Scientists from two Taiwanese universities have developed single fluorescent nanodiamonds and demonstrated their potential as biomarkers that track single particles in live-cell imaging, according to a study published this week.
The particles, originally developed for industrial surface-finishing applications, show promise as labels for long-term cellular imaging, drug or gene carriers, and even tools for targeting tumors in vivo, the scientists said. These potential benefits, however, are tempered by questions about the nanodiamonds’ size and photophysical properties.
Research on the nanodiamonds, published in the Jan. 9 online advance edition of the Proceeding of the National Academy of Sciences, was conducted by scientists from Academia Sinica and National Taiwan University, both in Taipei.
In their paper, the scientists described how the nanodiamonds stack up against other common cellular biomarkers such as organic fluorescent dyes, fluorescent proteins, and semiconductor nanocrystals, also known as quantum dots.
According to the researchers, good biological probes should absorb light at a wavelength longer then 500 nm and emit light at wavelengths longer than 600 nm to avoid interference from endogenous cellular fluorescence, which is usually in the range of 400 to 550 nm.
Though many organic fluorescent dyes and proteins have been engineered to such specifications, they typically suffer from rapid photobleaching and blinking, which hinders their use in long-term imaging studies.
The advantages of quantum dots as long-term imaging probes, meantime, have been well-established. But quantum dots are, at their core, toxic, and scientists are still working on reducing their cytotoxicity through complex surface chemistry.
Another disadvantage of quantum dots is that their stellar optical properties may be compromised by functionalizing their surfaces to label biological molecules.
The researchers believe that nanoparticles based on insulators such as nanodiamonds do not suffer from such limitations. In a previous paper, they showed that diamond crystallites 100 nm in size were capable of producing stable fluorescence after their surfaces were treated with acids. Depending on their preparation, the diamonds emit fluorescence in the range of 550 to 800 nm.
As described in the PNAS paper, the scientists more recently prepared nanodiamonds on the order of 35 nm and observed the particles on a glass substrate and in HeLa cells. For the cellular experiments, they incubated nanodiamonds with cultured HeLa cells and used confocal and wide-field epifluorescence microscopy to observe them.
The particles fluoresced approximately as intensely as quantum dots – that is, significantly more intensely than almost all fluorescent dyes – and were found to be distributed primarily in the cells’ cytoplasm. Although many of the nanodiamonds aggregated within the cytoplasm, the scientists were also able to detect some isolated particles.
Furthermore, the researchers demonstrated that the nanodiamonds’ surfaces could be functionalized so as to bind biomolecules without compromising their optical qualities, and specifically showed how they could be made to non-specifically bind DNA through electrostatic interactions.
Lastly, they were able to track single nanoparticles through the cytoplasm of live HeLa cells over time, and demonstrated that the nanodiamonds did not photobleach even after five minutes of continuous excitation.
In fact, in an e-mail to CBA News, corresponding author Wunshain Fann wrote that the particles’ “fluorescence never quenches, which means that you can track a single [fluorescent nanodiamond] in [a] cell [or] tissue forever.”
In the PNAS paper, the scientists wrote that all of their experiments showed that the nanodiamonds are “promising biomarker candidate[s] for in vivo imaging and diagnosis” and are a “promising material to be used as a fluorescent biomarker for in vitro as well as in vivo studies at the single-molecule level.”
Still in the Rough
But the particles are far from ready for prime time, according to some scientists. For example, even at 35 nm, the nanodiamonds are comparable in size to quantum dots, and thus may suffer from similar limitations in long-term live-cell imaging. And although they may not suffer from blinking as much as some fluorescent dyes, they still might not be perfect for long-term imaging studies.
Esther Conwell, a professor of chemistry at the University of Rochester and principal editor of the PNAS paper, cautioned that the particles, while promising, could not be considered a panacea for cellular imaging.
“The size [of the nanodiamonds] is a limitation, as is how rapidly they blink. Apparently, they don’t blink very rapidly, but if you wanted to look at something for a long time, that might be a limitation.”
“They are certainly capable of extending our knowledge to some extent,” Conwell told CBA News this week. “But they may not be the right thing for finding out everything that goes on in a cell. The size is a limitation, as is how rapidly they blink. Apparently, they don’t blink very rapidly, but if you wanted to look at something for a long time, that might be a limitation.”
However, in his e-mail to CBA News, Fann said that “there is no blinking, both for short- and long-term observation.”
Rochester’s Conwell also said that she collaborated with two referees to judge the research paper, and while one viewed the nanodiamonds as a positive development, the other was more skeptical.
“The main concern was that the crystals might be too large,” Conwell said.
It appears as if the Fann and colleagues are aware of the possible size limitations, and have plans to address them. “For future applications where smaller-sized particles are required, it is possible to employ standard separation methods to extract [fluorescent nanodiamonds] with a size that is in the range of 10 nm from the current sample,” they wrote. Each of these particles, they added, is expected to have similar photostability to the 35-nm particles.
“We are working on reducing the size,” Fann wrote in his e-mail to CBA News. “Actually, the quantum dot, [at about] 5 nm, is not that much smaller than 10 nm. Although I agree that it might not be easy to do [Förster resonance energy transfer] types of work with such size.”
If scientists are able to address some of the particles’ limitations, they may find utility in a variety of biomedical research applications, particularly long-term live-cell imaging both ex vivo and in vivo. A separate research article published in the January 11 issue of Journal of Physical Chemistry provided for the first time evidence that carbon-based nanomaterials, including diamond nanoparticles, are not toxic to a variety of cell types.
Therefore, the particles could be used for many of the applications that have shown potential with quantum dots but have been held back due to their toxicity, the Taiwanese researchers wrote in PNAS.
“A natural extension of the present application to in vivo studies includes the use of [fluorescent nanodiamonds] as a drug or gene carrier, as a device for tumor targeting, and as a fluorescent probe for two-photon confocal microscopy,” the researchers wrote.
Fann wrote that he and his colleagues are exploring avenues for commercializing the nanodiamonds as cellular probes, but declined to provide further detail. He also said that the group is eyeing several areas of continued research with the particles.
“We are pursuing several directions; however, it is not mature enough to talk about them,” Fann wrote.