechnology and molecular imaging have been struggling with.
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