Nano-Diamond-Enhanced Integrated Response of a Surface Plasmon Resonance Biosensor.

Surface plasmon resonance (SPR) sensing is a real-time detection technique for measuring biomolecular interactions on gold surfaces. This study presents a novel approach using nano-diamonds (NDs) on a gold nano-slit array to obtain an extraordinary transmission (EOT) spectrum for SPR biosensing. We used anti-bovine serum albumin (anti-BSA) to bind NDs for chemical attachment to a gold nano-slit array. The covalently bound NDs shifted the EOT response depending on their concentration. The number of ND-labeled molecules attached to the gold nano-slit array was quantified from the change in the EOT spectrum. The concentration of anti-BSA in the 35 nm ND solution sample was much lower than that in the anti-BSA-only sample (approximately 1/100). With the help of 35 nm NDs, we were able to use a lower concentration of analyte in this system and obtained better signal responses. The responses of anti-BSA-linked NDs had approximately a 10-fold signal enhancement compared to anti-BSA alone. This approach has the advantage of a simple setup and microscale detection area, which makes it suitable for applications in biochip technology.

Factors Related to COVID-19-Preventive Behaviors among Flight Attendants.

The health and safety of airline employees have been important issues during the 2019 coronavirus disease (COVID-19) pandemic. The exposure of flight attendants to passengers with severe acute respiratory syndrome coronavirus 2 infection without protective equipment is known to cause in-flight transmission and the worldwide spread of the virus. However, very few studies have focused on flight attendants' COVID-19-preventive behaviors and related factors. This cross-sectional study was performed to investigate relationships between COVID-19-preventive behaviors and relevant factors in a convenience sample of flight attendants. In total, 261 flight attendants working for two international airlines were recruited. A self-developed questionnaire was used to assess participants' COVID-19 information-seeking behavior, perceived susceptibility, severity, self-efficacy, and preventive behaviors. Analysis of variance and Pearson's correlation analysis were performed to analyze COVID-19 preventive behaviors according to socio-demographic and COVID-19-related factors. Multiple regression analysis was used to predict the flight attendants' COVID-19-preventive behaviors. The factors that primarily influenced participants' COVID-19-preventive behaviors were gender (women; ß = 0.17, p < 0.001), information-seeking behavior (ß = 0.39, p < 0.000), perceived severity (ß = 0.130, p < 0.05), and self-efficacy (ß = 0.17, p < 0.05). These factors explained 30.6% of the variance in COVID-19-preventive behaviors among flight attendants. Identification of the preventive behaviors performed by this population is important, as it aids the development of strategies to enhance such behaviors, thereby reducing the in-flight spread of COVID-19.

Manipulating the distribution of electric field intensity to effectively enhance the spatial and spectral fluorescence intensity of fluorescent nanodiamonds.

Fluorescent nanodiamonds (FNDs) having nitrogen-vacancy (NV) centers have drawn much attention for their biocompatibility and stable optical properties. Nevertheless, the NV centers are located in the interior of the FNDs, and it has not been possible to increase the fluorescence intensity of FNDs efficiently using previously developed enhancement methods. In this paper, we present a simple nanocavity structure that enhances the fluorescence intensity of FNDs. The designed Al/SiO2 nanocavities are stable and inexpensive, and provide a large region for efficient enhancement of fluorescence that can cover most 100 nm FNDs. By tuning the thickness of the capping SiO2 layer of the Al/SiO2 nanocavities, the distributions of both the spatial and spectral electric field intensities of the FNDs could be controlled and manipulated. In general, the FNDs were excited using a green-yellow laser; the broadband fluorescence of the FNDs comprised the emissions from neutral (NV0) and negatively charged (NV-) NV centers. To enhance the fluorescence intensity from the NV- centers of the FNDs, we designed an Al/70 nm SiO2 nanocavity to function at excitation and emission wavelengths of 633 and 710 nm, respectively, allowing the NV- centers to be excited efficiently; as a result, we achieved an enhancement in fluorescence intensity of 11.2-fold. Moreover, even when we covered 100 nm FNDs with polyglycerol (forming p-FND), the fluorescence intensities of the p-FND particles placed on the nanocavities remained greatly enhanced.

Simultaneous cathodoluminescence and electron microscopy cytometry of cellular vesicles labeled with fluorescent nanodiamonds.

Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ≈150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level.

Preparation and Characterization of Ion-Irradiated Nanodiamonds as Photoacoustic Contrast Agents.

Highly radiation-damaged or irradiated nanodiamonds (INDs) are a new type of nanomaterial developed recently as a potential photoacoustic (PA) contrast agent for deep-tissue imaging. This work characterized in detail the photophysical properties of these materials prepared by ion irradiation of natural diamond powders using various spectroscopic methods. For 40-nm NDs irradiated with 40-keV He+ at a dose of 3 x 10(15) ions/cm2, an average molar extinction coefficient of 4.2 M-1 cm-1 per carbon atom was measured at 1064 nm. Compared with gold nanorods of similar dimensions (10 nm x 67 nm), the INDs have a substantially smaller (by > 4 orders of magnitude) molar extinction coefficient per particle. However, the deficit is readily compensated by the much higher thermal stability, stronger hydrophilic interaction with water, and a lower nanobubble formation threshold (~30 mJ/cm2) of the sp3-carbon-based nanomaterial. No sign of photodamage was detected after high-energy (>100 mJ/cm2) illumination of the INDs for hours. Cell viability assays at the IND concentration of up to 100 µg/mL showed that the nanomaterial is non-cytotoxic and potentially useful for long-term PA bioimaging applications.

Targeted nanodiamonds as phenotype-specific photoacoustic contrast agents for breast cancer.

The aim is to develop irradiated nanodiamonds (INDs) as a molecularly targeted contrast agent for high-resolution and phenotype-specific detection of breast cancer with photoacoustic (PA) imaging. The surface of acid treated radiation-damaged nanodiamonds was grafted with PEG to improve its stability and circulation time in blood, followed by conjugation to an anti-HER2 peptide with a final nanoparticle size of approximately 92 nm. Immunocompetent mice bearing orthotopic HER2-positive or negative tumors were administered INDs and PA imaged using an 820-nm near-infrared laser. PA images demonstrated that INDs accumulate in tumors and completely delineated the entire tumor within 10 h. HER2 targeting significantly enhanced imaging of HER2-positive tumors. Pathological examination demonstrated INDs are nontoxic. PA technology is adaptable to low-cost bedside medicine, and with new contrast agents described herein, PA can achieve high-resolution (sub-mm) and phenotype-specific monitoring of cancer growth.

Unambiguous observation of shape effects on cellular fate of nanoparticles.

Cellular fate of nanoparticles is vital to application of nanoparticles to cell imaging, bio-sensing, drug delivery, suppression of drug resistance, gene delivery, and cytotoxicity analysis. However, the current studies on cellular fate of nanoparticles have been controversial due to complications of interplay between many possible factors. By well-controlled experiments, we demonstrated unambiguously that the morphology of nanoparticles independently determined their cellular fate. We found that nanoparticles with sharp shapes, regardless of their surface chemistry, size, or composition, could pierce the membranes of endosomes that carried them into the cells and escape to the cytoplasm, which in turn significantly reduced the cellular excretion rate of the nanoparticles. Such features of sharp-shaped nanoparticles are essential for drug delivery, gene delivery, subcellular targeting, and long-term tracking. This work opens up a controllable, purely geometrical and hence safe, degree of freedom for manipulating nanoparticle-cell interaction, with numerous applications in medicine, bio-imaging, and bio-sensing.

Multi-color imaging of fluorescent nanodiamonds in living HeLa cells using direct electron-beam excitation.

Multi-color, high spatial resolution imaging of fluorescent nanodiamonds (FNDs) in living HeLa cells has been performed with a direct electron-beam excitation-assisted fluorescence (D-EXA) microscope. In this technique, fluorescent materials are directly excited with a focused electron beam and the resulting cathodoluminescence (CL) is detected with nanoscale resolution. Green- and red-light-emitting FNDs were employed for two-color imaging, which were observed simultaneously in the cells with high spatial resolution. This technique could be applied generally for multi-color immunostaining to reveal various cell functions.

Creation of high density ensembles of nitrogen-vacancy centers in nitrogen-rich type Ib nanodiamonds.

This work explores the possibility of increasing the density of negatively charged nitrogen-vacancy centers ([NV(-)]) in nanodiamonds using nitrogen-rich type Ib diamond powders as the starting material. The nanodiamonds (10-100 nm in diameter) were prepared by ball milling of microdiamonds, in which the density of neutral and atomically dispersed nitrogen atoms ([N(0)]) was measured by diffuse reflectance infrared Fourier transform spectroscopy. A systematic measurement of the fluorescence intensities and lifetimes of the crushed monocrystalline diamonds as a function of [N(0)] indicated that [NV(-)] increases nearly linearly with [N(0)] at 100-200 ppm. The trend, however, failed to continue for nanodiamonds with higher [N(0)] (up to 390 ppm) but poorer crystallinity. We attribute the result to a combined effect of fluorescence quenching as well as the lower conversion efficiency of vacancies to NV(-) due to the presence of more impurities and defects in these as-grown diamond crystallites. The principles and practice of fabricating brighter and smaller fluorescent nanodiamonds are discussed.

Photoacoustic contrast imaging of biological tissues with nanodiamonds fabricated for high near-infrared absorbance.

Radiation-damaged nanodiamonds (DNDs) are potentially ideal optical contrast agents for photoacoustic (PA) imaging in biological tissues due to their low toxicity and high optical absorbance. PA imaging contrast agents have been limited to quantum dots and gold particles, since most existing carbon-based nanoparticles, including fluorescent nanodiamonds, do not have sufficient optical absorption in the near-infrared (NIR) range. A new DND by He+ ion beam irradiation with very high NIR absorption was synthesized. These DNDs produced a 71-fold higher PA signal on a molar basis than similarly dimensioned gold nanorods, and 7.1 fmol of DNDs injected into rodents could be clearly imaged 3 mm below the skin surface with PA signal enhancement of 567% using an 820-nm laser wavelength.

In vivo photoacoustic imaging of breast cancer tumor with HER2-targeted nanodiamonds.

Radiation-damaged nanodiamonds (NDs) are ideal optical contrast agents for photoacoustic (PA) imaging in biological tissues due to their good biocompatibility and high optical absorbance in the near-infrared (NIR) range. Acid treated NDs are oxidized to form carboxyl groups on the surface, functionalized with polyethylene glycol (PEG) and human epidermal growth factor receptor 2 (HER2) targeting ligand for breast cancer tumor imaging. Because of the specific binding of the ligand conjugated NDs to the HER2-overexpressing murine breast cancer cells (4T1.2 neu), the tumor tissues are significantly delineated from the surrounding normal tissue at wavelength of 820 nm under the PA imaging modality. Moreover, HER2 targeted NDs (HER2-PEG-NDs) result in higher accumulation in HER2 positive breast tumors as compared to non-targeted NDs after intravenous injection (i.v.). Longer retention time of HER-PEG-NDs is observed in HER2 overexpressing tumor model than that in negative tumor model (4T1.2). This demonstrates that targeting moiety conjugated NDs have great potential for the sensitive detection of cancer tumors and provide an attractive delivery strategy for anti-cancer drugs.

Photoacoustic emission from fluorescent nanodiamonds enhanced with gold nanoparticles.

Fluorescent nanodiamonds (FNDs) have drawn much attention in recent years for biomedical imaging applications due to their desired physical properties including excellent photostability, high biocompatibility, extended far-red fluorescence emission, and ease of surface functionalization. Here we explore a new feature of FNDs, i.e. their photoacoustic emission capability, which may lead to potential applications of using FNDs as a dual imaging contrast agent for combined fluorescence and photoacoustic imaging modalities. We observed significant enhancement of photoacoustic emission from FNDs when they were conjugated with gold nanoparticles (GNPs).

The exocytosis of fluorescent nanodiamond and its use as a long-term cell tracker.

Fluorescent nanodiamond (FND) has excellent biocompatibility and photostability, making it well suited for long-term labeling and tracking of cancer and stem cells. To prove the concept, the exocytosis of FND particles (size ≈100 nm) from three cell lines--HeLa cervical cancer cells, 3T3-L1 pre-adipocytes, and 489-2.1 multipotent stromal cells--is studied in detail. FND labeling is performed by incubating the cells in a serum-free medium containing 80 µg mL(-1) FND for 4 h. No significant alteration in growth or proliferation of the FND-labeled cells, including the multipotent stromal cells, is observed for up to 8 days. Flow cytometric analysis, in combination with parallel cell doubling-time measurements, indicates that there is little (≈15% or less) excretion of the endocytosed FND particles after 6 days of labeling for both HeLa and 489-2.1 cells, but exocytosis occurs more readily (up to 30%) for 3T3-L1 preadipocytes. A comparative experiment with FND and the widely used dye, carboxyfluorescein diacetate succinimidyl ester, demonstrates that the nanoparticle platform is a promising alternate probe for long-term cell labeling and tracking applications.

Receptor-mediated cellular uptake of folate-conjugated fluorescent nanodiamonds: a combined ensemble and single-particle study.

Fluorescent nanodiamonds (FNDs) are nontoxic and photostable nanomaterials, ideal for long-term in vivo imaging applications. This paper reports that FNDs with a size of approximately 140 nm can be covalently conjugated with folic acid (FA) for receptor-mediated targeting of cancer cells at the single-particle level. The conjugation is made by using biocompatible polymers, such as polyethylene glycol, as crosslinked buffer layers. Ensemble-averaged measurements with flow cytometry indicate that more than 50% of the FA-conjugated FND particles can be internalized by the cells (such as HeLa cells) through receptor-mediated endocytosis, as confirmed by competitive inhibition assays. Confocal fluorescence microscopy reveals that these FND particles accumulate in the perinuclear region. The absolute number of FNDs internalized by HeLa cells after 3 h of incubation at a particle concentration of 10 microg mL(-1) is in the range of 100 particles per cell. The receptor-mediated uptake process is further elucidated by single-particle tracking of 35-nm FNDs in three dimensions and real time during the endocytosis.

Mass production and dynamic imaging of fluorescent nanodiamonds.

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.