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...
Fluorescent nanodiamond is a new nanomaterial that possesses several useful properties, including good biocompatibility, excellent photostability and facile surface functionalizability. Moreover, when excited by a laser, defect centres within the nanodiamond emit photons that are capable of penetrating tissue, making them well suited for biological imaging applications. Here, we show that bright fluorescent nanodiamonds can be produced in large quantities by irradiating synthetic diamond nanocrystallites with helium ions. The fluorescence is sufficiently bright and stable to allow three-dimensional tracking of a single particle within the cell by means of either one- or two-photon-excited fluorescence microscopy. The excellent photophysical characteristics are maintained for particles as small as 25 nm, suggesting that fluorescent nanodiamond is an ideal probe for long-term tracking and imaging in vivo, with good temporal and spatial resolution.
The CA125 biomarker assay plays an important role in the diagnosis and management of primary invasive epithelial ovarian/tubal cancer (iEOC). However, a fundamental problem with CA125 is that it is not cancer-specific and may be elevated in benign gynecological conditions such as benign ovarian neoplasms and endometriosis. Aberrant O-glycosylation is an inherent and specific property of cancer cells and could potentially aid in differentiating cancer from these benign conditions, thereby improving specificity of the assay. We report on the development of a novel microarray-based platform for profiling specific aberrant glycoforms, such as Neu5Acα2,6GalNAc (STn) and GalNAc (Tn), present on CA125 (MUC16) and CA15-3 (MUC1). In a blinded cohort study of patients with an elevated CA125 levels (30-500 kU/L) and a pelvic mass from the UK Ovarian Cancer Population Study (UKOPS), we measured STn-CA125, ST-CA125 and STn-CA15-3. The combined glycoform profile was able to distinguish benign ovarian neoplasms from invasive epithelial ovarian/tubule cancer (iEOCs) with a specificity of 61.1% at 90% sensitivity. The findings suggest that microarray glycoprofiling could improve differential diagnosis and significantly reduce the number of patients elected for further testing. The approach warrants further investigation in other cancers.
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