Characterization and application of single fluorescent nanodiamonds as cellular biomarkers

Abstract

Type Ib diamonds emit bright fluorescence at 550 -800 nm from nitrogen-vacancy point defects, (N-V) 0 and (N-V) ؊ , produced by high-energy ion beam irradiation and subsequent thermal annealing. The emission, together with noncytotoxicity and easiness of surface functionalization, makes nano-sized diamonds a promising fluorescent probe for single-particle tracking in heterogeneous environments. We present the result of our characterization and application of single fluorescent nanodiamonds as cellular biomarkers. We found that, under the same excitation conditions, the fluorescence of a single 35-nm diamond is significantly brighter than that of a single dye molecule such as Alexa Fluor 546. The latter photobleached in the range of 10 s at a laser power density of 10 4 W/cm 2 , whereas the nanodiamond particle showed no sign of photobleaching even after 5 min of continuous excitation. Furthermore, no fluorescence blinking was detected within a time resolution of 1 ms. The photophysical properties of the particles do not deteriorate even after surface functionalization with carboxyl groups, which form covalent bonding with polyL-lysines that interact with DNA molecules through electrostatic forces. The feasibility of using surface-functionalized fluorescent nanodiamonds as single-particle biomarkers is demonstrated with both fixed and live HeLa cells.blinking ͉ photobleaching ͉ single-molecule detection ͉ single-particle tracking ͉ live cell O ne of the key avenues to understanding how biological systems function at the molecular level is to probe biomolecules individually and observe how they interact with each other directly in vivo. Laser-induced fluorescence is a technique widely adopted for this purpose owing to its ultrahigh sensitivity and capabilities of performing multiple-probe detection (1-3). However, in applying this technique to imaging and tracking a single molecule or particle in a biological cell, progress is often hampered by the presence of ubiquitous endogenous components such as flavins, nicotinamide adenine dinucleotides, collagens, and porphyrins that produce high fluorescence background signals (4-6). These biomolecules typically absorb light at wavelengths in the range of 300-500 nm and fluoresce at 400-550 nm (Fig. 1). To avoid such interference, a good biological fluorescent probe should absorb light at a wavelength longer than 500 nm and emit light at a wavelength longer than 600 nm, at which the emission has a long penetration depth through cells and tissues (5, 7). Organic dyes and fluorescent proteins are two types of molecules often used to meet such a requirement (1,8,9); however, the detrimental photophysical properties of these molecules, such as photobleaching and blinking, inevitably restrict their applications for long-term in vitro or in vivo observations. Fluorescent semiconductor nanocrystals (or quantum dots), on the other hand, have gained considerable attention in recent years because they hold a number of advantageous features including high photobleaching thresholds a...

What do diffusion measurements tell us about membrane compartmentalisation? Emergence of the role of interprotein interactions

“…This should allow a more accurate detection of the molecules in a longer time and could circumvent the main drawbacks of single-molecule tracking (see "Appendix 1"). The emergence of nanodiamonds is also a promising alternative since these particles emit bright fluorescence in the absence of photobleaching and, unlike quantum dots, they do not blink [74]. The improvement of fluorescent molecules will also benefit to Förster resonance energy transfer to detect interactions on a molecular scale [75].…”

Luminescent nanodiamonds for biomedical applications

“…In the context of bioimaging, the most noteworthy feature, of the more abundant, negatively charged NV centre is that it can be optically excited between 480 and 580 nm to emit a broad luminescence band centred at 690 nm ( Fig. 1b) with an excited state lifetime of approximately 17 ns (Fu et al 2007).…”

“…These preparative steps have enabled a variety of (Mohan et al 2010b) 0.99 (Mohan et al 2010b) 7.9 × 10 3 Photostable, only blinks when NV centre is close to surface (Bradac et al 2010) Non-toxic below 400 μg/mL ( biologically active molecules to be non-covalently (Table 4) or covalently (Table 5) attached to nanodiamonds. For instance, poly-L-lysine has been both physisorbed (Huang and Chang 2004;Kong et al 2005a;Huang et al 2008) and covalently bonded via carbodiimide chemistry (Fu et al 2007;Lim et al 2009) onto the surface of acid-oxidised nanodiamonds. Other nanodiamonds have been reduced with borane, had silanes grafted to their surfaces and then were covalently bonded to biotin, a widely used universal receptor for streptavidin in biological labelling techniques (Neugart et al 2007;.…”

“…2 A nanodiamond with a mixed composition of surface groups can be oxidised and terminated with oxygencontaining functional groups or reduced to produce a homogeneous hydrogen surface termination bead milling and sonication (Krüger et al 2005;Fu et al 2007;Faklaris et al 2008). Through a combination of these techniques we were first able to observe NV centres in isolated 5-nm nanodiamonds (Bradac et al 2010).…”

“…-apoobelin ) -aflatoxin B1 (AfB1) ) -DNA (Fu et al 2007) -luciferase Puzyr' et al 2004) -Alpha-bungarotoxin (Liu et al 2008 (Miller and Brown 1996;Knickerbocker et al 2003;Lu et al 2004;Strother et al 2002;Yang et al 2002;Takahashi et al 2003;Yeap et al 2008;Zhang et al 2009b;Zheng et al 2009;Chang et al 2010) (Strother et al 2002;Takahashi et al 2003;Ida et al 2003;Tsubota et al 2003;Härtl et al 2004;Christiaens et al 2006;Chang et al 2010) (Liu et al 2004;Ando et al 1996b;Khabashesku et al 2005;Ando et al 1996a;Ando et al 1993) (Ando et al 1996a;Ikeda et al 1998;Takahashi et al 2003;Miller and Brown 1996) Silanes Aryl Alkyl Amino (Zhang et al 2009b;Liang et al 2009;Krüger et al 2006;Yeap et al 2008 Zheng et al 2009) choice of biological buffer systems or media is also important, since both foetal bovine serum and phosphate buffered saline, two common biological buffer systems, tend to cause re-aggregation (Neugart et al 2007;Vaijayanthimala et al 2009). Despite the challenges involved in preparing and maintaining de-aggregated nanodiamonds, positive results strongly support their use in biological applications.…”